TGF-β trap

Abstract

Compositions and methods for inhibiting TGF-β are provided. A trap molecule is provided in which the ligand binding domain of the transforming growth factor-β receptor type 2 (TGFβRII) is fused to an immunoglobulin Fc domain containing an N-terminal immunoglobulin hinge region, wherein at least one unpaired cysteine ​​residue in the hinge region is replaced by a serine residue.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 887,272, filed on August 15, 2019, the entire contents of which are incorporated herein by reference.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing submitted as an electronic text file, entitled "8774-14-PCT_Seq_Listing_ST25.txt", which is 68 bytes in size and was created on August 14, 2020. Pursuant to 37 CFR §1.52(e)(5), the information contained in this electronic file is hereby incorporated by reference in its entirety. Technical Field

[0005] Compositions and methods for inhibiting transforming growth factor beta (TGF-β) activity are provided. Background Art

[0006] TGF-β is a multifunctional cytokine family, which participates in cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses. TGF-β expression has been studied in a variety of cancers, including prostate cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, skin cancer, and glioma. In early tumors, the level of elevated TGF-β is associated with good prognosis, while in late tumors, the level of elevated TGF-β is associated with tumor invasiveness and poor prognosis. More specifically, TGF-β promotes cancer cell movement, invasion, epithelial to mesenchymal transition (EMT) and cell stemness, which is conducive to tumor progression and metastasis.

[0007] There are three isoforms of human TGF-β: TGF-β1, TGF-β2, and TGF-β3. Each isoform occurs as a 25 kDa homodimer in which two 112 amino acid monomers are linked by an interchain disulfide bridge. TGF-β1 differs from TGF-β2 and TGF-β3 by 27 and 22 (mostly conservative) amino acid changes, respectively. Dysregulation of TGF-β is associated with many diseases, such as birth defects, cancer, chronic inflammatory diseases, autoimmune diseases, and fibrotic diseases.

[0008] TGF-β signaling occurs through a superfamily of receptors that can be divided into two groups of transmembrane proteins: type I and type II serine / threonine kinases. Whether a type I or type II receptor binds first is ligand-dependent, and a second type I or type II receptor is then recruited to form a heteromeric signaling complex. A functional receptor complex has a dimeric ligand that interacts with two type I and two type II receptors. The type I receptors are known as activin-like kinases (ALKs), while the type II receptors are named for the ligands they bind. The type II receptor binds TGF-β1 and TGF-β3 with high affinity, but binds TGF-β2 with much lower affinity. Together, the type I and type II receptors form a heterodimeric signaling complex that is essential for the transduction of the antiproliferative signal of TGF-β. There is also a TGF-β type III receptor, but its cytoplasmic domain lacks an obvious signaling motif, and this receptor is probably not directly involved in signal transduction.

[0009] Systemic inhibition of TGF-β has been achieved using a variety of approaches, including antibodies and so-called trap molecules containing a TGF-β receptor domain fused to an antibody Fc domain. See, for example, Gramont et al., Oncoimmunology 6:e1257453 (2017) and De Crescenzo et al., "Engineering TGF-β Traps: Artificially Dimerized Receptor Ectodomains as High-affinity Blockers of TGF-β Action" in Transforming Growth Factor-β in Cancer Therapy, Vol. II, pp. 671-684 (2008). Studies using knockout mice have shown that systemic loss of TGF-β function can lead to reduced wound healing, loss of immunoregulation, and increased inflammatory responses. Therefore, it would be preferable to inhibit TGF-β activity in a tumor-localized manner. Summary of the invention

[0010] Provided is a TGF-β trap containing an immunoglobulin Fc domain fused to a ligand binding domain of a transforming growth factor-β receptor type 2 (TGFβRII), wherein the trap does not contain a Sushi domain, and wherein the immunoglobulin Fc domain further contains an N-terminal immunoglobulin hinge region, wherein at least one unpaired cysteine ​​residue of the hinge region is replaced by a serine residue. In one aspect, the TGFβRII is connected to the Fc region via a flexible peptide linker portion. In certain non-limiting embodiments, the ligand binding domain of the TGFβRII is connected to the carboxyl terminus (C-terminus) of the Fc domain via the flexible peptide linker portion, and in other non-limiting embodiments, the ligand binding domain is connected to the amino terminus (N-terminus) of the hinge region of the Fc domain via the flexible peptide linker portion. In some aspects, the C27 residue of the hinge region is replaced by a serine residue.

[0011] In certain embodiments, the trap may have the structure: NH2-hinge-Fc-linker-TGFβRII-CO2H. In certain embodiments, the trap may have the structure NH2-TGFβRII-linker-hinge-Fc-CO2H. In one aspect, the flexible linker comprises G4S repeats, such as three, four, five, or more G4S repeats.

[0012] In one embodiment, the trap comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:24 or at least 85% identical to SEQ ID NO:25.

[0013] Also provided is a nucleic acid molecule encoding a trap as described above, wherein the trap can be optionally fused to an N-terminal peptide signal sequence. The nucleic acid molecule can be contained in an expression vector. The promoter in the expression vector can be operably linked to the nucleic acid molecule encoding the trap. In certain embodiments, the promoter can be an inducible promoter. In one aspect, the inducible promoter is a TGF-β inducible promoter. In certain aspects, the promoter can be constitutively active, such as a cytomegalovirus (CMV) promoter.

[0014] Also provided is a host cell transformed with a vector as described above.In certain non-limiting embodiments, the host cell can be a mammalian cell, such as a CHO cell or a human cell such as a NK-92 cell.

[0015] In other embodiments, the present invention relates to methods for inhibiting the activity of TGF-β in a subject or patient, the methods comprising administering to a subject in need thereof an effective amount of a trap as described above.

[0016] In further embodiments, the invention relates to methods for inhibiting the activity of TGF-β in a subject, comprising administering to a subject in need thereof a composition comprising a host cell as described above in an amount sufficient to produce an effective amount of a TGF-β trap.

[0017] In other embodiments, the invention relates to methods for treating neoplasia in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a composition containing a host cell as described above. In one aspect, the host cells can be administered parenterally, intravenously, peritumorally, or by infusion.

[0018] In any of the methods of treatment disclosed herein, an additional therapeutic agent may be administered to the subject or patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A and 1B Inhibition of TGF-β response by constitutively active TGF-β trap constructs is shown. These CMV-driven expression constructs contain the Sushi domain (Sushi), unmodified hinge (AltH), Fc domain (Fc) with or without TGFBRII (trap), compared to modified hinge ((C27S)H), Fc with or without TGFBRII (trap).

[0020] Figure 2A and 2B Inhibition of TGF-β response by TGF-β inducible trap constructs is shown. A 293T cell line stably expressing TGF-β-induced luciferase was transfected with an expression construct driven by a TGF-β response element (TGFBRE) and the Sushi domain (Sushi), unmodified hinge (AltH), Fc domain (Fc) with or without TGFBRII (trap) was compared to a modified hinge ((C27S)H), Fc with or without TGFBRII (trap).

[0021] Figure 3 Testing of TGF-β trap expression constructs in stably transfected 293T cells is shown. A 293T cell line stably expressing TGF-β-induced luciferase was transfected with TGF-β response element (TGFBRE) driven expression constructs with the Sushi domain (Sushi), unmodified hinge (AltH), Fc with or without TGFBRII (trap) compared to modified hinge ((C27S)H), Fc with or without TGFBRII (trap).

[0022] Figure 4Shown is testing of N-terminal and C-terminal fusions to the TGF-β trap in stably transfected 293T cells. A 293T cell line stably expressing TGF-β-induced luciferase was transfected with a CMV-driven expression construct to compare the C-terminal (C-terminal) versus the N-terminal (N-terminal) TGF-βRII (trap) Fc fusion protein with a modified hinge ((C27S)H).

[0023] Figure 5A and 5B The ability of the TGF-β trap to neutralize TGF-β2 is shown. A 293T cell line stably expressing TGF-β-induced luciferase was transfected with either CMV-driven or TGF-β response element (TGFBRE)-driven expression constructs and then stimulated with titrations of TGF-β2.

[0024] Fig. 6A and 6B The ability of TGF-β trap to neutralize mouse TGF-β2 is shown. A 293T cell line stably expressing TGF-β-induced luciferase was transfected with CMV-driven or TGF-β response element (TGFBRE)-driven expression constructs (DNA sequences are shown in SEQ ID NOs: 8 and 16) and then stimulated with titrations of mouse TGF-β1 (mTGF-β1). The cells were incubated overnight and the resulting luciferase activity was measured after 24 hours.

[0025] Figure 7 The effects of the Sushi domain and hinge modification on the production of TGF-β-trap are shown. 293T cell lines were transfected with CMV-driven TGFBRII trap Fc fusion protein expression constructs containing the Sushi domain and the original unmodified hinge sequence (AltH), no Sushi domain with AltH, or no Sushi with a modified hinge ((C27S)H). The cells were stationary and cultured in serum-free medium for 24 hours, and the resulting supernatants were concentrated and the levels of human IgG FC were measured. DETAILED DESCRIPTION

[0026] TGF-β trap molecules are provided, which contain an immunoglobulin Fc domain connected to the ligand binding domain of the type 2 transforming growth factor-β receptor (TGFβRII). The immunoglobulin Fc domain contains an improved N-terminal immunoglobulin hinge region, wherein at least one unpaired cysteine ​​residue of the hinge region is replaced, for example, by a serine residue. In certain embodiments, the trap molecule does not contain a Sushi domain. Nucleic acid molecules encoding the trap molecules are provided, together with vectors and expression constructs, which can be used to transfect cells and express the trap molecules. Cells transfected with nucleic acid constructs are also provided, wherein the expression of these trap molecules is under the control of a TGF-β inducible promoter. Methods of using these traps, nucleic acid molecules and transfected cells to treat diseases (such as cancer) are also provided.

