Patent Description:
TGF-β is a multifunctional cytokine with diverse biological effects on cellular processes, including cell proliferation, migration, differentiation, and apoptosis. The three mammalian TGF-β isoforms, TGF-β1, -β2 and -β3, exert their functions through a cell surface receptor complex composed of type I (TβRI) and type II (TβRII) serine/threonine kinase receptors. Receptor activation induces both SMAD proteins and other downstream targets, including Ras, RhoA, TAK1, MEKK1, PI3K, and PP2A, to produce the full spectrum of TGF-β responses (<NPL>; <NPL>; <NPL>).

TGF-β proteins are known to promote the progression of fibrotic disorders and certain types of cancer. In the context of fibrotic disorders, TGF-β potently stimulates the expression of extracellular matrix (ECM) proteins. Dysregulation of the ECM remodeling can lead to pathological fibrosis. The role of TGF-β in cancer is multi-faceted. TGF-β isoforms, TGF-β1, - β2 and -β3 are also known to suppress host immune surveillance and to stimulate epithelial-to-mesenchymal transitions, which drive cancer progression and metastasis.

The invention is defined in the claims. Any features which are disclosed herein, but which are not claimed do not form part of the invention. Described herein are engineered TGF-β monomers that are capable of blocking TGF-β signaling. The engineered monomers inhibit TGF-β signaling by preventing TGF-β dimerization and recruitment of TβRI.

Provided herein is a recombinant human TGF-β monomer selected from TGF-β1, TGF-β2, and TGF-β3 that includes a cysteine to serine substitution at amino acid residue <NUM>; a deletion of amino acid residues <NUM>-<NUM>; and at least one amino acid substitution relative to a wild-type TGF-β monomer that increases the net charge of the recombinant human TGF-β monomer, wherein.

wherein wild-type TGF-β <NUM>, TGF-β2, and TGF-β3 monomers have the amino acid sequence of SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>, respectively.

In some embodiments, the recombinant human TGF-β monomer further includes at least one amino acid substitution relative to a wild-type TFG-β2 monomer that increases affinity of the TGF-β monomer for TGF-β type II receptor (TβRII), wherein the at least one amino acid substitution that increases affinity of the monomer for TβRII comprises a substitution at an amino acid residue corresponding to residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> of SEQ ID NO: <NUM>, or any combination of two or more residues thereof. The TGF-β monomer is a human TGF-β2, TGF-β1 or TGF-β3 monomer. In some embodiments, the recombinant human TGF-β monomer is a human TGF-β2 monomer. In some embodiments, the recombinant human TGF-β2 monomer comprises at least one amino acid substitution that increases net charge of the monomer which comprises:.

In some embodiments, the recombinant human TGF-β2 monomer comprises at least one amino acid substitution that increases affinity of the monomer for TβRII comprises at least one substitution at residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>, and at least one substitution at residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>.

In some embodiments, the recombinant human TGF-β2 monomer comprises at least one amino acid substitution that increases affinity of the monomer for TβRII comprises a lysine to arginine at residue <NUM>, an arginine to lysine at residue <NUM>, a leucine to valine at residue <NUM>, an isoleucine to valine at residue <NUM>, an asparagine to arginine at residue <NUM>, a threonine to lysine at residue <NUM>, an isoleucine to valine at residue <NUM>; or any combination of two or more thereof.

In some embodiments, the recombinant human TGF-β2 monomer comprises at least one amino acid substitution that increases affinity of the monomer for TβRII comprises a lysine to arginine at residue <NUM>, an arginine to lysine at residue <NUM>, a leucine to valine at residue <NUM>, an isoleucine to valine at residue <NUM>, an asparagine to arginine at residue <NUM>, a threonine to lysine at residue <NUM>, and an isoleucine to valine at residue <NUM>. In some embodiments, the recombinant human TGF-β2 monomer comprises the amino acid sequence of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>.

In some embodiments, the recombinant human TGF-β monomer is a human TGF-β1 monomer.

In some embodiments, the recombinant human TGF-β1 monomer comprises at least one amino acid substitution that increases net charge of the monomer comprises:.

In some embodiments, the recombinant human TGF-β1 monomer comprises the amino acid sequence of SEQ ID NO: <NUM>. In some embodiments, the recombinant human TGF-β monomer is a human TGF-β3 monomer.

In some embodiments, the recombinant human TGF-β3 monomer comprises at least one amino acid substitution that increases net charge of the monomer comprises:.

In some embodiments, the recombinant human TGF-β3 monomer comprises the amino acid sequence of SEQ ID NO: <NUM>. In some embodiments, the recombinant human TGF-β monomer is PEGylated, or which is glycosylated or hyper-glycosylated.

Fusion proteins that include a recombinant human TGF-β monomer and a heterologous protein are also provided, particularly wherein the heterologous protein comprises a protein tag,.

In some embodiments, the recombinant human TGF-β monomer or the fusion protein comprises a radiotherapy agent, a cytotoxic agent for chemotherapy, or a drug. In some embodiments, the fusion protein comprises an imaging agent, a fluorescent dye, or a fluorescent protein tag. Further provided are compositions that include a recombinant human TGF-β monomer or fusion protein disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

Further provided are in vitro methods of inhibiting TGF-β signaling in a cell by contacting the cell with a recombinant human TGF-β monomer, fusion protein or composition disclosed herein. In other embodiments, the recombinant human TGF-β monomer, fusion protein or composition are for use in inhibiting TGF-β signaling in a cell by contacting the cell with a recombinant human TGF-β monomer, fusion protein, or composition disclosed herein. In some embodiments, the use further includes administering the recombinant human TGF-β monomer, fusion protein or composition to a subject having a disease or disorder associated with aberrant TGF-β signaling.

In other embodiments, the recombinant human TGF-β monomer, fusion protein, or composition are for or use in treating a disease or disorder associated with aberrant TGF-β signaling in a subject, comprising administering to the subject the recombinant human TGF-β monomer, fusion protein, or composition disclosed herein, particularly wherein the disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder,.

or wherein the disease or disorder associated with aberrant TGF-β signaling is a genetic disorder of connective tissue. Also provided are isolated nucleic acid molecules, particularly wherein the nucleic acid is operably linked to a promoter, and vectors that encode a recombinant human TGF-β monomer disclosed herein. Further provided are isolated cells, such as isolated T lymphocytes, that comprise the recombinant human TGF-β monomer-encoding nucleic acid molecule or vector.

An isolated cell for use in treating a disease or disorder associated with aberrant TGF-β signaling in a subject by administering to the subject an isolated cell (such as a T cell) comprising the disclosed nucleic acids or vectors are further provided, particularly wherein the disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder,.

The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids. The Sequence Listing is submitted as an ASCII text file, created on November <NUM>, <NUM>, <NUM> KB. In the accompanying sequence listing:.

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in <NPL>); <NPL>); and <NPL>).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:.

Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below.

Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, serine or threonine, is substituted for (or by) a hydrophobic residue, for example, leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, for example, glutamine or aspartic acid; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. <NPL>, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.

In general, the nature of the carrier will depend on the particular mode of administration being employed. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease.

Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. The term recombinant includes nucleic acids and proteins that have been altered by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein.

Sequence identity/similarity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>;<NPL>; and <NPL>. <NPL>, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (<NPL>) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

Subject: Living multi-cellular organisms, including vertebrate organisms, a category that includes both human and non-human mammals.

Tag: A molecule that can be attached to a protein or nucleic acid, such as for labeling, detection, or purification purposes. In some embodiments, the tag is a protein tag. In some embodiments, the protein tag is an affinity tag (for example, Avitag, hexahistidine, chitin binding protein, maltose binding protein, or glutathione-S-transferase), an epitope tag (for example, V5, c-myc, HA or FLAG) or a fluorescent tag (e.g., GFP or another well-known fluorescent protein).

Therapeutically effective amount: A quantity of compound or composition, for instance, a recombinant human TGF-β monomer, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or block TGF-β signaling in a cell.

Transforming growth factor-β (TGF-β): A secreted, multi-functional protein that regulates proliferation, cellular differentiation, and a number of other cellular functions. Many cells synthesize TGF-β and nearly all cells express receptors for TGF-β. The term "TGF-β" refers to three different protein isoforms, TGF-β1, TGF-β2 and TGF-β3, encoded by the genes TGFB1, TGFB2, TGFB3, respectively.

TGF-β signaling pathway: A signaling pathway involved in a number of cellular processes, such as cell proliferation, differentiation and apoptosis. Members of the TGF-β pathway include, but are not limited to, TGF-β1, TGF-β2, TGF-β3 and TGF-β receptor type I and TGF-β receptor type II.