[0027] Ligand binding domain

[0028] These trap molecules contain a TGF-β binding domain derived from the extracellular ligand binding portion of the transforming growth factor β receptor II (TGFβRII). The amino acid sequence of TGFβRII is:

[0029]

[0030] Wherein the extracellular domain of the receptor is underlined. These trap molecules may contain some or all of the extracellular domain, provided that the domain retains the ability to bind to the ligand. Advantageously, the trap molecule contains the ligand binding sequence shown below. The skilled person will recognize that amino acids may be added or deleted from the N- and / or C-terminus of the domain, provided that the ligand binding properties of the domain are retained.

[0031]

[0032] Immunoglobulin Fc domain

[0033] The ligand binding domain is connected to a stabilizing protein domain that prolongs the in vivo plasma half-life of the ligand binding domain. Although in principle any extended length amino acid can be used as a stabilizing domain, in certain preferred embodiments, the stabilizing domain is an immunoglobulin constant (Fc) domain. The Fc is advantageously a human Fc and can be of any isotype, although the most commonly used are IgG1 and IgG2 isotypes. Suitable Fc domains for this purpose are well known in the art. See, for example, US 5,428,130; Economides et al., Nature Medicine [Natural Medicine] 9:47 (2003); and Czajkowsky et al., EMBO Mol Med. [European Molecular Biology Organization Molecular Medicine] 4:1015-28 (2012). Suitable Fc sequences are shown below. The technician will recognize that amino acids can be added or deleted from the N- and / or C-terminus of the domain, provided that the stabilizing properties of the domain are retained.

[0034]

[0035] Hinge area

[0036] The ligand binding domain of a naturally occurring immunoglobulin is connected to the Fc domain via a flexible hinge region, and the trap molecules as described herein also contain a modified hinge region fused to the N-terminus of the Fc region. The hinge region can act as a flexible tether and also contains a cysteine ​​moiety that forms an interchain disulfide bond. The naturally occurring hinge region also contains unpaired cysteine ​​residues. It has been surprisingly found that replacing at least one of these unpaired cysteine ​​residues with a hydrophilic residue (e.g., serine) not only provides higher levels of protein expression when these trap molecules are produced in recombinant host cells, but also provides increased activity of the trap in terms of binding to TGF-β. Advantageously, at least the first (closest to the N-terminus) cysteine ​​of the hinge region is replaced with a hydrophilic residue. This corresponds to position C27 of the naturally occurring hinge region or position C5 of SEQ ID NO:29 shown below. This modification of the modified hinge is referred to herein as ((C27S)H). An exemplary modified hinge region is shown below, where the underlined serine residues indicate the position of the cysteine ​​to serine substitution:

[0037] EPKS S DKTHTCPPCP (SEQ ID NO: 4)

[0038] An exemplary unmodified hinge sequence of naturally occurring human IgG1 is: EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 29) (Nezlin, R. General Characteristics of Immunoglobulin Molecules, The Immunoglobulins, 1998)

[0039] Flexible joints

[0040] The trap molecule contains a flexible hydrophilic linker domain placed between the ligand binding domain and the Fc region. Advantageously, as in the well-known (G4S) commonly used for example for ScFv molecules, n In the linker, the linker also lacks secondary structure and is composed of groups of glycine and serine residues. The linker can be about 5-35 amino acids long, and advantageously about 15-30 amino acids long. An exemplary linker contains 5 repeats of G4S. As described in more detail below, the linker can directly connect the C-terminus of the Fc to the N-terminus of the ligand binding domain, or can connect the C-terminus of the ligand binding domain to the N-terminus of the hinge region.

[0041] secretion signal sequence

[0042] The trap molecules as described herein are produced in recombinant eukaryotic host cells (either in cell culture or in an in vivo host cell administered to a patient or subject). Thus, the nucleic acid molecule encoding the trap molecule also encodes an N-terminal signal peptide that directs the newly synthesized protein into the secretory pathway of the host cell. The signal peptide is cleaved from the rest of the protein during the secretion process to produce the mature trap protein. Suitable signal peptides are well known in the art. See, for example, von Heijne, Eur. J. Biochem. 133: 17-21 (1983); Martoglio and Dobberstein, Trends Cell Biol. 8: 410-15 (1988); Hegde and Bernstein., Trends Biochem Sci 31: 563-71 (2006). Advantageously, the signal peptide is an immunoglobulin signal peptide. Exemplary signal peptide sequences are shown below:

[0043] MDWIWRILFLLVGAATGAHSAQPA(SEQ ID NO:5)

[0044] Structure of the trap molecule

[0045] As described above, the hinge region is fused to the N-terminus of the Fc domain, and the ligand binding domain is fused to the hinge-Fc structure via a flexible linker peptide. The ligand binding domain can be located at the N-terminus or the C-terminus relative to the Fc region. When the ligand binding domain is located at the C-terminus, the mature trap protein has the following domain structure:

[0046] NH2-hinge-Fc-linker-TGFβRII-CO2H.

[0047] An exemplary trap molecule having such a structure is given herein as SEQ ID NO: 24. In certain embodiments, the trap molecule may have at least 85% identity to SEQ ID NO: 24, such as 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% identity to SEQ ID NO: 24, provided that a molecule having "X%" identity to SEQ ID NO: 24 is always understood to have a serine at the position corresponding to position 5 of SEQ ID NO: 24, regardless of which other amino acids may have changed relative to the SEQ ID NO: 24 sequence.

[0048] When the ligand binding domain is located at the N-terminus, the mature SNARE protein has a domain structure:

[0049] NH2-TGFβRII-Linker-Hinge-Fc-CO2H.

[0050] An exemplary trap molecule having such a structure is given herein as SEQ ID NO: 25. In certain embodiments, the trap molecule may have at least 85% identity to SEQ ID NO: 25, such as 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% identity to SEQ ID NO: 25, provided that a molecule having "X%" identity to SEQ ID NO: 25 is always understood to have a serine at the position corresponding to position 166 of SEQ ID NO: 25, regardless of which other amino acids may have changed relative to the SEQ ID NO: 25 sequence.

[0051] Nucleic acids and vectors

[0052] Nucleic acid molecules and vectors encoding the trap molecule are provided. Methods for synthesizing these nucleic acid molecules are well known in the art. The nucleic acid sequence can be contained in a vector suitable for extrachromosomal replication, such as a bacteriophage, virus, plasmid, phagemid, cosmid, YAC, or episome. For protein expression, the vector is an expression vector, that is, a vector containing control elements required for transcription and translation of the inserted protein coding sequence. Suitable expression systems include mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g., baculovirus); microorganisms (such as yeast containing yeast vectors, or bacteria transformed with phage DNA, plasmid DNA, or cosmid DNA). Transcription and translation elements suitable for such host-vector systems are well known in the art. General techniques for preparing nucleic acid molecules, cloning, and protein expression are described in, for example, "Molecular Cloning: A Laboratory Manual," Second Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology [Contemporary Laboratory Manual of Immunology]" (Coligan, 1991).

[0053] In certain embodiments, an inducible promoter containing a TGF-β response element controls the expression of the trap molecule. A suitable vector encoding the trap molecule under the control of the inducible promoter is introduced into a suitable host cell. In certain embodiments, the host cell is selected based on the fact that it can be introduced into a patient or subject to be treated with the trap molecule. The expression of TGF-β by the tumor induces the production of the trap in the vicinity of the tumor, reducing or eliminating the TGF-β activity in the vicinity. This method advantageously suppresses or inhibits TGF-β activity only in the vicinity of the tumor expressing TGF-β, while avoiding potential undesirable systemic effects. Suitable TGF-β response elements are known in the art. See, e.g., Zhang and Derynck, J. Biol. Chem. 275:16979 (2000); Grotendorst et al., Cell Growth Differ. 7:469-80 (1996); Riccio et al., Mol. Cell Biol. 12:1846-55 (1992); see also SEQ ID NOs:26-28. Expression vectors containing suitable response elements are commercially available. See pGL4.28 (Promega, Madison, Wisconsin), which contains a TGF-β response element as part of the minP promoter element and is described in further detail below.

[0054] The trap molecule can be produced by introducing a DNA expression vector encoding a trap molecule with an N-terminal signal sequence into a host cell, culturing the host cell in a culture medium under conditions sufficient to express the trap molecule and allow dimerization of the trap molecule, and purifying the dimer soluble trap molecule from the host cells or the culture medium. When the trap molecule is produced and purified in vitro, the expression vector advantageously contains DNA encoding a polypeptide under the control of a CMV constitutive promoter.

[0055] Alternatively, the host cell may be a mammalian cell, in particular a human cell line, which can be introduced into a patient or subject and produce the trap molecule in situ. In this case, the host cell advantageously contains an expression vector encoding the trap molecule under the control of a promoter containing a TGF-β response element.