TGF-β receptor: The term "TGF-β receptor" includes TGF-β receptor type I (encoded by TGFBR1) and TGF-β receptor type II (encoded by TGFBR2). TGF-β receptors are serine/threonine protein kinases. The type I and type II TGF-β receptors form a heterodimeric complex when bound to TGF-β, transducing the TGF-β signal from the cell surface to the cytoplasm.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. "Comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Disclosed herein are recombinant human transforming growth factor (TGF)-β monomers that are modified to inhibit dimerization and type I receptor binding, but retain the capacity to bind the high affinity TGF-β type II receptor (TβRII). The recombinant human TGF-β monomers disclosed herein can be used to inhibit TGF-β signaling, such as for the treatment of diseases or disorders characterized by aberrant TGF-β signaling, for example fibrotic disorders, ocular diseases, certain types of cancer, or a genetic disorder of connective tissue. In addition, nucleic acid molecules encoding a recombinant human TGF-β monomer can be used to reprogram T cells to overproduce the recombinant protein. T cells engineered to overexpress the recombinant human TGF-β monomer can be used in gene therapy applications, such as for the treatment of diseases or disorders characterized by aberrant TGF-β signaling.

Provided herein is a recombinant human TGF-β monomer selected from TGF-β1, TGF-β2, and TGF-β3 that includes a cysteine to serine substitution at amino acid residue <NUM> of SEQ ID NO: <NUM>; a deletion of amino acid residues <NUM>-<NUM> of SEQ ID NO: <NUM>; and at least one amino acid substitution (for example, a substitution proximal to the deleted residues) relative to a wild-type TFG-β monomer that increases net charge of the monomer, wherein.

wherein wild-type TGF-β1, TGF-β2, and TGF-β3 monomers have the amino acid sequence of SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>, respectively.

The cysteine to serine substitution prevents disulfide bond formation between TGF-β monomers. The deletion of amino acid residues <NUM>-<NUM> removes the α-helical <NUM> (α3) region (the primary dimerization motif), as well as a few flanking residues (<FIG>). When residues <NUM>-<NUM> are removed, the remaining residues form a loop that contains polar and charged residues (<FIG> and <FIG>).

In some embodiments, the TGF-β monomer further includes at least one amino acid substitution relative to a wild-type TFG-β2 monomer that increases affinity of the TGF-β monomer for TβRII.

In some embodiments, the TGF-β monomer is a human TGF-β2 monomer. In some examples, the at least one amino acid substitution that increases net charge of the human TGF-β2 monomer includes a leucine to arginine substitution at residue <NUM>; an alanine to lysine substitution at residue <NUM>; or both a leucine to arginine substitution at residue <NUM> and an alanine to lysine substitution at residue <NUM> (with reference to SEQ ID NO: <NUM>).

In some embodiments, the at least one amino acid substitution that increases affinity of the human TGF-β2 monomer for TβRII includes a substitution at an amino acid residue corresponding to residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> of SEQ ID NO: <NUM>, or any combination of two or more residues thereof. In some examples, the at least one amino acid substitution that increases affinity of the monomer for TβRII comprises at least one substitution at residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>, and at least one substitution at residue <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. In specific examples, the at least one amino acid substitution that increases affinity of the human TGF-β2 monomer for TβRII includes a lysine to arginine at residue <NUM>, an arginine to lysine at residue <NUM>, a leucine to valine at residue <NUM>, an isoleucine to valine at residue <NUM>, an asparagine to arginine at residue <NUM>, a threonine to lysine at residue <NUM>, an isoleucine to valine at residue <NUM>, or any combination of two or more thereof, such as three or more, four or more, five or more, or six or more. In one non-limiting examples, the recombinant human TGF-β2 monomer includes a lysine to arginine at residue <NUM>, an arginine to lysine at residue <NUM>, a leucine to valine at residue <NUM>, an isoleucine to valine at residue <NUM>, an asparagine to arginine at residue <NUM>, a threonine to lysine at residue <NUM>, and an isoleucine to valine at residue <NUM>.

In some examples, the amino acid sequence of the human TGF-β2 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM> or SEQ ID NO: <NUM>. In some instances, the human TGF-β2 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM> or SEQ ID NO: <NUM> and contains only conservative amino acid substitutions. In particular non-limiting examples, the amino acid sequence of the human TGF-β2 monomer comprises or consists of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>.

In other embodiments, recombinant human TGF-β monomer is a human TGF-β1 monomer. In some examples, the at least one amino acid substitution that increases net charge of the human TGF-β1 monomer includes an isoleucine to arginine substitution at residue <NUM>; an alanine to lysine substitution at residue <NUM>; an alanine to serine substitution at residue <NUM>; or an isoleucine to arginine substitution at residue <NUM>, an alanine to lysine substitution at residue <NUM> and an alanine to serine substitution at residue <NUM> (with reference to SEQ ID NO: <NUM>).

In some examples, the amino acid sequence of the human TGF-β1 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM>. In some instances, the human TGF-β1 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM> and contains only conservative amino acid substitutions. In particular non-limiting examples, the amino acid sequence of the human TGF-β1 monomer comprises or consists of SEQ ID NO: <NUM>.

In other embodiments, the recombinant human TGF-β monomer is a human TGF-β3 monomer. In some examples, the at least one amino acid substitution that increases net charge of the human TGF-β3 monomer includes a leucine to glutamate substitution at residue <NUM>; an alanine to glutamate substitution at residue <NUM>; an alanine to aspartate substitution at residue <NUM>; or a leucine to glutamate substitution at residue <NUM>, an alanine to glutamate substitution at residue <NUM> and an alanine to aspartate substitution at residue <NUM> (with reference to SEQ ID NO: <NUM>).

In some examples, the amino acid sequence of the human TGF-β3 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM>. In some instances, the human TGF-β3 monomer is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% identical to SEQ ID NO: <NUM> and contains only conservative amino acid substitutions. In particular non-limiting examples, the amino acid sequence of the human TGF-β3 monomer comprises or consists of SEQ ID NO: <NUM>.

In some embodiments herein, the recombinant human TGF-β monomer is PEGylated, glycosylated, hyper-glycosylated, or includes another modification that prolongs circulatory time.

Also provided herein are fusion proteins that include a TGF-β monomer and a heterologous protein. In some embodiments, the heterologous protein is a protein tag. In some examples, the protein tag is an affinity tag (for example, Avitag, hexahistidine, chitin binding protein, maltose binding protein, or glutathione-S-transferase), an epitope tag (for example, V5, c-myc, HA or FLAG) or a fluorescent tag (e.g., GFP or another well-known fluorescent protein). In other embodiments, the heterologous protein comprises an Fc domain, such as a mouse or human Fc domain. In specific embodiments, the heterologous protein promotes intermolecular association into homodimeric (for example, Fc domain from human IgG1, IgG2, IgG3), heterodimeric (for example, an engineered Fc domain, E/K coiled-coil), or multimeric (for example, pentabodies, nanoparticles) states of the fusion protein. In other embodiments, the heterologous protein is albumin, an albumin-binding protein or agent, or another protein that increases circulatory time of the TGF-β monomer in vivo.

Also provided are recombinant human TGF-β monomers or fusion proteins comprising a radiotherapy agent, a cytotoxic agent for chemotherapy, or a drug. Further provided are recombinant human TGF-β monomers or fusion proteins comprising an imaging agent, a fluorescent dye, or a fluorescent protein tag.

Further provided herein is a composition, such as a pharmaceutical composition, that includes a recombinant human TGF-β monomer or fusion protein disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

Also provided herein is a recombinant human TGF-β monomer, fusion protein or composition disclosed herein for use in a method of inhibiting TGF-β signaling in a cell, comprising contacting the cell with a recombinant human TGF-β monomer, fusion protein or composition disclosed herein.

In some embodiments, the method is an in vitro method of inhibiting TGF-β signaling in a cell, comprising contacting the cell with the recombinant human TGF-β monomer, the fusion protein, or the composition disclosed herein.

In some examples, the use includes administering the recombinant human TGF-β monomer, fusion protein or composition to a subject having a disease or disorder associated with aberrant TGF-β signaling. In some examples, the recombinant human TGF-β monomer, fusion protein or composition is administered by injection, such as by subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous or intratumoral injection.

Also provided is a recombinant human TGF-β monomer, fusion protein or composition disclosed herein for use in a method of treating a disease or disorder associated with aberrant TGF-β signaling. In some embodiments, the use in the method includes administering a recombinant human TGF-β monomer, fusion protein or composition disclosed herein to a subject.

In some embodiments, the disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder, such as but not limited to, pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, liver cirrhosis, kidney fibrosis (such as from damage caused by diabetes), atrial fibrosis, endomyocardial fibrosis, atherosclerosis, restenosis, scleroderma, or fibrosis caused by a surgical complication, chemotherapeutic drugs, radiation, injury or burns.

In other embodiments, the disease or disorder associated with aberrant TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, skin cancer, bladder cancer, liver cancer, ovarian cancer, renal cancer, endometrial cancer, colorectal cancer, gastric cancer, skin cancer (such as malignant melanoma), or thyroid cancer.

In other embodiments, the disease or disorder associated with aberrant TGF-β signaling is an ocular disease.

In other embodiments, the disease or disorder associated with aberrant TGF-β signaling is a genetic disorder of connective tissue.

Further provided are isolated nucleic acid molecules encoding a recombinant human TGF-β monomer disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter, such as a T cell specific promoter.

Also provided are vectors that include a TGF-β monomer-encoding nucleic acid molecule. In some embodiments, the vector is a viral vector, such as a lentiviral vector.

Isolated cells, such as, but not limited to, isolated T cells comprising a nucleic acid molecule or vector encoding a recombinant human TGF-β monomer disclosed herein are further provided. The cells can be autologous to the subject, or they can be heterologous (allogeneic). Compositions that include the isolated cells and a pharmaceutically acceptable carrier are also provided.

Further provided is a nucleic acid molecule, vector or isolated cell disclosed herein for use in methods of treating a disease or disorder associated with aberrant TGF-β signaling in a subject, comprising administering to the subject a nucleic acid molecule, vector or isolated cell disclosed herein. In some examples, the disease or disorder associated with aberrant TGF-β signaling is a fibrotic disorder. In other examples, the disease or disorder associated with aberrant TGF-β signaling is breast cancer, brain cancer, pancreatic cancer, prostate cancer, or skin cancer. In other examples, the disease or disorder associated with aberrant TGF-β signaling is an ocular disease. In yet other examples, the disease or disorder associated with aberrant TGF-β signaling is a genetic disorder of connective tissue.

Compositions, such as pharmaceutical compositions, that include a recombinant human TGF-β monomer or fusion protein, are provided herein. Also provided are compositions that include an isolated cell, such as a T cell, comprising a vector encoding a recombinant human TGF-β monomer. The composition includes a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers and excipients useful in this disclosure are conventional. See, e.g., <NPL>). For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.

With regard to administration of cells, a variety of aqueous carriers can be used, for example, buffered saline and the like, for introducing the cells. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.

The dosage form of the composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral and suppository formulations can be employed. Topical preparations can include eye drops, ointments, sprays, patches and the like. Inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like. Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in a suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

The compositions, such as pharmaceutical compositions, that include a recombinant human TGF-β monomer, can be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of TGF-β monomer administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.

The TGF-β monomers, or compositions thereof, can be administered to humans or other animals on whose tissues they are effective in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.

The following examples are provided to illustrate certain particular features and/or embodiments.

This example describes an engineered TGF-β monomer that is capable of blocking TGF-β signaling. The engineered TGF-β monomer, referred to herein as mmTGF-β2-<NUM>, has three changes relative to the monomer of wild type dimeric TGF-β2:.

The features of mmTGF-β2-<NUM> and other engineered TGF-β variants disclosed herein are described below and listed in Table <NUM>. The sequences of all engineered TGF-β variants are shown in <FIG> and set forth as SEQ ID NOs: <NUM>-<NUM>.

Previous studies showed that wild type TGF-β1 and TGF-β3 monomers (that is TGF-β1 and TGF-β3 monomers with the cysteine residue that normally forms the interchain disulfide, Cys77, substituted to serine) were about <NUM>-<NUM> fold less potent compared to the naturally occurring disulfide-linked homodimers, yet they nonetheless retained significant signaling activity, with midpoint stimulatory potencies (EC<NUM>s) of about <NUM> pM (<NPL>;<NPL>).

Based on structural studies, it was not clear why TGF-β1 Cys77→Ser and TGF-β3 Cys77→Ser variants would retain such significant signaling activity since one of the two essential receptors that binds to the growth factor, the TGF-β type I receptor (TβRI) was shown to bind by straddling the TGF-β homodimer interface (<FIG>) (<NPL>).

It was hypothesized that the TGF-β monomers were signaling by non-covalently dimerizing and binding the receptors, which in turn stabilized the noncovalent dimers (by virtue of the fact that at least one of them, TβRI, binds across the dimer interface). To generate a TGF-β monomer that would function as an inhibitor, rather than a stimulator of TGF-beta signaling, an engineered monomer was produced in which the primary dimerization motif, the interfacial α-helix, α3, was replaced with a flexible loop (<FIG> and <FIG>). It was reasoned that this would interfere with the ability of TGF-β to dimerize and recruit TβRI by (a) limiting the potential of the monomers to non-covalently dimerize due to hydrophobic contacts (<FIG>) and (b) by eliminating a significant portion of the contact surface for the TGF-β type I receptor, TβRI, that binds by straddling the TGF-beta dimer interface (<FIG>).

TGF-β1 was expressed as a secreted protein bound to its prodomain in stably transfected Chinese hamster ovary (CHO) cells. The cell line used to produce TGF-β1, and the accompanying procedure to isolate the mature disulfide-linked TGF-β1 homodimer from the conditioned medium has been previously described (<NPL>). Human homodimeric TGF-β2 (TGF-β2), human homodimeric TGF-β3 (TGF-β3), and variants, including avi-tagged (<NPL>) homodimeric TGF-β3 (TGF-β3-avi), monomeric TGF-β2 (mTGF-β2), monomeric TGF-β2 (mTGF-β3), mini monomeric TGF-β1 (mmTGF-β1), mini monomeric TGF-β2 (mmTGF-β2), mini monomeric TGF-β3 (mmTGF-β3), mini monomeric TGF-β2 with seven substitutions to enable high affinity TβRII binding (mmTGF-β2-<NUM>), and avi-tagged (<NPL>) mini monomeric TGF-β2 with seven substitutions to enable high affinity TβRII binding (mmTGF-β2-<NUM>) were expressed in E. coli, refolded from inclusion bodies into native folded disulfide-linked homodimers (TGF-β2, TGF-β3, TGF-β3-avi) or monomers (mTGF-β1, mTGF-β2, mTGF-β3, mmTGF-β1, mmTGF-β2, mmTGF-β3, mmTGF-β2-<NUM>, mmTGF-β2-<NUM>-avi), and purified to homogeneity using high resolution cation exchange chromatography (Source Q, GE Healthcare, Piscataway, NJ) as previously described (<NPL>). The nomenclature and major features of the dimeric and monomeric TGF-β used in this study are summarized in Table <NUM>, and the complete sequences are shown in <FIG>.

The human TβRI ectodomain (TβRI), spanning residues <NUM>-<NUM> of the mature receptor, or a variant spanning residues <NUM>-<NUM> of the mature receptor with a <NUM> amino acid avitag (<NPL>) appended to the C-terminus (TβRI-ΔC-Avi) was expressed in E. coli, refolded from inclusion bodies, and purified to homogeneity as previously described (<NPL>). The human TβRII ectodomain (TβRII), spanning residues <NUM>-<NUM> of the mature receptor, or the same but with a C-terminal hexahistidine tag (TβRII-His) was expressed in E. coli, refolded from inclusion bodies, and purified to homogeneity as previously described (<NPL>).