[0056] In obtaining variant bioactive TGFβRII, hinge, linker, or Fc domain encoding sequences, one of ordinary skill in the art will recognize that these polypeptides can be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, it is well known that conservative amino acid substitutions, i.e., substitution of one amino acid for another amino acid of similar size, charge, polarity, and conformation, are unlikely to significantly alter protein function. The 20 standard amino acids that are components of proteins can be roughly classified into four groups of conservative amino acids as follows: the non-polar (hydrophobic) group includes alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; the polar (uncharged, neutral) group includes asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine; the positively charged (basic) group contains arginine, histidine, and lysine; and the negatively charged (acidic) group contains aspartic acid and glutamic acid. Substitution of one amino acid in a protein by another amino acid in the same group is unlikely to adversely affect the biological activity of the protein. In other cases, the amino acid positions can be modified to reduce or enhance the biological activity of the protein. Such changes can be introduced randomly or via site-specific mutations based on the known or assumed structural or functional properties of one or more target residues. After expressing the variant protein, changes in biological activity caused by the modification can be easily assessed using binding or functional assays.

[0057] Sequence identity between nucleotide sequences can be determined by DNA hybridization analysis, where the stability of double-stranded DNA hybrids depends on the degree of base pairing that occurs. Conditions of high temperature and / or low salt content reduce the stability of the hybrid and can be changed to prevent annealing of sequences with less than a selected degree of identity. For example, for sequences with about 55% GC content, hybridization and washing conditions of 40-50°C, 6x SSC (sodium chloride / sodium citrate buffer) and 0.1% SDS (sodium dodecyl sulfate) show about 60%-70% identity; hybridization and washing conditions of 50°C-65°C, 1x SSC and 0.1% SDS show about 82%-97% identity; and hybridization and washing conditions of 52°C, 0.1x SSC and 0.1% SDS show about 99%-100% identity. Various computer programs for comparing nucleotide and amino acid sequences (and measuring degree of identity) are also available, and a list providing commercially available and free software sources is found in the literature of Ausubel et al. (1999).

[0058] Protein expression

[0059] For the production of purified trap proteins, mammalian cells are advantageously used, in particular CHO, J558, NSO, or SP2-0 cells. Other suitable hosts include, for example, insect cells, such as Sf9. Non-limiting examples of mammalian cell lines that can be used include CHO dhfr cells (Urlaub and Chasm, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59 (1977)) or myeloma cells, like SP2 or NSO (Galfre and Milstein, Meth. Enzymol., 73(B):3 (1981)). Conventional culture conditions are employed. See, Sambrook, supra. Stably transformed or transfected cell lines can then be selected. Cells expressing the trap molecule can be identified by known procedures. For example, the expression of the trap molecule can be determined by ELISA specific for a domain of the trap molecule (such as a binding domain or an Fc domain) and / or by immunoblotting.

[0060] For host cells introduced into a patient and expressing the trap molecule in a TGF-β-dependent manner, human host cells are used. A variety of human host cells can be used, including the patient's own cells that can be removed from the patient. However, advantageously, the host cell is a non-MHC restricted natural killer (NK) cell, and therefore it can be used in essentially any patient without causing an immune response against the host cell in the patient. A suitable NK cell line is the NK-92 cell line known in the art. See, for example, U.S. Patent No. 7,618,817 and Zhang et al., Frontiers in Immunol. [Immunology Frontier], Vol. 8, Article 533 (2017). In a specific embodiment, NK-92 cells are transfected with a suitable nucleic acid construct encoding a trap molecule under the control of a TGF-β inducible control element, and then the NK-92 cells are introduced into the patient by, for example, intravenous or intraperitoneal infusion, or direct injection into a solid tumor or other cancerous lesion.

[0061] Nucleic acids encoding the trap molecule can be introduced into host cells by standard techniques for transfecting cells. The term "transfection" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acids into host cells, including calcium phosphate coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, viral transduction, and / or integration. Suitable methods for transfecting host cells can be found in Sambrook et al., supra, and other laboratory texts.

[0062] Various promoters (transcription initiation regulatory regions) can be used to control the expression of the molecules described herein. The selection of suitable promoters depends on the proposed expression host. Promoters from heterologous sources can be used as long as they work in the selected host. As described above, promoters or CMV promoters that incorporate TGF-β response elements are advantageously used.

[0063] Selective markers are usually used, which can be a part of an expression construct or separated therefrom (for example, carried by an expression vector) so that the marker can be integrated at a site different from the target gene. Examples include conferring antibiotic resistance (for example, for Escherichia coli (E.coli) host cells, bla conferring ampicillin resistance; for a variety of prokaryotic and eukaryotic cells, nptII conferring kanamycin resistance) or allowing the host to grow on a minimal medium (for example, HIS4 makes Pichia pastoris (P.pastoris) or His- Saccharomyces cerevisiae (S.cerevisiae) grow in the absence of histidine) markers. Selective markers have their own transcription and translation initiation and termination regulatory regions to allow independent expression of markers. If antibiotic resistance is used as a marker, the concentration of the antibiotic used for selection will vary according to the antibiotic, and the range is generally from 10 to 600 μg antibiotics / mL medium.

[0064] Expression constructs can be assembled using known recombinant DNA techniques (Sambrook et al., 1989; Ausubel et al., 1999). Restriction enzyme digestion and connection are basic steps for connecting two fragments of DNA. The ends of the DNA fragments may need to be modified before connection, and this can be achieved by filling in overhangs, using nucleases (e.g., ExoIII) to make the end portions of one or more fragments missing, site-directed mutagenesis, or adding new base pairs by PCR. Polymer linkers and adapters can be used to promote the connection of selected fragments. Expression constructs are typically assembled in the stage of using multiple rounds of restriction, connection, and E. coli transformation.

[0065] Many cloning vectors suitable for constructing expression constructs are known in the art (λZAP and pBLUESCRIPTSK-1, Stratagene, La Jolla, California; pET, Novagen Inc., Madison, Wisconsin, cited in Ausubel et al., 1999). The choice of cloning vector will be influenced by the gene transfer system selected for introducing the expression construct into the host cell. At the end of each stage, the resulting construct can be analyzed by restriction, DNA sequence, hybridization and PCR analysis.

[0066] The expression construct can be transformed into the host as a cloning vector construct (linear or circular), or can be removed from the cloning vector and used as is or introduced into the delivery vector. The delivery vector helps to introduce and maintain the expression construct in the selected host cell type. By any of many known gene transfer systems (for example, natural competence, chemically mediated conversion, protoplast transformation, electroporation, biolistic transformation, transfection or conjugation), the expression construct is introduced into the host cell (see Ausubel or Sambrook, the same). Depend on the host cell used and the vector system selection gene transfer system.

[0067] Standard protein purification techniques can be used to isolate the desired trap molecule protein from the culture medium or from harvested cells. In particular, purification techniques can be used to express and purify the desired fusion protein on a large scale (i.e., in at least milligram quantities) from a variety of implementation devices, including spinner bottles, spinner flasks, tissue culture plates, bioreactors, or fermenters.

[0068] The expressed trap proteins can be isolated and purified by known methods. Typically, the culture medium is centrifuged or filtered, and the supernatant is then purified by affinity chromatography or immunoaffinity chromatography (e.g., protein-A or protein-G affinity chromatography or immunoaffinity protocols involving the use of antibodies or other binding molecules that bind to the expressed trap molecules). The trap molecules can be isolated and purified by appropriate combinations of known techniques. These methods include, for example, methods utilizing solubility (such as salt precipitation and solvent precipitation); methods utilizing molecular weight differences (such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis); methods utilizing charge differences (such as ion exchange column chromatography); methods utilizing specific affinity (such as affinity chromatography); methods utilizing hydrophobicity differences (such as reverse phase high performance liquid chromatography); and methods utilizing isoelectric point differences (such as isoelectric focusing electrophoresis), metal affinity columns (such as Ni-NTA). See generally Sambrook or Ausubel, supra.

[0069] The trap molecule is advantageously substantially pure. That is, the fusion protein has been separated from the cellular replacements that naturally accompany it, so that these fusion proteins are preferably present in at least 80% or 90% to 95% homogeneity (w / w). Fusion proteins with at least 98% to 99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified, the fusion protein should be substantially free of contaminants for therapeutic applications. Once partially purified or reaching a substantial purity, the soluble fusion protein can be used for treatment, or for performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques (such as chromatography and gel electrophoresis).

[0070] Pharmacotherapeutic agents

[0071] Pharmaceutical compositions containing trap molecules for use as therapeutic agents are provided. These trap molecules can be administered as protein therapeutics or can be provided in situ by secretion from host cells in a TGF-dependent manner.

[0072] In one aspect, the trap molecule is administered systemically, e.g., formulated in a pharmaceutically acceptable buffer such as saline. Preferred routes of administration include, e.g., instillation into the bladder, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral or intradermal injection, which provide continuous, sustained or effective levels of the composition in the patient. Treatment of human patients or other animals is performed using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. Suitable carriers and formulations thereof are described, e.g., in Remington's Pharmaceutical Sciences, edited by EW Martin.

[0073] The amount of therapeutic agent to be administered varies depending on the mode of administration, the age and weight of the patient, and the clinical symptoms of neoplasia. For example, the dosage may vary from about 1 μg trap / kg body weight to about 5000 mg trap / kg body weight; or from about 5 mg / kg body weight to about 4,000 mg / kg body weight or from about 10 mg / kg body weight to about 3,000 mg / kg body weight; or from about 50 mg / kg body weight to about 2000 mg / kg body weight; or from about 100 mg / kg body weight to about 1000 mg / kg body weight; or from about 150 mg / kg body weight to about 500 mg / kg body weight. For example, the dosage is about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 mg / kg body weight. Alternatively, the dosage is in the range of about 5 mg compound / kg body weight to about 20 mg compound / kg body weight. In another example, the dosage is about 8, 10, 12, 14, 16 or 18 mg / kg body weight. Preferably, the trap molecule is administered at 0.5 mg / kg to about 10 mg / kg (e.g., 0.5, 1, 3, 5, 10 mg / kg). In certain embodiments, the trap is administered at a dosage that enhances the immune response of the subject, or reduces the proliferation, survival, or invasiveness of neoplastic cells, infected cells, or autoimmune cells, as determined by methods known to those skilled in the art.