TGF-β dimers and monomers were concentrated in <NUM> acetic acid to concentrations of <NUM> or higher and diluted to the desired concentration in either <NUM> acetic acid or phosphate buffered saline (PBS, <NUM> Na<NUM>HPO<NUM>, <NUM> KH<NUM>PO<NUM>, <NUM> NaCl, <NUM> KCl, pH <NUM>). The pH of the samples diluted into PBS were adjusted with small aliquots of NaOH to ensure a final pH of <NUM>. The light scattering at <NUM> of the samples were measured using a HP <NUM> diode array spectrophotometer (HP, Palo Alto, CA). The samples were transferred to a microfuge tube, centrifuged at <NUM> × g for <NUM> minutes and the absorbance at <NUM> of the supernatant was measured using a NANODROP™ spectrophotometer (ThermoFisher, Waltham, MA). Results of solubility assays are shown in <FIG>.

mmTGF-β2 and mmTGF-β2-<NUM> samples isotopically labeled with <NUM>N or <NUM>N and <NUM>C for NMR were prepared by growing bacterial cells in M9 media containing <NUM> % (w/v) <NUM>NH<NUM>Cl or <NUM> % (w/v) <NUM>NH<NUM>Cl and <NUM>% (w/v) <NUM>C labeled glucose. All NMR samples were prepared in <NUM> sodium phosphate, <NUM> <NUM>-[(<NUM>-cholamidopropyl)dimethylammonio]-<NUM>-propanesulfonate (CHAPS), <NUM>% <NUM>H<NUM>O, <NUM>% w/v sodium azide at a protein concentration of <NUM> - <NUM>, pH <NUM>. All NMR data was acquired at a sample temperature of <NUM> at either <NUM> or <NUM>. Backbone resonance assignments of mmTGF-β2 and mmTGF-β2-<NUM> were obtained by collecting and analyzing sensitivity-enhanced HNCACB (<NPL>), CBCA(CO)NH (<NPL>), C(CO)NH (<NPL>), HNCO (<NPL>), data sets with <NUM>% non-uniform sampling (NUS) of the points in the <NUM>C,<NUM>N acquisition grid. Backbone amide <NUM>N T<NUM> relaxation parameters were measured in an interleaved manner at <NUM>°K at a <NUM>N frequency of <NUM> using <NUM>H-detected pulse schemes previously described (<NPL>). The T<NUM> data sets were each collected using <NUM> - <NUM> delay times, varying between <NUM> - <NUM>. The T<NUM> relaxation times were obtained by fitting relative peak intensities as a function of the T<NUM> delay time to a two parameter decaying exponential. Data was processed using NMRPipe (<NPL>), with the SMILE algorithm used for prediction of the missing points in the <NUM>C and <NUM>N dimensions of the NUS data sets (<NPL>). Data analysis was performed using NMRFAM-SPARKY (<NPL>).

SPR measurements with TGF-β2 and mmTGF-β2 shown in <FIG> were performed using a BIACORE™ <NUM> SPR (G. Healthcare, Piscataway, NJ) instrument with direct immobilization of TGF-β2 or mmTGF-β2 on the surface of a CM5 sensor chip (G. Healthcare, Piscataway, NJ) using an amine (carbodiimide-based) coupling kit (G. Healthcare, Piscataway, NJ). SPR experiments shown in <FIG>, <FIG> and <FIG> and <FIG>, <FIG> with TGF-β3 and mmTGF-β2-<NUM>, respectively, were performed using a BIACORE™ X100 SPR instrument (G. Healthcare, Piscataway, NJ) with biotinylated ligands captured at a moderate density (<NUM> - <NUM> RU) onto a streptavidin-coated CM5 sensor chip (GE Healthcare, Piscataway, NJ). Biotinylated TGF-β3 or mmTGF-β2-<NUM> was generated by expressing TGF-β3 or mmTGF-β2-<NUM> with an N-terminal <NUM> amino acid avitag (<NPL>). TGF-β3-avi or mmTGF-β2-<NUM>-avi was bound to TβRII in <NUM> bicine at pH <NUM> and biotinylated by incubating with a catalytic amount of bacterially expressed BirA recombinase, biotin, and ATP at <NUM> for <NUM> hours as described (<NPL>). Biotinylated avi-tagged TGF-β3 or avi-tagged TGF-β2-<NUM> were bound to a C4 reverse phase column equilibrated with <NUM>% water/<NUM>% acetonitrile/<NUM>% triflouroacetic acid and eluted with a linear acetonitrile gradient.

SPR measurements shown in <FIG> were performed in HBS-EP buffer (<NUM> HEPES, pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% surfactant P20; GE Healthcare, Piscataway, NJ) with the receptor indicated injected over a series of two-fold dilutions over the concentration range shown. Injections were carried out in duplicate and included <NUM> buffer blank injections at the start of the experiment. Binding was allowed to associate for <NUM> - <NUM> minutes at a flow rate of <NUM> min-<NUM>, followed by dissociation for <NUM> minute or longer. Each cycle of injection was followed by <NUM> of regeneration with <NUM> guanidine•HCl, <NUM> NaCl. Data was processed by subtracting both the response from a blank flow cell and buffer blanks using the program Scrubber2 (Biologic software, Campbell, Australia). Kinetic fitting of the data was performed with Scrubber2 assuming a simple <NUM>:<NUM> binding model. SPR measurements shown in <FIG> were performed similarly, except <NUM> TβRII was included in both the running buffer and the injected samples. The results of SPR measurements are shown in Table <NUM> and <FIG>.

Crystals of mmTGF-β2 were formed in sitting drops at <NUM> by combining <NUM>µL of a <NUM> mL-<NUM> protein stock solution in <NUM> MES pH <NUM> with <NUM>µL of the precipitant from the well, <NUM>% PEG <NUM>, <NUM> sodium thiocyanate. Harvested crystals were mounted in undersized nylon loops with excess mother liquor wicked off, followed by flash-cooling in liquid nitrogen prior to data collection. Data were acquired at the Advanced Photon Source NE-CAT beamline <NUM>-ID-C and integrated and scaled using XDS (<NPL>). The structure was determined by the molecular replacement method implemented in PHASER (<NPL>) using a truncated version of PDB entry 2TGI (<NPL>) as the search model. Coordinates were refined using PHENIX (<NPL>), including simulated annealing with torsion angle dynamics, and alternated with manual rebuilding using COOT (<NPL>). Data collection and refinement statistics are shown in Table <NUM>.

Crystals of the mmTGF-β2-<NUM>:TβRII complex were formed in hanging drops at <NUM> by combining <NUM>µL of a <NUM> mL-<NUM> stock solution of the complex in <NUM> Tris, pH <NUM> with <NUM>µL of <NUM> HEPES, pH <NUM>, <NUM> % v/v (+/-)-<NUM>-Methyl-<NUM>,<NUM>-pentanediol. Harvested crystals were mounted in nylon loops, followed by flash-cooling in liquid nitrogen prior to data collection. Data were acquired at the Advanced Photon Source <NUM>-ID-C and integrated and scaled using HKL2000 (<NPL>). The structure was determined by the molecular replacement method implemented in PHASER (<NPL>) using TβRII (PDB 1M9Z;<NPL>) and mmTGF-β2 as search models. Coordinates were refined using PHENIX (<NPL>), alternated with manual rebuilding using COOT (<NPL>). Data collection and refinement statistics are shown in Table <NUM>.

Crystals of mmTGF-β2-<NUM> were formed in hanging drops at <NUM> by combining <NUM>µL of a <NUM> mL-<NUM> protein stock solution in <NUM> acetic acid with <NUM>µL of the precipitant from the well, <NUM> sodium acetate dibasic trihydrate, pH <NUM>, <NUM>% <NUM>-propanol, and <NUM> calcium chloride dehydrate, and <NUM>µL <NUM>% n-ocyl-β-D-glucoside. Harvested crystals were mounted in nylon loops and cryoprotected in well buffer containing <NUM>% glycerol and flash-cooled in a nitrogen stream. Data was collected at <NUM> using a Rigaku FR-E Superbright generator equipped with a Saturn <NUM> CCD detector and processed using MOSFLM (<NPL>) in CCP4 (<NPL>). The structure of mmTGF-β2-<NUM> was solved via molecular replacement using the structure of mmTGF-β2-<NUM> from its co-crystal structure with TβRII. Iterative model building and refinement were performed using COOT (<NPL>) and PHENIX, respectively. Data collection and refinement statistics are shown in Table <NUM>.

Results of structural studies are shown in <FIG>, <FIG>, <FIG> and <FIG>.

Human embryonic kidney <NUM> (HEK293) cells stably transfected with the CAGA<NUM> TGF-β reporter were used for the luciferase reporter assays (<NPL>). HEK293 cells containing the stably transfected CAGA<NUM> TGF-β reporter were maintained in Dulbecco's modified eagles medium (DMEM) containing <NUM>% fetal bovine serum (FBS) and <NUM>% penicillin/streptomycin. Cells were treated for <NUM> hours with a TGF-β (TGF-β1, mTGF-β3 or mmTGF-β2-<NUM>) concentration series or an mmTGF-β2-<NUM> concentration series in the presence of a constant sub-saturating concentration of TGF-β (TGF-β1, <NUM> pM; TGF-β2, <NUM> pM; TGF-β3, <NUM> pM). Proteins were diluted in DMEM containing <NUM>% w/v BSA. After <NUM> hours, cells were lysed with Tropix lysis buffer (ThermoFisher, Waltham, MA) and luciferase activity was read with a Promega GloMax luminometer (Promega, Madison, WI). Luciferase activity was normalized to total protein levels determined by bicinchoninic acid (BCA) protein assay. Graphpad Prism <NUM> was used to fit the data to standard models for ligand activity (EC<NUM>) and ligand inhibitory activity (IC<NUM>) (Graphpad, La Jolla, CA). Results are shown in <FIG>.