[0074] As described above, host cells that secrete trap molecules in a TGF-β-dependent manner can be administered to a patient. These cells can be delivered via intravenous or intraperitoneal infusion, or by other methods known in the art, such as by direct injection into a solid tumor or other lesion. In certain embodiments, the patient is administered at least 10 3 cells, for example at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , or at least 10 9 cells.

[0075] Formulation of pharmaceutical compositions

[0076] Administration of the trap molecule can be performed by any suitable method that results in a concentration of the therapeutic agent in combination with the other components that is effective to inhibit TGF-β activity. The trap molecule can be contained in any suitable amount in any suitable carrier substance, and is generally present in an amount of 1%-95% by weight of the total weight of the composition (e.g., at least 10% w / w, at least 15% w / w, at least 20% w / w, at least 25% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, and 95% w / w or about 95% w / w). The composition can be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intracystic, intratumoral or intraperitoneal) administration routes. For example, pharmaceutical compositions are formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.) A. R. Gennaro, Lippincott Williams & Wilkins, eds., 2000, and Encyclopedia of Pharmaceutical Technology, J. Swarbrick and J. C. Boylan, eds., 1988-1999, Marcel Dekker, New York).

[0077] Human dosages are initially determined by extrapolation from the amount of compound used in mice or non-human primates, as the skilled artisan recognizes that it is routine in the art to modify dosages in humans compared to animal models. For example, dosages may vary from about 1 μg compound / kg body weight to about 5000 mg compound / kg body weight; or from about 5 mg / kg body weight to about 4,000 mg / kg body weight or from about 10 mg / kg body weight to about 3,000 mg / kg body weight; or from about 50 mg / kg body weight to about 2000 mg / kg body weight; or from about 100 mg / kg body weight to about 1000 mg / kg body weight; or from about 150 mg / kg body weight to about 500 mg / kg body weight. For example, the dosage is about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 mg / kg body weight. Alternatively, the dosage is in the range of about 5 mg compound / kg body weight to about 20 mg compound / kg body weight. In another example, the dosage is about 8, 10, 12, 14, 16 or 18 mg / kg body weight. Preferably, the trap molecule is administered at 0.5 mg / kg to about 10 mg / kg (e.g., 0.5, 1, 3, 5, 10 mg / kg). Of course, depending on the results of the initial clinical trials and the needs of the particular patient, the dosage can be adjusted upward or downward, as is routinely done in such treatment regimens.

[0078] In certain embodiments, the trap molecules as described herein can be formulated with appropriate excipients into pharmaceutical compositions that release the trap molecules in a controlled manner after administration. Examples include single or multi-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. Preferably, the trap molecules are formulated in excipients suitable for parenteral administration.

[0079] Parenteral compositions

[0080] The pharmaceutical composition comprising the trap molecule is administered parenterally by injection, infusion or implantation (subcutaneously, intravenously, intramuscularly, intratumorally, intracystically, intraperitoneally), in a dosage form, formulation or via a suitable delivery device or implant containing a conventional non-toxic pharmaceutically acceptable carrier and adjuvant. Formulations and preparations of such compositions are well known to those skilled in the art of pharmaceutical formulations. Formulations can be found in Remington: The Science and Practice of Pharmacy (supra). Compositions comprising trap molecules for parenteral use are provided in unit dosage form (e.g., in single-dose ampoules). Alternatively, the composition is provided in a vial containing several doses, and a suitable preservative may be added thereto. The composition is in the form of a solution, suspension, emulsion, infusion device or delivery device for implantation, or is presented as a dry powder to be reconstituted with water or another suitable vehicle before use. The composition may include suitable parenterally acceptable carriers and / or excipients, and may include suspending agents, solubilizers, stabilizers, pH adjusters, tonicity adjusters, and / or dispersing agents.

[0081] As indicated above, the pharmaceutical composition containing the trap molecule can be in a form suitable for sterile injection. To prepare such a composition, the trap molecule is dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used are water, water adjusted to a suitable pH by adding an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methylparaben, ethylparaben or n-propylparaben). In the case where one of the compounds is only trace or slightly soluble in water, a solubility enhancer or solubilizer may be added, or the solvent may include 10%-60% w / w propylene glycol.

[0082] The present disclosure provides methods of inhibiting TGF-β activity or treating neoplasia, infectious diseases, or autoimmune diseases, or symptoms thereof, comprising administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition comprising a trap molecule as described herein. Thus, one embodiment is a method of treating a subject suffering from or susceptible to neoplasia, infectious diseases, or autoimmune diseases, or symptoms thereof. The method comprises the steps of administering to the mammal a therapeutic amount of a trap molecule in an amount sufficient to treat the disease or disorder, or symptoms thereof, under conditions that treat the disease or disorder. A method of inhibiting TGF-β activity is also provided by administering to the mammal an amount of a trap molecule sufficient to inhibit TGF-β activity in the patient or subject to a degree that provides a desired therapeutic benefit, such as slowing or inhibiting tumor growth. Identifying a subject in need of such treatment can be a judgment of the subject or a health care professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0083] These therapeutic and prophylactic methods generally comprise administering a therapeutically effective amount of a trap molecule to a subject (e.g., an animal, a human, including a mammal, particularly a human) in need thereof. Such treatment will be appropriately administered to a subject (particularly a human) in which inhibition of TGF-β activity is desired. Such a patient may be suffering from, have, or be susceptible to or be at risk of a neoplasia, an infectious disease, an autoimmune disease, a disorder, or a symptom thereof. Determination of those subjects who are "at risk" may be made by any objective or subjective determination by diagnostic testing or opinion of the subject or health care provider (e.g., genetic testing, enzyme or protein markers, biomarkers, family history, etc.). These trap molecules may be used to treat any other disorder in which it is desirable to reduce TGF-β activity.

[0084] Methods of monitoring the progress of treatment are also provided. These methods include, for example, determining the level of a diagnostic marker or diagnostic measurement (e.g., TGF-β level) in a subject to which a therapeutic amount of a trap molecule or a host cell secreting a trap molecule has been administered.

[0085] The level of the diagnostic marker or measured value determined in the method can be compared with the known level of the marker in a healthy normal control or other sick patient to establish the disease state of the subject. In some cases, the second level of the marker in the subject is determined at a later time point than the determination of the first level, and the two levels are compared to monitor the course of the disease or the efficacy of the therapy. In certain aspects, the pre-treatment level of the marker in the subject is determined before treatment is started according to the method disclosed herein; the pre-treatment level of the marker can then be compared with the level of the marker in the subject after treatment starts to determine the efficacy of treatment.

[0086] Combination therapy

[0087] Optionally, the trap molecule is administered in combination with any other standard therapy; such methods are known to those skilled in the art and are described in Remington's Pharmaceutical Sciences by EW Martin. If desired, the trap molecule is administered in combination with any conventional anti-tumor therapy or other therapy, including but not limited to immunotherapy, therapeutic antibodies, targeted therapy, surgery, radiation therapy, or chemotherapy.

[0088] Kit or drug system

[0089] The pharmaceutical composition comprising the trap molecule or the host cell expressing the trap molecule can be assembled into a kit or a pharmaceutical system for inhibiting TGF-β in a subject or patient and thereby improving a disease (such as neoplasia, infectious disease or autoimmune disease). The kit or pharmaceutical system can include a carrier (such as a box, a carton, a tube) having one or more containers (such as vials, tubes, ampoules, bottles, etc.) in a defined space therein. These kits or pharmaceutical systems can also include relevant instructions for using the trap molecule.

[0090] definition

[0091] "Improvement" means to reduce, suppress, attenuate, alleviate, halt or stabilize the development or progression of a disease.

[0092] "Analogs" refer to molecules that are not identical but have similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications relative to the naturally occurring polypeptide that enhance the function of the analog. Such biochemical modifications may increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. Analogs may include non-natural amino acids.

[0093] "Binding to a molecule" means having a physicochemical affinity for that molecule.

[0094] "Detecting" refers to identifying the presence, absence, or amount of an analyte.

[0095] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia, autoimmune reactions, and viral infections.

[0096] The terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component refer to an amount of a formulation or component, alone or in combination, sufficient to provide the desired effect. For example, an "effective amount" means the amount of a compound, alone or in combination, required to improve disease symptoms relative to an untreated patient. The effective amount of one or more active compounds used to practice the methods disclosed herein to therapeutically treat a disease varies with the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0097] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany it as found in nature. "Isolated" means the degree of separation from the original source or surrounding environment. "Purified" means a degree of separation greater than isolation.

[0098] A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide as described herein is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). The term "purified" can mean that a nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated proteins that can be purified individually.

[0099] Similarly, "substantially pure" means a nucleotide or polypeptide that has been separated from components that naturally accompany it. Typically, nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95% or even 99% free by weight of proteins and naturally occurring organic molecules with which they are naturally associated.