The following purified proteins were used to address the ligand requirements for the formation of complexes containing TβRI and TβRII: TGF-β3, mTGF-β3, mmTGF-β2-<NUM>, biotinylated TβRI-ΔC-Avi and TβRII-His. Initially <NUM> binary complexes of TGF-β3:TbRII-His (<NUM>:<NUM>), mTGF-β3:TβRII-His (<NUM>:<NUM>), and mmTGF-β2-<NUM>:TβRII-His (<NUM>:<NUM>) were formed in a <NUM> Tris, pH <NUM> buffer and stored at <NUM>. A time-resolved fluorescence resonance energy transfer (TR-FRET) assay based on the proximity-dependent transfer of fluorescence from the donor terbium cryptate labeled anti-His mAb (Tb-anti-His, CisBio, Bedford, MA) to the acceptor XL665 labeled streptavidin (SA-<NUM>, CisBio, Bedford, MA) was used to monitor the assembly of ternary ligand:TβRII-His:biotinylated TβRI-ΔC-Avi complexes. Fifty µL assays containing <NUM> or <NUM> TGF-β3:TβRII-His (<NUM>:<NUM>), mTGF-β3:TβRII-His (<NUM>:<NUM>), and mmTGF-β2-<NUM>:TβRII-His (<NUM>:<NUM>) complexes were incubated with <NUM> biotinylated TβRI-ΔC-Avi. Each <NUM>µl ternary complex formation assay also contained <NUM> Tb-anti-His and <NUM> SA-<NUM> and was incubated at room temperature for <NUM> hours. Each condition was tested in replicates of six. Buffer control (n=<NUM>) contained only <NUM> Tb-anti-His and <NUM> SA-XL665. The buffer conditions for each assay were <NUM> Tris, <NUM> NaCl, pH <NUM>. The assays were performed in Corning black <NUM> well low flange microplates (ThermoFisher, Waltham, MA). After a <NUM>-hour incubation, the assay plate was measured for terbium/XL-<NUM> TR-FRET on a BMG Labtech Pherastar FS multimode plate reader (BMG Labtech Inc. , Cary, NC). An optic module containing <NUM>, <NUM> and <NUM> filters was used to monitor TR-FRET producing raw data for <NUM>/<NUM> (terbium emission) and <NUM>/<NUM> (XL-<NUM>) emission. The ratio of <NUM> emission/<NUM> emission was determined for each condition and was subsequently used to calculate ΔF, which is a measure that reflects the signal of the sample versus the background. ΔF was calculated using the following equation: (Ratiosignal-Rationegative/Rationegative) × <NUM>. The Ratiosignal refers to the assays containing the trimeric complexes or buffer control. The Rationegative refers to two assays buffer control (<NUM> Tb-anti-His and <NUM> SA-<NUM>). For the buffer control, <NUM> out of the <NUM> replicates were assigned as negative controls for the purpose of calculating ΔF. ΔF was calculated for the remaining <NUM> buffer control replicates. Results are shown in <FIG> and <FIG>.

mTGF-β3, mmTGF-β2, and mmTGF-β2-<NUM> were analyzed by sedimentation velocity to establish equilibrium constants for self-association of monomeric TGF-βs to form homodimers. mTGF-β3, mmTGF-β2, and mmTGF-β2-<NUM> were each measured at <NUM> in an epon two channel centerpiece fitted with quartz windows, and centrifuged at <NUM> and <NUM>,<NUM> rpm for <NUM> hours in a <NUM> sodium phosphate buffer adjusted to pH <NUM>, containing <NUM> NaCl. Three hundred scans were collected in intensity mode on a Beckman Optima XL-I analytical ultracentrifuge at the CAUMA facility at the UTHSCSA. Data analysis was performed with UltraScan release <NUM> (<NPL>; <NPL>), calculations were performed at the Texas Advanced Computing Center on Lonestar-<NUM>. The sedimentation velocity data were initially fitted with the two-dimensional spectrum analysis as described in (<NPL>) to remove time- and radially invariant noise from the raw data, and to fit the meniscus position. Subsequently, the data were fitted to a discrete monomer-dimer model using the adaptive space-time finite element method (<NPL>) and genetic algorithms for the parameter optimization (<NPL>). The monomer-dimer model accounts for mass action and the reversible association behavior, fitting the KD, hydrodynamic parameters, as well as the partial specific volume while assuming the predicted molar mass for either wildtype or mutant. A Monte Carlo analysis (<NPL>) with <NUM> iterations was performed for each dataset to obtain fitting statistics. Buffer density and viscosity were estimated with UltraScan based on buffer composition and all hydrodynamic values were corrected for standard conditions (<NUM> and water). The fitting results provided an excellent fit with random residuals and very low RMSD values. All results are summarized in Table <NUM>, and <FIG>.

The structures of the TGF-β receptor complexes (<NPL>; <NPL>), as well as accompanying binding and cross-linking studies with TGF-β3 C77S (<NPL>; <NPL>; <NPL>), suggested that the signaling capacity of monomeric TGF-βs (TGF-β1 C77S or mTGF-β1 and TGF-β3 C77S or mTGF-β3) arise from their ability to non-covalently dimerize and in turn bind their receptors (<FIG>). This led to the hypothesis that it should be possible to diminish or completely eliminate receptor complex assembly with monomeric TGF-βs by removing or altering residues responsible for dimer formation and binding of TβRI. The structural motif that likely contributes the greatest to self-association of the monomers is the "heel" α-helix, α-helix <NUM> (<FIG>). This helix is highly amphipathic and has numerous hydrophobic interactions with residues that line the "palm" of the opposing monomer (<FIG>). This helix also forms a large portion of the binding surface for TβRI (<FIG>). Thus, it was hypothesized that elimination of α-helix <NUM> would interfere with both self-association of the monomers and binding of TβRI, but would not impair TβRII binding as this occurs through the ligand fingertips far away from α-helix <NUM> (<FIG>).

To evaluate this hypothesis, bacterial expression constructs were generated for TGF-β1, TGF-β2, and TGF-β3 in which residues <NUM>-<NUM> were eliminated and Cys-<NUM> was substituted with serine. This corresponds to deletion of all of α-helix <NUM>, as well as five flanking residues on the N-terminal end and three flanking residues on the C-terminal end (<FIG>). The length of the deletion was chosen so as to leave a sufficient number of residues between the last residue of β-strand <NUM> (Gly-<NUM>) and the first residue of β-strand <NUM> (Cys-<NUM>/Ser-<NUM>) to form an unconstrained loop that bridges β-strands <NUM> and <NUM>. Although a secondary consideration, either two (TGF-β2) or three (TGF-β1 and -β3) of the loop-forming residues were also substituted to increase the net overall charge at pH <NUM> for the full-length TGF-β1, -β2, and -β3 monomers from -<NUM>, +<NUM>, and +<NUM> to -<NUM>, +<NUM>, and +<NUM>, respectively, for the constructs in which α-helix <NUM> was deleted (<FIG>). The rationale for this was that the solubility of the monomers, which like the homodimers are poor from pH <NUM> to <NUM> (see <FIG>), might be improved by both removing hydrophobic α-helix <NUM> and by artificially increasing the net charge at pH <NUM>.

The TGF-β1, -β2, and -β3 "mini-monomers" described above, designated mmTGF-β1, mmTGF-β2, and mmTGF-β3, were expressed in Escherichia coli and accumulated in the form of insoluble inclusion bodies. The inclusion bodies were isolated, and after reconstitution and purification in denaturant, the mini-monomers were renatured by dilution into CHAPS-containing buffer at pH <NUM> as described previously (<NPL>). The folding of the mini-monomers differed greatly; a large portion of the mmTGF-β2 remained soluble during the folding and yielded large amounts of monomeric protein after purification by cation exchange chromatography, whereas only a small amount of mmTGF-β1 and mmTGF-β3 remained soluble during the folding, and either no monomeric protein (TGF-β1) or a very small amount of monomeric protein (TGF-β3) was obtained after purification by cation exchange chromatography. This pattern mirrors the pattern previously observed for the folding of TGF-β homodimers from full-length wild type monomers (<NPL>) and likely reflects differences in the intrinsic propensity of the monomers to properly form the four intramolecular disulfides characteristic of each monomer. mmTGF-β2 was the least desired variant, due to the expected low affinity for binding TβRII. However, this was considered an addressable concern based on prior studies, which demonstrated that substitution of Lys-<NUM>, Ile-<NUM>, and Lys-<NUM> in TGF-β2 with the corresponding residues in TGF-β1 and TGF-β3 engendered TGF-β2 with the ability to bind TβRII with high affinity (<NPL>; <NPL>).

To determine whether mmTGF-β2 was suitable for further development in the manner described above, it was characterized in terms of its folding, solubility, and receptor binding properties. To assess folding, a <NUM>N-labeled sample of mmTGF-β2 was prepared and examined by recording a two-dimensional <NUM>H-<NUM>N shift correlation spectrum (<FIG>). This revealed a highly dispersed spectrum characteristic of natively folded protein. The spectrum could be fully assigned, and analysis of the assigned chemical shifts to identify secondary structure propensities showed that the protein had the expected secondary structure, particularly in the palm region formed by the cysteine knot and the finger region where TβRII binds (<FIG>). This analysis further showed that the newly created loop between residues <NUM> and <NUM> had near zero probability of forming either an α-helix or β-strand, suggesting that it is likely flexible as would be expected for a loop of this length connecting two antiparallel β-strands. This was directly confirmed by an analysis of backbone <NUM>N T<NUM> values. These values provide information about motions on fast (nanosecond-picosecond) and intermediate (microsecond-millisecond) time scales and were significantly elevated in the region corresponding to the newly created loop relative to the other parts of the protein (<FIG>), which, except for the N terminus and the short loop connecting α-helix <NUM> and β-strand <NUM>, are expected to be structurally well-ordered.

To directly examine the three-dimensional structure, mmTGF-β2 was crystallized, and its structure was determined to a resolution of <NUM>Å using molecular replacement (Table <NUM>). The overall fold of mmTGF-β2 was shown to be highly similar to that previously determined for TGF-β2, with the exception of the newly created loop, which was shown to take the place of α-helix <NUM> as anticipated (<FIG>). Superimposition of the mmTGF-β2 with the monomer from the structure of TGF-β2 shows that there is a systematic displacement of up to about <NUM>Å of the finger region of mmTGF-β2 relative to TGF-β2. Such differences appear to be the result of bending of the monomer near the center of the finger region and not a change in the structure of the finger region, as superimposition of the fingers alone show that they correspond closely, with a backbone root-mean-square deviation of under <NUM>Å and similar orientations of the side chains of several residues that pack and stabilize the fingers (<FIG>). Such bending is also supported by an overlay of the two molecules of mmTGF-β2 present in the crystallographic asymmetric unit, which also exhibit a smaller but still noticeable displacement of the finger regions relative to one another (<FIG>). Consistent with the NMR analysis, not only was the electron density noticeably weaker in the region corresponding to the newly created loop, but also it was shown to adopt different orientations for the two molecules from the asymmetric unit <FIG>).

The similar folding of mmTGF-β2 relative to TGF-β2, especially in the TβRII-binding finger region, suggested that it would also bind TβRII in a similar manner. To evaluate this, surface plasmon resonance (SPR) experiments were performed in which the same concentration series of TβRII was injected over TGF-β2 and mmTGF-β2 immobilized on separate flow cells (<FIG>). Although it was not possible to quantitate affinity due to weak binding, the sensorgrams nonetheless showed similar shapes and concentration dependence. These sensorgrams showed that mmTGF-β2 binds TβRII weakly, consistent with earlier reports <NPL>), and that it did so in a manner qualitatively similar to TGF-β2.

The solubility of mmTGF-β2 appeared to be significantly better than that of TGF-β2 and the full-length TGF-β2 monomer, mTGF-β2, as samples of the former could be readily prepared at concentrations of <NUM>-<NUM> ml-<NUM> without noticeable precipitation at pH <NUM>, whereas samples of the latter two proteins were completely precipitated under these same conditions. To quantitate solubility, TGF-β2, mTGF-β2, and mmTGF-β2 were prepared as concentrated stocks in <NUM> acetic acid, pH <NUM>, where they were readily soluble and then diluted into PBS, pH <NUM>. The light scattering at <NUM> was measured to assess precipitation, and then the samples were centrifuged, and the absorbance at <NUM> was measured to assess the protein concentration. This demonstrated that TGF-β2 and mTGF-β2 were both effectively insoluble at neutral pH over the entire concentration range evaluated (<NUM>-<NUM>) (<FIG>). This is consistent with the known poor solubility of the TGF-β homodimers (<NPL>), but it shows that this property also extends to full-length monomeric TGF-βs. The mini-monomeric TGF-β2 (mmTGF-β2) in contrast, exhibited modest light scattering and a corresponding modest reduction in the amount of soluble protein relative to that expected when the protein concentration was <NUM> or higher, indicating that indeed mmTGF-β2 was reasonably soluble at neutral pH, although not perfectly so. This was reflected in NMR spectra, which showed that although <NUM>-<NUM> <NUM>N mmTGF-β2 samples could be readily prepared, the spectrum was nonetheless poor, with the only detectable signals arising from residues in the flexible parts of the protein, namely the N terminus, the exposed loop between α-helix <NUM> and β-strand <NUM>, and the newly created loop between β-strands <NUM> and <NUM>. The fact that signals could only be detected from the flexible parts of the protein suggested that mmTGF-β2 forms large soluble aggregates under these conditions. Through trial and error, it was found that these soluble aggregates could be eliminated by addition of the zwitterionic detergent CHAPS, with the majority of the NMR signals appearing at the concentration of <NUM> CHAPS and all of the NMR signals appearing at <NUM> CHAPS. Thus, all NMR spectra, including that shown in <FIG>, were recorded in the presence of <NUM> CHAPS.

The results presented above show that whereas mmTGF-β2 is natively folded, it nonetheless possesses low intrinsic affinity for binding TβRII. To confer mmTGF-β2 with the ability to bind TβRII with high affinity comparable with that of TGF-β1 and TGF-β3, the three residues in mouse TGF-β2 shown previously to differ in the interface with TβRII, Lys-<NUM>, Ile-<NUM>, and Asn-<NUM> (<NPL>; <NPL>), were substituted with the corresponding residues from TGF-β1 and -β3, Arg-<NUM>, Val-<NUM>, and Arg-<NUM> (<FIG>). In previous studies, substitution of these three residues was shown to be sufficient to confer TGF-β2 with a TβRII binding affinity comparable with TGF-β1 and TGF-β3 (<NPL>; <NPL>). Despite this, four additional residues peripheral to the TβRII-binding site that differed in TGF-β2 relative to TGF-β1 were also substituted with the corresponding residues from TGF-β1 (R26K, L89V, T95K, and I98V) (<FIG>). Although previous results suggested this was not strictly necessary, it was nonetheless done to ensure that the precise orientation of residues in the mmTGF-β2-binding site for TβRII matched as closely as possible with that in the high affinity TGF-β isoforms, TGF-β1 and TGF-β3. The resulting construct bearing these seven amino acid substitutions, designated mmTGF-β2-<NUM> (<FIG>, <FIG> and Table <NUM>), was expressed in E. coli in the form of insoluble inclusion bodies. As with mmTGF-β2, most of the protein remained in solution after reconstitution and dilution into native folding buffer, and large amounts of homogenous monomer could be isolated (<NUM>-<NUM>/liter of E. coli culture medium).

The folding and homogeneity of the isolated mmTGF-β2-<NUM> was evaluated by NMR, and as with mmTGF-β2, the protein was found to have the expected number of signals in a 2D <NUM>H-<NUM>N shift correlation spectrum (<FIG>) as well as secondary structure, as determined by an analysis of the NMR secondary shifts (<FIG>). The solubility of mmTGF-β2-<NUM> was evaluated as before, and as shown, its behavior was comparable or perhaps slightly better than that of mmTGF-β2 (<FIG>). This slight improvement in the macroscopic solubility did not however change the microscopic solubility as NMR analysis showed that it was still necessary to include <NUM> CHAPS in the sample buffer to detect signals from all of the backbone amide resonances in the protein.

The three-dimensional structure of mmTGF-β2-<NUM> was determined by crystallography to a resolution of <NUM>Å (Table <NUM>), and as before the overall fold was preserved relative to TGF-β2, with the only difference being a slight hinge bending of the monomer as described for mmTGF-β2 (<FIG>). The increase in the <NUM>N T<NUM> relaxation times in the region corresponding to the newly formed loop in mmTGF-β2-<NUM> was comparable with that in mmTGF-β2 (<FIG>). This suggested that the missing density in the region corresponding to the newly formed loop in mmTGF-β2-<NUM>, which among the three molecules in the asymmetric unit was observed for part of chain A and most of chain C, was not due to increased dynamics, but other factors, most likely the lower resolution of the mmTGF-β2-<NUM> structure compared with the mmTGF-β2 structure (Table <NUM>).