[0100] "Isolated nucleic acid" means a nucleic acid that does not contain the nucleotides flanking it in the naturally occurring genome of the organism from which it is derived. The term encompasses, for example: (a) DNA that is part of a naturally occurring genomic DNA molecule, but is not flanked by two nucleic acid sequences that flank the part of the molecule in the genome of the organism in which the part of the molecule is naturally present; (b) nucleic acid that is incorporated into a vector or into the genomic DNA of a prokaryotic or eukaryotic organism in a manner such that the resulting molecule is different from any naturally occurring vector or genomic DNA; (c) a separate molecule, such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein). Nucleic acid molecules separated according to the present disclosure further include synthetically produced molecules, as well as any nucleic acid that has been chemically altered and / or has a modified backbone. For example, an isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid (mRNA) molecules.

[0101] "Isolated polypeptide" means a polypeptide that has been separated from the components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% free of proteins and naturally occurring organic molecules that are naturally associated with it by weight. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% polypeptide by weight. An isolated polypeptide can be obtained, for example, by extraction from a natural source, by expressing a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method (e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis).

[0102] "Marker" means any protein or polynucleotide or other molecule having a change in expression level or activity that is associated with a disease or disorder.

[0103] "Neoplasia" means a disease or disorder characterized by excessive proliferation. Exemplary tumors for which the trap molecules disclosed herein can be used include, but are not limited to, leukemias (e.g., acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias, acute myeloblastic leukemias, acute promyelocytic leukemias, acute myelomonocytic leukemias, acute monocytic leukemias, acute erythroleukemias, chronic leukemias, chronic myeloid leukemias, chronic lymphocytic leukemias), polycythemia vera, lymphomas (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain diseases, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcomas, osteogenic sarcomas, chordomas, angiosarcomas, endotheliosarcomas, lymphangiosarcomas, lymphangioendotheliosarcomas, synoviomas, mesotheliomas). In some embodiments, the neoplasia is a type of cancer that is not selected from the group consisting of leukemia, medullary cancer, ovarian cancer, pancreatic cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In particular embodiments, the neoplasia is multiple myeloma, beta cell lymphoma, urothelial / bladder cancer, or melanoma. As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing or otherwise obtaining an agent.

[0104] By "reduced" is meant a decrease of at least 5%, such as at least 10%, at least 25%, at least 50%, at least 75%, or even 100%.

[0105] "Reference" means a standard or control condition.

[0106] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will typically be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will typically be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer near or between them.

[0107] "Specifically binds" means that the compound or antibody recognizes and binds to the polypeptide, but does not substantially recognize and bind to other molecules in a sample (eg, a biological sample that naturally includes the polypeptide).

[0108] Nucleic acid molecules useful in the methods disclosed herein include any nucleic acid molecules encoding polypeptides or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but will typically show basic identity. Polynucleotides having "basic identity" with endogenous sequences are typically capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods disclosed herein include any nucleic acid molecules encoding polypeptides or fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but will typically show basic identity. Polynucleotides having "basic identity" with endogenous sequences are typically capable of hybridizing with at least one chain of a double-stranded nucleic acid molecule. "Hybridization" means pairing under different stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. [Enzymology Methods] 152: 399; Kimmel, AR (1987) Methods Enzymol. [Enzymology Methods] 152: 507).

[0109] For example, stringent salt concentrations will typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30° C., more preferably at least about 37° C., and most preferably at least about 42° C. Different additional parameters, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Different stringency levels are achieved by combining these different conditions as needed. In a preferred embodiment, hybridization will occur at 30° C., in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 mu.g / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0110] For most applications, the washing step after hybridization will also be different in stringency. Washing stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by reducing salt concentration or by increasing temperature. For example, the stringent salt concentration for the washing step will be preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions for the washing step will generally include a temperature of at least about 25°C, more preferably at least about 42°C and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur at 25°C, at 30mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C, at 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions should be readily apparent to those of ordinary skill in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0111] A polypeptide or nucleic acid molecule is "substantially identical" when it exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid. In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window. The degree of amino acid or nucleic acid sequence identity for the purposes of this disclosure is determined using the BLAST algorithm described in Altschul et al. (199) J. Mol. Biol. 215:403-10, which is publicly available through software provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The algorithm identifies high scoring sequence pairs (HSPS) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). The initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (a reward score for a pair of matching residues; always >0) and N (a penalty for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction terminates when the following occurs: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. For determining the percent identity of an amino acid sequence or a nucleic acid sequence, the default parameters of the BLAST program can be used. For the analysis of amino acid sequences, the BLASTP defaults are: word length (W), 3; expectation (E), 10; and BLOSUM62 scoring matrix. For the analysis of nucleic acid sequences, the BLASTN program defaults are word length (W), 11; expectation (E), 10; M=5; N=-4; and comparison of both chains. The TBLASTN program (querying a nucleotide sequence database using a protein sequence) uses the following as defaults: word length (W) of 3, expectation (E) of 10, and BLOSUM62 scoring matrix.(See Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[0112] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90:5873-87). The smallest sum probability (P(N)) provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.01.

[0113] The terms "treating" and "treatment" refer to the administration of an agent or formulation to a clinically symptomatic individual suffering from an undesirable condition, disorder, or disease in order to achieve a decrease in the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, and / or promote improvement or remediation of damage. It should be understood that treatment of a disorder or condition does not require, although it cannot be excluded, complete elimination of the disorder, condition, or symptoms associated therewith.

[0114] The terms "preventing" and "prevention" refer to the administration of an agent or composition to a clinically asymptomatic individual susceptible or susceptible to a particular undesirable condition, disorder or disease, and thus involve preventing the occurrence of symptoms and / or their underlying causes.

[0115] Unless specifically stated or obvious from the context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from the context, as used herein, the terms "a, an," and "the" are understood to be singular or plural.

[0116] Unless specifically specified or obvious from the context, as used herein, the term "about" is understood to be within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value.

[0117] The following examples are given to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use assays, screening and treatment methods. These examples are not intended to limit the scope of the invention as claimed herein.

[0118] Examples

[0119] Materials and methods:

[0120] TGF-β reporter cell line. TGF-β-responsive stable cell lines were created by transfecting HEK-293T cells with pGL4.28 (Promega), an expression plasmid containing luciferase driven by a TGF-β response element, using Lipofectamine according to the manufacturer's recommended protocol. Transfected cells were selected with hygromycin for two months.

[0121] Transfection. TGF-β trap constructs were transiently transfected into the TGF-β reporter 293T cell line using Lipofectamine (Thermo Fisher) using the recommended protocol and then incubated overnight. The cells were then stimulated for 18 hours with TGF-β1, TGF-β2 or mouse TGF-β1 (Cell Signaling Technology) at concentrations as indicated in the figures. After stimulation, responses were determined using the luciferase assay system (Promega) according to the recommended protocol.

[0122] IgG titer measurement: TGF-β trap IgG fusion titers were measured using a protein A biosensor on a ForteBio Octet Red96 instrument. HEK-293T cells were transfected with the TGF-β trap construct and incubated for 18 hours, then the cell culture supernatant was collected and concentrated. The concentrated supernatant was diluted 10-fold in 1x PBS to a final volume of 200 μl and placed in a 96-well plate. Prior to measurement, the protein A biosensor was incubated in 1x PBS for 10 minutes. The assay was performed and read at 25°C, and the IgG concentration of each sample was determined by comparison with a standard curve generated with known concentrations of purified antibodies diluted in PBS.

[0123] Example 1: Inhibition of TGF-β Response by Constitutively Active TGF-β Trap Constructs

[0124] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with a CMV-driven expression construct, comparing the Sushi domain (Sushi), unmodified hinge (AltH), Fc domain (Fc) with or without TGFBRII (trap) and modified hinge ((C27S)H), Fc with or without TGFBRII (trap) (SEQ ID NOs: 15, 17, 19 and 21; with corresponding DNA sequences of SEQ ID NOs: 14, 16, 18 and 20). Cells were incubated overnight, washed and stimulated with TGF-β at the indicated concentrations. The resulting luciferase activity was measured 18 hours later. Figure 1A and 1B The data shown in show that these trap constructs inhibit TGF-β at low levels (0-1 ng / ml), but only the construct with a modified hinge is effective at high concentrations.

[0125] Example 2: Inhibition of TGF-β response by TGF-β inducible trap constructs

[0126] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with an expression construct driven by a TGF-β response element (TGFBRE) comparing the Sushi domain (Sushi), unmodified hinge (AltH), Fc domain (Fc) with or without TGFBRII (trap) compared to modified hinge ((C27S)H), Fc with or without TGFBRII (trap) (SEQ ID NOs: 7, 9, 11 and 13; with corresponding DNA sequences of SEQ ID NOs: 6, 8, 10 and 12). Cells were incubated overnight, washed and stimulated with TGF-β at the indicated concentrations. The resulting luciferase activity was measured 18 hours later and the data are shown in Figure 2A and 2B The original construct was unable to block TGF-β activity in an induced form, whereas the modified construct was effective at low concentrations (0.1 ng / ml) and still exhibited neutralizing activity at medium to high concentrations (1-10 ng / ml).

[0127] Example 3: Testing of TGF-β Trap Expression Constructs with the 293T-TGF-β Stability System

[0128] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with expression constructs driven by a TGF-β response element (TGFBRE) with a Sushi domain (Sushi), unmodified hinge (AltH), Fc with or without TGFBRII (trap) compared to a modified hinge ((C27S)H), Fc with or without TGFBRII (trap) (SEQ ID NOs: 7, 17, 11, and 13; with corresponding DNA sequences of SEQ ID NOs: 6, 16, 10, and 12). Cells were incubated overnight, washed, and stimulated with TGF-β at a "lower" titration as indicated. The resulting luciferase activity was measured 24 hours later. Figure 3 The data shown in indicate that the construct with the modified hinge is significantly more efficient.