To determine whether mmTGF-β2-<NUM> bound TβRII with high affinity, variants of mmTGF-β2-<NUM> and TGF-β3 were produced bearing an N-terminal avitag, and after biotinylation and immobilization onto a streptavidin-coated SPR sensor, their binding affinity for TβRII was measured by performing kinetic SPR experiments (<FIG>). The sensorgrams obtained differed greatly from that previously obtained for mmTGF-β2 and TGF-β2, in that they exhibited a clear pattern of saturation. The sensorgrams were furthermore shown to have similar shapes as well as fitted parameters, including KD values (Table <NUM>), which were within experimental error of one another and consistent, although on the high end, with KD values reported earlier for TβRII binding to TGF-β1 and TGF-β3 (<NPL>; <NPL>; <NPL>).

To determine whether the interactions that enabled high affinity TβRII binding were preserved in mmTGF-β2-<NUM> compared with TGF-β1 and TGF-β3, the mmTGF-β2-<NUM>·TβRII complex was crystallized, and its structure was determined to a resolution of <NUM>Å (Table <NUM>). The overall structure of the mmTGF-β2-<NUM>·TβRII complex was shown to be very similar to that of one of the TβRII-bound monomers from the structure of the TGF-β3·TβRII·TβRI complex, with TβRII bound to the mmTGF-β2-<NUM> fingertips in a manner that is essentially indistinguishable from that of TGF-β3 (<FIG>). The interactions known to contribute most significantly to high affinity binding are furthermore shown to be fully preserved in the mmTGF-β2-<NUM>·TβRII complex relative to TGF-β1·TβRII and TGF-β3·TβRII complexes that have been previously determined (the TGF-β3·TβRII complex determined to <NUM>Å (<NPL>) is shown as this is the highest resolution structure determined to date) (<FIG>). This includes the packing of Ile-<NUM> from TβRII in the hydrophobic pocket between the TGF-β fingers, and the hydrogen-bonded ion pairs formed between TGF-β Arg-<NUM> and Arg-<NUM> on the tips of the loops connecting fingers <NUM>/<NUM> and <NUM>/<NUM>, respectively, and the carboxylate groups of Glu-<NUM> and Asp-<NUM> on TβRII (<FIG>).

The results presented above show that mmTGF-β2-<NUM> possesses one of the essential attributes required to function as a dominant negative inhibitor of TGF-β signaling, which is the ability to bind TβRII with high affinity comparable with that of TGF-β1 and TGF-β3. To directly assess whether mmTGF-β2-<NUM> might signal and, if not, whether it might function as an inhibitor, TGF-β signaling was assessed by treating HEK293 cells stably transfected with a TGF-β luciferase reporter under the control of a CAGA<NUM> promoter (<NPL>) with increasing concentrations of TGF-βs. The results showed that dimeric TGF-β1 (TGF-β1) and full-length monomeric TGF-β3 (mTGF-β3) resulted in a sigmoidal increase in the luciferase response, with concentrations of roughly <NUM> pM TGF-β1 and <NUM> pM mTGF-β3 leading to no further increase in the measured luciferase response. This is consistent with earlier reports that showed that (full-length) monomeric TGF-β1 and -β3 were <NUM>-<NUM>-fold less potent than their dimeric counterparts (<NPL>; <NPL>). The normalized luciferase responses could be readily fitted to a standard model for ligand-dependent activation and yielded EC<NUM> values of <NUM> ± <NUM> pM for TGF-β1 and <NUM> ± <NUM> pM for mTGF-β3. The values for TGF-β1 and mTGF-β3 were in close accord with the values previously reported by <NPL>) for TGF-β1 and by <NPL>) for mTGF-β3. The potent sub-nanomolar signaling activity observed for TGF-β1 and mTGF-β3 stands in contrast to that of mmTGF-β2-<NUM>, which had no detectable signaling activity at the concentration that led to a saturating response for mTGF-β3 (ca. <NUM> pM) or at concentrations that were up to four orders of magnitude higher (<FIG>). Thus, mmTGF-β2-<NUM> was either completely devoid of signaling activity or it possessed signaling activity, but with a potency more than a <NUM>,<NUM>-fold less than that of mTGF-β3.

To further investigate the properties of mmTGF-β2-<NUM>, a competition experiment was performed in which the same HEK293 luciferase reporter cell line was stimulated with a constant sub-EC<NUM> concentration of dimeric TGF-β1 (<NUM> pM) and increasing concentrations of mTGF-β3 or mmTGF-β2-<NUM>. The results showed that mTGF-β3 further stimulated signaling with a midpoint concentration similar to that of mTGF-β3 alone (<FIG>). The fitted EC<NUM> values confirm this, with an EC<NUM> of <NUM> ± <NUM> pM for the data shown in <FIG> and EC<NUM> of <NUM> ± <NUM> pM for the data shown in <FIG>. The behavior of mmTGF-β2-<NUM> was very different, with no detectable change in the signaling activity when added up to concentrations of <NUM>, but with a sharp decrease to no detectable signaling activity when the concentration was increased to <NUM> (<FIG>). This shows that mmTGF-β2-<NUM> indeed possesses no signaling activity and that it can function to completely block and inhibit TGF-β signaling. The normalized luciferase responses could be readily fitted to a standard model for ligand-dependent inhibition and yielded an IC<NUM> value of <NUM> ± <NUM>. Similar experiments showed that mmTGF-β2-<NUM> also functioned as a potent competitive inhibitor against the other TGF-β isoforms, TGF-β2 and TGF-β3, with measured IC<NUM> values (TGF-β2 IC<NUM> <NUM> ± <NUM> and TGF-β3 IC<NUM> <NUM> ± <NUM>) within a factor of <NUM>-<NUM> of that measured for TGF-β1 (<FIG>). These IC<NUM> values are on the lower end of the range of affinities that have been reported for binding of the high affinity TGF-β isoforms to TβRII, including mmTGF-β2-<NUM> reported herein (Table <NUM>). This suggests that mmTGF-β2-<NUM> functions to inhibit TGF-β signaling by binding to and blocking endogenous TβRII. The fact that the measured potency is greater than the greatest affinity previously reported for TGF-β1 and TGF-β3 binding to TβRII (<NUM>) (<NUM>), suggests that other factors, such as nonspecific association of mmTGF-β2-<NUM> with the plasma membrane, may serve to potentiate its inhibitory activity.

The finding that mmTGF-β2-<NUM> possesses no apparent signaling activity, and functions as a low nanomolar inhibitor of TGF-β signaling, suggests that the elimination of α-helix <NUM> diminished non-covalent association of the monomers and greatly attenuated or abrogated TβRI binding. To assess this directly, SPR experiments were performed to determine whether mmTGF-β2-<NUM> could recruit TβRI in the presence of TβRII. To accomplish this, increasing concentrations of TβRI and the same concentration series of TβRI in the presence of near-saturating amounts of TβRII (<NUM>) were injected over the same TGF-β3 and mmTGF-β2-<NUM> SPR chip surfaces used for the TβRII binding measurements described above. This showed that TβRI alone binding is negligible to both TGF-β3 and mmTGF-β2-<NUM> (<FIG>), but unlike TGF-β3, TβRII-bound mmTGF-β2-<NUM> is unable to recruit TβRI (<FIG>). This is consistent with the earlier result reported by <NPL>) that TβRII-bound mTGF-β3 was significantly or completely impaired in terms of its ability to bind and recruit TβRI. This also provides further evidence that TβRII-bound TGF-β monomers are incapable of binding and recruiting TβRI, but because the mmTGF-β2-<NUM> was immobilized on the surface of the sensor, it alone does not provide any insight as to whether mmTGF-β2-<NUM> might be capable of non-covalently dimerizing and binding and recruiting TβRI.