[0129] Example 4: Testing N-terminal versus C-terminal fusion TGF-β traps using the 293T-TGF-β stabilization system

[0130] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with a CMV-driven expression construct comparing the C-terminus (C-terminus) versus the N-terminus (N-terminus) TGF-βRII (trap) Fc fusion proteins (SEQ ID NOs: 17, 21, and 23; with corresponding DNA sequences of SEQ ID NOs: 16, 20, and 22). Cells were incubated overnight, washed, and stimulated with TGF-β as indicated. The resulting luciferase activity was measured 24 hours later. Figure 4 The data in show no observable differences in activity between the C-terminal compared to the N-terminal fusion proteins.

[0131] Example 5: Determining the ability of TGF-β traps to neutralize TGF-β2

[0132] A 293T cell line stably expressing TGF-β-induced luciferase was transfected with either CMV-driven or TGF-β response element (TGFBRE)-driven expression constructs and then stimulated with titrations of TGF-β 2. Cells were incubated overnight and the resulting luciferase activity was measured after 24 hours. Figure 5A and 5B The data in show partial ability of the CMV-driven construct and minimal ability of the TGFBRE-driven construct to inhibit TGF-β2; however, the TGFBRE-driven construct had no discernible ability to neutralize TGF-β2.

[0133] Example 6: Determining the ability of TGF-β traps to neutralize mouse TGF-β2

[0134] 293T cell lines stably expressing TGF-β-induced luciferase were transfected with CMV-driven or TGF-β response element (TGFBRE)-driven expression constructs (DNA sequences are shown in SEQ ID NOs 8 and 16) and then stimulated with titrations of mouse TGF-β1 (mTGF-β1). Cells were incubated overnight and the resulting luciferase activity was measured after 24 hours. Fig. 6A and 6B The data shown in indicate that both CMV-driven and TGFBRE-driven trap expression constructs are able to inhibit mouse TGF-β-induced luciferase activity.

[0135] Example 7: Determining the effects of Sushi domain and hinge modifications on the generation of TGF-β-traps

[0136] 293T cell lines were transfected with CMV-driven TGFBRII trap Fc fusion protein expression constructs containing the Sushi domain and the original unmodified hinge sequence (AltH), no Sushi domain with AltH, or no Sushi with a modified hinge ((C27S)H). The cells were stationary and cultured in serum-free medium for 24 hours, and the resulting supernatants were concentrated and measured for human IgG Fc levels. Figure 7 The data shown in show that the C27S hinge product without Sushi exhibited the highest concentration.