To address these questions directly, two solution-based techniques were used, analytical ultracentrifugation (AUC) and time-resolved fluorescence resonance energy transfer (TR-FRET). The AUC experiments were performed by measuring the total UV absorbance at <NUM> as a function of the radial position and time as mTGF-β3, mmTGF-β2, and mmTGF-β2-<NUM> were sedimented under acidic conditions, pH <NUM>, where the monomers are fully soluble. The AUC data revealed parabolically shaped van Holde-Weischet sedimentation coefficient distribution plots for all three monomers, consistent with each undergoing reversible self-association to form a dimer or other higher order oligomer. To determine more precisely which species might be present in solution, the data were fitted to the simplest model possible, a discrete monomer-dimer equilibrium, using finite element analysis. The fitting procedure resulted in near-perfect fits for all three monomers to the simple monomer-dimer model, as shown by (a) the close overlays between the fitted curves (red) with the raw data, after the time and radially-invariant noise was removed (black) and (b) the absence of any systemic deviations in the residuals (<FIG>). The fitted parameters further showed that KD for self-association was <NUM> order of magnitude greater for mTGF-β3 compared with mmTGF-β2 and mmTGF-β2-<NUM>. Thus, the removal of the heel helix, α3, does diminish self-association of the monomers to form dimers, but it does not completely abrogate dimer formation.

TR-FRET was used to assess the ability of dimeric and monomeric TGF-βs to bind and bring TβRI and TβRII together. This was accomplished by generating differentially tagged forms of TβRII and TβRI and in turn binding to these tags with proteins labeled with fluorescent donors and acceptors. TβRII was tagged with a C-terminal His tag and was bound by a terbium cryptate-labeled anti-His monoclonal antibody fluorescent donor, and TβRI was tagged with an N-terminal avitag, which after enzymatic biotinylation was bound to a dye-labeled (XL-<NUM>) streptavidin fluorescent acceptor (<FIG>). The addition of TGF-β to the tagged receptors brings them together and leads to a large increase in the ΔF value, which is defined as the ratio of the acceptor and donor emission fluorescent intensities. The TR-FRET assay is demonstrated by the data presented in <FIG> and was used here to compare the ability of the TGF-β3 full-length monomer, mTGF-β3, and the TGF-β2 mini-monomer that binds TβRII with high affinity, mmTGF-β2-<NUM>, to bind and bring TβRI and TβRII together. The TR-FRET signal for mTGF-β3 was shown to be comparable with that of TGF-β3, and this did not depend on whether the TGF-β concentration was <NUM> or <NUM> (<FIG>). The TR-FRET signal of mmTGF-β2-<NUM> was, in contrast, within the error limits of the buffer control, and this did not depend on the TGF-β concentration (<FIG>). These results demonstrate that under these conditions, mTGF-β3 retains full capacity to assemble a non-covalent dimeric complex with TβRI and TβRII, but under these same conditions, mmTGF-β2-<NUM> has no capacity to do so. These results, together with the AUC results, indicate that the removal of the heel helix had the effects hypothesized; its removal reduced, but did not eliminate, dimer formation, and even though dimers are still formed, they are unable to bind and recruit TβRI.

The TGF-βs are responsible for promoting the progression of numerous human diseases (<NPL>; <NPL>; <NPL>; <NPL>), yet despite nearly two decades of preclinical studies and clinical trials, no inhibitors have been approved for use in humans. The results presented herein demonstrate that an engineered TGF-β monomer, lacking Cys-<NUM> and the heel α-helix (α3), functions to potently block and inhibit signaling of the TGF-β1, -β2, and -β3 with IC<NUM> values in the range of <NUM>-<NUM> (<FIG> and <FIG>). This novel inhibitor has several attributes that overcome limitations that have been encountered with other classes of inhibitors, for example the natural high specificity of TGF-β and thus the inhibitor for TβRII may engender it with much greater specificity, and thus fewer undesirable side effects, compared with the much more promiscuous TGF-β kinase inhibitors. The small size of the inhibitor (~<NUM> kDa) further confers a much greater ability to penetrate tumors and other dense tissues where the TGF-βs drive disease progression, a distinct advantage compared with IgG antibodies, which are much larger (~<NUM> kDa) and tend to occupy only the vascular and interstitial space of well perfused organs (<NPL>; <NPL>). The other advantages of this novel inhibitor include its high intrinsic stability, because of the four intramolecular disulfide bonds that tie the four fingers together, and the fact that it is highly soluble in water at neutral pH, unlike native TGF-β dimers or full-length TGF-β monomers.

The structures of TGF-β receptor complexes, together with the previously published chemical cross-linking data, suggested that the potent signaling activity of TGF-β1 C77S and TGF-β3 C77S was due to the ability of the monomers to non-covalently dimerize and in turn assemble a (TβRI·TβRII)<NUM> heterotetramer. The results presented here, namely the AUC experiments that were used to assess non-covalent dimer formation and the TR-FRET experiments that were used to assess assembly of complexes with TβRI and TβRII, provided further evidence for this. The AUC data showed that full-length monomeric TGF-β3, mTGF-β3, self-associates to form dimers with a dimerization constant of <NUM> (Table <NUM>). The TR-FRET data showed that at a concentration of <NUM> or <NUM> and in the presence of comparable concentrations of the TβRI and TβRII ectodomains, mTGF-β3 assembles TβRI·TβRII complexes to the same extent as dimeric TGF-β3 (<FIG>). That this occurs, even under conditions where the mTGF-β3 concentrations (<NUM>-<NUM>, <FIG>) were more than an order of magnitude below the KD value for self-association (<NUM>, Table <NUM>), indicates that receptor binding also contributes significantly to assembly of TβRI·TβRII complexes. The assembly of TβRI·TβRII complexes with mTGF-β3, and presumably mTGF-β1 as well, therefore appears to be a cooperative process, much like protein folding, in which multiple weaker interactions, including monomer-monomer, non-covalent dimer-receptor, and receptor-receptor interactions, cooperate to enable formation of a thermodynamically stable TGF-β·TβRI·TβRII complex. This manner of cooperative assembly is likely responsible for the ability of mTGF-β1 and mTGF-β3 to induce signaling at concentrations that are more than <NUM> orders of magnitude below the KD value for self-association of the monomers (EC<NUM> values of about <NUM> versus KD values for self-association of <NUM>).

The elimination of the heel helix from the TGF-β monomer was shown to be very effective in terms of blocking the cooperative assembly of TβRI·TβRII complexes as shown by the TR-FRET data (<FIG>) and the cell based signaling data (<FIG>). The AUC data showed that elimination of the heel helix led to the weakening of the monomer-monomer interaction by one order of magnitude (Table <NUM>). The SPR data shown in <FIG>, further showed that the TβRII-bound form of mmTGF-β2-<NUM> was incapable of binding and recruiting TβRI, which is expected based on published structures of TGF-β receptor complexes that show that TβRI binds to a composite interface formed by both chains of TGF-β, as well as TβRII (<NPL>; <NPL>). Thus, the data show that the reduced propensity of the engineered monomer to self-associate, together with what would be expected to be very weak binding of TβRI to any dimers that do form, is responsible for the inability of mmTGF-β2-<NUM> to assemble a TβRI·TβRII complex. This accounts for the lack of signaling activity, and this together with the retention of high affinity TβRII binding accounts for the inhibitory activity.

Claim 1:
A recombinant human transforming growth factor (TGF)-β monomer selected from TGF-β1, TGF-β2, and TGF-β3, comprising:
(i) a cysteine to serine substitution at an amino acid residue corresponding to residue <NUM> of SEQ ID NO: <NUM>;
(ii) a deletion of the α3 helix corresponding to amino acid residues <NUM>-<NUM> of SEQ ID NO: <NUM>; and
(iii) at least one amino acid substitution relative to a wild-type TGF-β monomer that increases net charge of the recombinant TGF-β monomer, wherein
(a) for a TGF-β2 monomer, the at least one amino acid substitution is selected from a leucine to arginine substitution at an amino acid residue corresponding to residue <NUM> and an alanine to lysine substitution at an amino acid residue corresponding to residue <NUM> of SEQ ID NO: <NUM>;
(b) for a TGF-β1 monomer, the at least one amino acid substitution is selected from an isoleucine to arginine substitution at an amino acid residue corresponding to residue <NUM>, an alanine to lysine substitution at an amino acid residue corresponding to residue <NUM> and an alanine to serine substitution at an amino acid residue corresponding to residue <NUM> of SEQ ID NO: <NUM>; or
(c) for a TGF-β3 monomer, the at least one amino acid substitution is selected from a leucine to glutamate substitution at an amino acid residue corresponding to residue <NUM>, an alanine to glutamate substitution at an amino acid residue corresponding to residue <NUM> and an alanine to aspartate substitution at an amino acid residue corresponding to residue <NUM> of SEQ ID NO: <NUM>,
wherein wild-type TGF-β1, TGF-β2, and TGF-β3 monomers have the amino acid sequence of SEQ ID NO: <NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>, respectively.