[0137] Other embodiments

[0138] While the invention has been particularly shown and described with reference to certain embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope covered by the appended claims. Sequence Listing <110> NantBio Inc. Liu, Philip Higashide, Wendy Olson, C. Anders Niazi, Kayvan <120> TGF-β trap <130> 8774-14-PCT <140> not specified <141> 2020-08-14 <150> 62 / 887,272 <151> 2019-08-15 <160> 29 <170> PatentIn Version 3.5 <210> 1 <211> 566 <212> PRT <213> Homo sapiens <400> 1 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gly 405 410 415 Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Arg 420 425 430 Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser 435 440 445 Methionine Alanine Leucine Valine Leucine Tryptophan Glutamic acid Methionine Threonine Serine Arginine Cysteine Asparagine Alanine Valine Glycine 450 455 460 Glutamic acid Valine Lysine Aspartic acid Tyrosine Glutamic acid Proline Proline Phenylalanine Glycine Serine Lysine Valine Arginine Glutamic acid Histidine 465 470 475 480 Proline Cysteine Valine Glutamic acid Serine Methionine Lysine Asparagine Valine Leucine Arginine Aspartic acid Arginine Glycine Arginine 485 490 495 Proline Glutamic acid Isoleucine Proline Serine Phenylalanine Tryptophan Leucine Asparagine Histidine Glutamine Glycine Isoleucine Glutamine Methionine Valine 500 505 510 Cysteine Glutamic acid Threonine Leucine Threonine Glutamic acid Cysteine Tryptophan Aspartic acid Histidine Aspartic acid Proline Glutamic acid Alanine Arginine Leucine 515 520 525 Threonine Alanine Glutamine Cysteine Valine Alanine Glutamic acid Arginine Phenylalanine Serine Glutamic acid Leucine Glutamic acid Histidine Leucine Aspartic acid 530 535 540 Arginine Leucine Serine Glycine Arginine Serine Cysteine Serine Glutamic acid Glutamic acid Lysine Isoleucine Proline Glutamic acid Aspartic acid Glycine 545 550 555 560 Serine Leucine Asparagine Threonine Threonine Lysine 565 <210> 2 <211> 136 <212> PRT <213> Homo sapiens <400> 2 Isoleucine Proline Proline Histidine Valine Glutamine Lysine Serine Valine Asparagine Asparagine Aspartic acid Methionine Isoleucine Valine Threonine 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 3 <211> 217 <212> PRT <213> Homo sapiens <400> 3 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 4 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Mutation variants of the Fc hinge region of Homo sapiens IgG <400> 4 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 5 <211> twenty three <212> PRT <213> Homo sapiens <400> 5 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala 20 <210> 6 <211> 1557 <212> DNA <213> Artificial sequence <220> <223> TGF-β RE minP - Sushi - Unmodified hinge - IgG1 Fc - TGFBRII <400> 6 60 agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt tggtaaagcc 120 accatggact ggatctggcg gattctgttt ctcgtgggag ctgccacagg cgctcattct 180 gctcagcctg ccatcacgtg tcctcctcct atgtccgtgg aacacgcaga catctgggtc 240 300 gccggcacgt ccagcctgac ggagtgcgtg ttgaacaagg ccacgaatgt cgccactgg 360 acaaccccca gtctcaaatg cattagagag ccgaaatctt gtgacaaaac tcacacatgc 420 ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttctctt ccccccaaaaa 480 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 540 600 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 660 accgtcctgc accaggactg gctgaatggc aaggagtca agtgcaaggt ctccaacaaa 720 gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 780 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 840 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 900 ccggagaaca actacaagac cacgcctcccc gtgctggact ccgacggctc cttcttctc 960 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1020 gtgatgcatg aggctctgca caaccactac acgcaagaga gcctctccct gtctcctggt 1080 aaaggagg gtggctccgg aggcggtggc tccggtggag gtggctccgg aggtggcggt 1140 tccggtatcc ccccccacgt gcagaagtcc gttaacaacg acatgatcgt gaccgacaac 1200 aacggcgccg tgaagttccc ccagctgtgc aagttctgcg acgtgaggtt ctccacctgc 1260 gaaccaga agtcctgcat gtccaactgc tccatcacct ccatctgcga gaagcctcag 1320 gaggtgtgcg tggctgtgtg gcggaagaac gacgaagaa tcaccctgga gaccgtgtgc 1380 cacgacccca agctgcccta ccacgacttc atcctggagg acgccgcctc ccccaagtgc 1440 atcatgaagg agaagaagaa gcccggcgag accttcttta tgtgctcctg ctccagcgac 1500 gagtgcaacg acaacatcat cttctccgag gagtacaaca cctccaaccc cgactga 1557 <210> 7 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> TGF-β RE minP - Sushi - Unmodified Hinge - IgG1 Fc - TGFBRII <400> 7 Met Glu Ala Arg Leu Pro Ala Trp Gln Ser Gly Thr Val Gly Lys Ala 1 5 10 15 Thr Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr 20 25 30 Gly Ala His Ser Ala Gln Pro Ala Ile Thr Cys Pro Pro Pro Met Ser 35 40 45 Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg 50 55 60 Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser 65 70 75 80 Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp 85 90 95 Thr Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Cys Asp Lys 100 105 110 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 115 120 125 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 130 135 140 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 145 150 155 160 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 165 170 175 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 180 185 190 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 195 200 205 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 210 215 220 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 225 230 235 240 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 245 250 255 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 260 265 270 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 275 280 285 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 290 295 300 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 305 310 315 320 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 330 335 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 340 345 350 Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn 355 360 365 Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln 370 375 380 Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys 385 390 395 400 Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln 405 410 415 Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu 420 425 430 Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu 435 440 445 Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro 450 455 460 Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp 465 470 475 480 Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 485 490 <210> 8 <211> 1362 <212> DNA <213> Artificial Sequence <220> <223> TGF-β RE minP - C27S hinge - IgG1 Fc - TGFBRII <400> 8 agtatgtcta gactgaagta tgtctagact gaagtatgtc tagactgaag cttagacact 60 agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt tggtaaagcc 120 accatggact ggatctggcg gattctgttt ctcgtgggag ctgccacagg cgctcattct 180 gctcagcctg ccgagccgaa atcttctgac aaaactcaca catgcccacc gtgcccagca 240 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 360 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacg cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 480 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 540 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gggatgagct caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 780 gtggacaaga gcaggtggca gcaggggac gtcttctcat gctccgtgat gcatgaggct 840 ctgcacaacc actaccgca gaagagcctc tccctgtctc ctggtaaagg aggaggtggc tccggaggcg gtggctccgg tggaggtggc tccggaggtg gcggttccgg tatcccccccc 960 cacgtgcaga agtccgttaa caacgacatg atcgtgaccg acaacaacgg cgccgtgaag 1020 ttcccccagc tgtgcaagtt ctgcgacgtg aggttctcca cctgcgacaa ccagaagtcc 1080 tgcatgtcca actgctccat cacctccatc tgcgagaagc ctcaggaggt gtgcgtggct 1140 gtgtggcgga agaacgacga gaacatcacc ctggagaccg tgtgccacga ccccaagctg 1200 ccctaccacg acttcatcct ggaggacgcc gcctccccca agtgcatcat gaaggagaag 1260 aagaagcccg gcgagacctt ctttatgtgc tcctgctcca gcgacgagtg caacgacaac 1320 atcatcttct ccgaggagta caacacctcc aaccccgact ga 1362 <210> 9 <211> 429 <212> PRT <213> Artificial sequence <220> <223> TGF-β RE minP - C27S hinge - IgG1 Fc - TGFBRII <220> <221> Features not yet classified <222> (41)..(55) <223> Hinge area <220> <221> Features not yet classified <222> (56)..(272) <223> Fc region <220> <221> Features not yet classified <222> (294)..(429) <223> TGFBRII binding domain <400> 9 Met Glu Ala Arg Leu Pro Ala Trp Gln Ser Gly Thr Val Gly Lys Ala 1 5 10 15 Thr Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr 20 25 30 Gly Ala His Ser Ala Gln Pro Ala Glu Pro Lys Ser Ser Asp Lys Thr 35 40 45 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 50 55 60 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 65 70 75 80 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 85 90 95 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 100 105 110 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 115 120 125 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 130 135 140 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 145 150 155 160 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 165 170 175 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 180 185 190 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 195 200 205 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 210 215 220 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 225 230 235 240 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 245 250 255 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275 280 285 Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 290 295 300 Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 305 310 315 320 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 325 330 335 Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu 340 345 350 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 355 360 365 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 370 375 380 Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 385 390 395 400 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 405 410 415 Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 420 425 <210> 10 <211> 1082 <212> DNA <213> Artificial Sequence <220> <223> TGF-β RE minP - Sushi - Unmodified Hinge - IgG1 Fc <400> 10 ggtatgtcta gactgaagta tgtctagact gagattgtc tagactgaag cttagacact 60 agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt tggtaaagcc 120 accatggact ggatctggcg gattctgttt ctcgtgggag ctgccacagg cgctcattct 180 gctcagcctg ccatcacgtg tcctcctcct atgtccgtgg aacacgcaga catctgggtc 240 300 gccggcacgt ccagcctgac ggagtgcgtg ttgaacaagg ccacgaatgt cgccactgg 360 acaaccccca gtctcaaatg cattagagag ccgaaatctt gtgacaaaac tcacacatgc 420 ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttctctt ccccccaaaaa 480 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 540 600 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 660 accgtcctgc accaggactg gctgaatggc aaggagtca agtgcaaggt ctccaacaaa 720 gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 780 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 840 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 900 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 960 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1020 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctcctggt 1080 aa 1082 <210> 11 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> TGF-β RE minP - Sushi - Unmodified Hinge - IgG1 Fc <400> 11 Met Glu Ala Arg Leu Pro Ala Trp Gln Ser Gly Thr Val Gly Lys Ala 1 5 10 15 Thr Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr 20 25 30 Gly Ala His Ser Ala Gln Pro Ala Ile Thr Cys Pro Pro Pro Met Ser 35 40 45 Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg 50 55 60 Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser 65 70 75 80 Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp 85 90 95 Thr Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Cys Asp Lys 100 105 110 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 115 120 125 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 130 135 140 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 145 150 155 160 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 165 170 175 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 180 185 190 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 195 200 205 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 210 215 220 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 225 230 235 240 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 245 250 255 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 260 265 270 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 275 280 285 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 290 295 300 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 305 310 315 320 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 330 335 Lys <210> 12 <211> 888 <212> DNA <213> Artificial sequence <220> <223> TGF-β RE minP - C27S hinge - IgG1 Fc <400> 12 agtatgtcta gactgaagta tgtctagact gaagtatgtc tagactgaag cttagacact 60 agagggtata taatggaagc tcgacttcca gcttggcaat ccggtactgt tggtaaagcc 120 accatggact ggatctggcg gattctgttt ctcgtgggag ctgccacagg cgctcattct 180 gctcagcctg ccgagccgaa atcttctgac aaaactcaca catgcccacc gtgcccagca 240 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 300 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 360 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 420 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 480 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 540 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 600 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 660 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 720 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 780 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 840 ctgcacaacc actacacgca gaagagcctc tccctgtctc ctggtaaa 888 <210> 13 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> TGF-β RE minP - C27S hinge - IgG1 Fc <400> 13 Met Glu Ala Arg Leu Pro Ala Trp Gln Ser Gly Thr Val Gly Lys Ala 1 5 10 15 Thr Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr 20 25 30 Gly Ala His Ser Ala Gln Pro Ala Glu Pro Lys Ser Ser Asp Lys Thr 35 40 45 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 50 55 60 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 65 70 75 80 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 85 90 95 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 100 105 110 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 115 120 125 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 130 135 140 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 145 150 155 160 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 165 170 175 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 180 185 190 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 195 200 205 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 210 215 220 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 225 230 235 240 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 245 250 255 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 265 270 <210> 14 <211> 2414 <212> DNA <213> Artificial Sequence <220> <223> CMV - Sushi - Unmodified Hinge - IgG1 Fc - TGFBRII <400> 14 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720 780 tgctaacgca gtcagtgctc gactgatcac aggtaagt caaggttaca agacaggttt 840 aaagggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960 tagcgctacc ggtcgccacc atggactgga tctggcggat tctgtttctc gtgggagctg 1020 ccacaggcgc tcattctgct cagcctgcca tcacgtgtcc tcctcctatg tccgtggaac 1080 acgcagacat ctgggtcaag agctacagct tgtactccag ggagcggtac atttgtaact 1140 ctggtttcaa gcgtaaagcc ggcacgtcca gcctgacgga gtgcgtgttg aacaaggcca 1200 cgaatgtcgc ccactggaca acccccagtc tcaaatgcat tagagagccg aaatcttgtg 1260 acaaaactca cacatgccca ccgtgcccag cacctgaact cctggggga ccgtcagtct 1320 tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacat 1380 gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg 1440 gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc 1500 gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt 1560 gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag 1620 ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga 1680 accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt 1740 gggagagcaa tgggcagccg gagaacaact acaagaccac gcctccccgtg ctggactccg 1800 acggctcctt cttctcttac agcaagctca ccgtggacaa gagcaggtgg cagcaggggga 1860 acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc 1920 tctccctgtc tcctggtaaa ggaggaggtg gctccggagg cggtggctcc ggtggaggtg 1980 gctccggagg tggcggttcc ggtatccccc cccacgtgca gaagtccgtt aacaacgaca 2040 tgatcgtgac cgacaacaac ggcgccgtga agttccccca gctgtgcaag ttctgcgacg 2100 tgaggttctc cacctgcgac aaccagaagt cctgcatgtc caactgctcc atcacctcca 2160 tctgcgagaa gcctcaggag gtgtgcgtgg ctgtgtggcg gaagaacgac gagaacatca 2220 ccctggagac cgtgtgccac gaccccaagc tgccctacca cgacttcatc ctggaggacg 2280 ccgcctcccc caagtgcatc atgaaggaga agaagaagcc cggcgagacc ttctttatgt 2340 gctcctgctc cagcgacgag tgcaacgaca acatcatctt ctccgaggag tacaacacct 2400 ccaaccccga ctga 2414 <210> 15 <211> 477 <212> PRT <213> Artificial Sequence <220> <223> CMV - Sushi - Unmodified Hinge - IgG1 Fc - TGFBRII <400> 15 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Ile Thr Cys Pro Pro Pro Met Ser Val 20 25 30 Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu 35 40 45 Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser 50 55 60 Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr 65 70 75 80 Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Cys Asp Lys Thr 85 90 95 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 100 105 110 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 115 120 125 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 130 135 140 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 145 150 155 160 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 165 170 175 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 180 185 190 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 195 200 205 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 210 215 220 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 225 230 235 240 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 245 250 255 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 260 265 270 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 275 280 285 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 290 295 300 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 305 310 315 320 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 325 330 335 Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn 340 345 350 Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu 355 360 365 Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser 370 375 380 Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu 385 390 395 400 Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu 405 410 415 Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu 420 425 430 Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly 435 440 445 Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn 450 455 460 Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 465 470 475 <210> 16 <211> 2219 <212> DNA <213> Artificial Sequence <220> <223> CMV - C27S hinge - IgG1 Fc - TGFBRII <400> 16 acaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720 780 tgctaacgca gtcagtgctc gactgatcac aggtaagt caaggttaca agacaggttt 840 aaagggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960 tagcgctacc ggtcgccacc atggactgga tctggcggat tctgtttctc gtgggagctg 1020 ccacaggcgc tcattctgct cagcctgccg agccgaaatc ttctgacaaa actcacacat 1080 gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc ttccccccaa 1140 aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg gtggtggacg 1200 tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata 1260 atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg gtcagcgtcc 1320 1380 aagccctccc agcccccatc gagaaacca tctccaaagc caaagggcag ccccgagaac 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc 1560. agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg cacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctcctg gtaaaggagg aggtggctcc ggaggcggtg gctccggtgg aggtggctcc ggaggtggcg gttccggtat ccccccccac gtgcagaagt ccgttaacaa cgacatgatc gtgaccgaca acaacggcgc cgtgaagttc ccccagctgt gcaagttctg cgacgtgagg ttctccacct gcgacaacca gaagtcctgc atgtccaact gctccatcac ctccatctgc gagaagcctc aggaggtgtg cgtggctgtg tggcggaga acgacgaga catcaccctg gagaccgtgt gccacgaccc caagctgccc taccacgact tcatcctgga ggacgccgcc tcccccaagt gcatcatgaa ggagaagaag aagcccggcg agaccttctt tatgtgctcc tgctccagcg 2160 acgagtgcaa cgacaacatc atcttctccg aggagtacaa cacctccaac cccgactga 2219 <210> 17 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> CMV - C27S hinge - IgG1 Fc - TGFBRII <400> 17 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Glu Pro Lys Ser Ser Asp Lys Thr His 20 25 30 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 35 40 45 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 50 55 60 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 65 70 75 80 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 85 90 95 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 100 105 110 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 115 120 125 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 130 135 140 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 145 150 155 160 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 165 170 175 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 180 185 190 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 195 200 205 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 210 215 220 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 225 230 235 240 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 260 265 270 Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp 275 280 285 Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys 290 295 300 Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 305 310 315 320 Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 325 330 335 Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 340 345 350 Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp 355 360 365 Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu 370 375 380 Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 385 390 395 400 Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 405 410 <210> 18 <211> 1940 <212> DNA <213> Artificial sequence <220> <223> CMV - Sushi - Unmodified hinge - IgG1 Fc <400> 18 gcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720 780 tgctaacgca gtcagtgctc gactgatcac aggtaagt caaggttaca agacaggttt 840 aaagggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960 tagcgctacc ggtcgccacc atggactgga tctggcggat tctgtttctc gtgggagctg 1020 ccacaggcgc tcattctgct cagcctgcca tcacgtgtcc tcctcctatg tccgtggaac 1080 acgcagacat ctgggtcaag agctacagct tgtactccag ggagcggtac atttgtaact 1140 ctggtttcaa gcgtaaagcc ggcacgtcca gcctgacgga gtgcgtgttg aacaaggcca 1200 cgaatgtcgc ccactggaca acccccagtc tcaaatgcat tagagagccg aaatcttgtg 1260 acaaaactca cacatgccca ccgtgcccag cacctgaact cctggggga ccgtcagtct 1320 tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacat 1380 gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg 1440 gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc 1500 gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt 1560 gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag 1620 ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga 1680 accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt 1740 gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg 1800 acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga 1860 acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc 1920 tctccctgtc tcctggtaaa 1940 <210> 19 <211> 320 <212> PRT <213> Artificial Sequence <220> <223> CMV - Sushi - Unmodified Hinge - IgG1 Fc <400> 19 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Ile Thr Cys Pro Pro Pro Met Ser Val 20 25 30 Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu 35 40 45 Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser 50 55 60 Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr 65 70 75 80 Thr Pro Ser Leu Lys Cys Ile Arg Glu Pro Lys Ser Cys Asp Lys Thr 85 90 95 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 100 105 110 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 115 120 125 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 130 135 140 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 145 150 155 160 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 165 170 175 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 180 185 190 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 195 200 205 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 210 215 220 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 225 230 235 240 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 245 250 255 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 260 265 270 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 275 280 285 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 290 295 300 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 305 310 315 320 <210> 20 <211> 1745 <212> DNA <213> Artificial Sequence <220> <223> CMV - C27S Hinge - IgG1 Fc <400> 20 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720 780 tgctaacgca gtcagtgctc gactgatcac aggtaagt caaggttaca agacaggttt 840 aaagggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960 tagcgctacc ggtcgccacc atggactgga tctggcggat tctgtttctc gtgggagctg 1020 ccacaggcgc tcattctgct cagcctgccg agccgaaatc ttctgacaaa actcacacat 1080 gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc ttccccccaa 1140 aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg gtggtggacg 1200 tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata 1260 atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc 1320 tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca 1380 aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac 1440 cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga 1500 cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc 1560 agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc 1620 tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct 1680 ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctcctg 1740 gtaaa 1745 <210> 21 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> CMV - C27S hinge - IgG1 Fc <400> 21 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Glu Pro Lys Ser Ser Asp Lys Thr His 20 25 30 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 35 40 45 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 50 55 60 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 65 70 75 80 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 85 90 95 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 100 105 110 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 115 120 125 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 130 135 140 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 145 150 155 160 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 165 170 175 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 180 185 190 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 195 200 205 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 210 215 220 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 225 230 235 240 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 255 <210> 22 <211> 2228 <212> DNA <213> Artificial Sequence <220> <223> CMV - TGFBRII - (G4S)25 Polylinker - C27S Hinge - IgG1 Fc <400> 22 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagg 720 780 tgctaacgca gtcagtgctc gactgatcac aggtaagt caaggttaca agacaggttt 840 aaagggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg 900 cacctattgg tcttactgac atccactttg cctttctctc cacaggggta ccgaagccgc 960 tagcgctacc ggtcgccacc atggactgga tctggcggat tctgtttctc gtgggagctg 1020 ccacaggcgc tcattctgct cagcctgcca tcccccccca cgtgcagaag tccgttaaca 1080 acgacatgat cgtgaccgac aacaacggcg ccgtgaagtt cccccagctg tgcaagttct 1140 gcgacgtgag gttctccacc tgcgacaacc agaagtcctg catgtccaac tgctccatca 1200 cctccatctg cgagaagcct caggaggtgt gcgtggctgt gtggcggaag aacgacgaga 1260 acatcaccct ggagaccgtg tgccacgacc ccaagctgcc ctaccacgac ttcatcctgg 1320 aggacgccgc ctcccccaag tgcatcatga aggagaagaa gaagcccggc gagaccttct 1380 ttatgtgctc ctgctccagc gacgagtgca acgacaacat catcttctcc gaggagtaca 1440 acacctccaa ccccgacgga ggaggtggct ccggaggcgg tggctccggt ggaggtggct 1500 ccggaggtgg cggttccggt ggcggtggct ccgagccgaa atcttctgac aaaactcaca 1560 catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc ctcttccccc 1620 caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc gtggtggtgg 1680 acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc 1740 ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg 1800 tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca 1860 acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg cagccccgag 1920 aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac caggtcagcc 1980 tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg 2040 ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct 2100 tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat 2160 gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc tccctgtctc 2220 ctggtaaa 2228 <210> 23 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> CMV - TGFBRII - (G4S)25 linker - C27S hinge - IgG1 Fc <400> 23 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 165 170 175 Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr 180 185 190 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 195 200 205 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 210 215 220 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 225 230 235 240 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 245 250 255 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 260 265 270 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 275 280 285 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 290 295 300 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 305 310 315 320 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 325 330 335 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 340 345 350 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 355 360 365 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 370 375 380 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 385 390 395 400 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 24 <211> 389 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Trap Molecule <400> 24 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro 245 250 255 His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn 260 265 270 Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe 275 280 285 Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr 290 295 300 Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys 305 310 315 320 Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu 325 330 335 Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile 340 345 350 Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys 355 360 365 Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn 370 375 380 Thr Ser Asn Pro Asp 385 <210> 25 <211> 393 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Trap Molecule <400> 25 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 165 170 175 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 180 185 190 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 195 200 205 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 210 215 220 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 225 230 235 240 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 245 250 255 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 260 265 270 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 275 280 285 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 290 295 300 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 305 310 315 320 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 325 330 335 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 340 345 350 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 355 360 365 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 370 375 380 Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 <210> 26 <211> 38 <212> DNA <213> House mouse <400> 26 ccaccacagc cagaccacag gccagacatg acgtggag 38 <210> 27 <211> 50 <212> DNA <213> Homo sapiens <400> 27 tggagtgtgc cagctttttc agacggagga atgctgagtg tcaaggggtc 50 <210> 28 <211> 67 <212> DNA <213> Homo sapiens <400> 28 caagtcctag acagacaaaa cctagacaat cacgtggctg gctgcatgcc ctgtggctgt 60 tgggctg 67 <210> 29 <211> twenty three <212> PRT <213> Homo sapiens <220> <221> Features not yet classified <222> (1)..(23) <223> IgG1 Fc hinge region <400> 29 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro 20

Claims

1. A TGF-β trap comprising an immunoglobulin constant (Fc) domain fused to a ligand binding domain of transforming growth factor-β receptor type 2 (TGFβRII), wherein the trap does not contain a Sushi domain, wherein the immunoglobulin Fc domain further comprises an N-terminal immunoglobulin hinge region, wherein at least one unpaired cysteine ​​residue in the hinge region is replaced by a serine residue, and wherein the trap has the structure NH2-hinge-Fc-linker-TGFβRII-COOH or NH2-TGFβRII-linker-hinge-Fc-COOH, and wherein the trap comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:

25.

2. The trap of claim 1, wherein the trap comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 23 or SEQ ID NO:

25.

3. A nucleic acid molecule encoding a trap according to any one of the preceding claims. An expression vector comprising the nucleic acid according to claim 3 .

5. The vector of claim 4, wherein the nucleic acid is operably linked to an inducible promoter. The vector of claim 5 , wherein the inducible promoter comprises a TGF-β inducible promoter.

7. The vector of claim 4, wherein the nucleic acid is operably linked to a constitutively active promoter.

8. The vector according to claim 7, wherein the constitutively active promoter is a CMV promoter.

9. A host cell comprising the vector according to any one of claims 4 to 8.

10. The host cell according to claim 9, wherein the host cell is an IL-2-dependent natural killer cell.

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