HUMANIZED FN14-BINDING PROTEINS AND USES THEREOF

RELATED APPLICATION DATA

The present application claims priority from Australian Patent Application No. 2021900117 filed on 20 January 2021 entitled “Humanized Fn14-binding proteins and uses thereof’ and Australian Patent Application No. 2021902355 filed 30 July 2021 entitled “Humanized Fn14-binding proteins and uses thereof”. The entire contents of both applications are hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present disclosure relates to humanized Fn14-binding proteins comprising an antibody variable region that specifically binds to Fn14 and uses thereof and methods of treating, preventing, diagnosing or prognosing various conditions including wasting disorders, such as cachexia.

BACKGROUND

Fibroblast growth factor inducible 14 (Fn14 or TNFRSF12A), is a member of the Tumor Necrosis Factor receptor superfamily. Expression of Fn14 is up-regulated by growth factors in vitro and in vivo in response to tissue injury, regeneration, and inflammation.

Fn 14- mediated signaling is involved in pathways that play important roles in human diseases. Fn14-mediated signaling has been suggested to play a role in numerous diseases, including, cancer, metastasis, immunological disorders (including autoimmune diseases, graft rejection and graft versus host disease, and chronic and acute neurological conditions [including stroke]).

Fn14 is expressed by many non-lymphoid cell types (epithelial, mesenchymal, endothelial cells and neurons), by many tissue progenitor cells, including all progenitor cells of the mesenchymal lineage. This protein is highly inducible by growth factors e.g., in serum that are encountered in vivo at sites of tissue injuries and/or tissue remodelling. As a consequence, Fn14 expression is relatively low in most healthy tissues, but increased in injured and/or diseased tissues.

The role of Fn14 in wound repair and muscle development has also been extended to the aetiology of cachexia, with tumor expressed Fn14 shown to be responsible for the induction of cachexia and tumor induced inflammation. Treatment of Fn14-expressing tumor with murine derived anti-Fn14 antibodies has also been shown to prevent tumor induced inflammation and loss of fat and muscle mass (Johnston et al., 2015, Cell 162, 1365-1378).

It is well known that the use of animal (e.g., murine) derived antibodies therapeutically can trigger the generation of antibodies directed at the foreign protein as well as adverse events, such as cytokine release syndrome. The development of humanized and fully human monoclonal antibodies has, to some extent, addressed these issues. However, humanization of murine antibodies is not always successful with reductions in clinical effectiveness as a result of reduced antigen-binding and protein stability.

Based on the foregoing description, the skilled artisan will be aware that humanized therapeutic antibodies that bind to Fn14 are desirable. In particular, stable humanized antibodies that bind to Fn14 are desirable. These antibodies can be used to treat, prevent and/or reduce progression of Fn14-mediated conditions.

SUMMARY

The present disclosure is based on the inventors’ production of a humanized antibody that binds specifically to Fn14. Following humanization of both the variable light and variable heavy chains, the inventors found that the stability of the antibody on human IgGi and lgG4 frameworks was reduced and the antibodies had a propensity to aggregate. Accordingly, the inventors performed a second round of humanization including selected conservative and non-conservative back mutations to the murine amino acid sequence to improve the stability of the antibody. Surprisingly, certain conservative amino acid mutations in the light chain variable region (VL) back to the parental murine residues improved the stability and reduced aggregation of the humanized antibody on both human IgGi and lgG ₄ frameworks.

Thus, the present disclosure is broadly directed to a Fn14-binding protein comprising humanized antibody variable regions that specifically binds to Fn14.

The present disclosure provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a heavy chain variable region (V _H ) comprising an amino acid sequence set forth in SEQ ID NO: 8 and a light chain variable region (VL) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16 or 18.

The present disclosure also provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 12.

The present disclosure further provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 13.

The present disclosure also provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 14.

The present disclosure provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 16.

The present disclosure further provides a humanized Fn14-binding protein comprising an antibody variable region that specifically binds to Fn14, wherein the antibody variable region comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 18. Such a protein has been shown by the inventors to advantageously be stable.

In one example, the humanized Fn14-binding protein of the present disclosure binds to recombinant human Fn14 with an affinity dissociation constant (KD) of 3nM or less.

For example, the humanized Fn14-binding protein binds to recombinant human Fn14 with an KD of 3nM or less, such as, 2.5nM or less, for example, 2nM or less, or 1.5nM or less. In one example, the KD is between about 2nM and 2.5nM, such as between about 2.1 nM and 2.3nM, for example, between about 2.2nM and 2.3nM. For example, the KD is about 2.23nM.

In one example, the humanized Fn14-binding protein binds to cynomolgus Fn14 with a KD of 2.5nM or less, such as 2nM or less, for example, 1.5nM or less, or 1 nM or less.

In one example, the humanized Fn14-binding protein binds to rat Fn14 with a KD of 5.5nM or less, such as 5nM or less, for example, 4.5nM or less or 4nM or less.

In one example, the humanized Fn14-binding protein binds to murine Fn14 with a KD of 5nM or less, such as 4.5nM or less, for example, 4nM or less or 3.5nM or less.

Methods of determining binding of the Fn14-binding protein to recombinant human Fn14 will be apparent to the skilled person and/or are described herein. In one example, the KD is assessed by immobilizing the e.g., recombinant human Fn14 and assessing binding of the Fn14-binding protein to the immobilized Fn14 using surface plasmon resonance. In one example, the humanized Fn14-binding protein of the present disclosure comprises a VH and a VL wherein:

(i) the V _H and the V _L are in a single polypeptide chain and the protein is selected from the group consisting of:

(a) a single chain fragment variable (Fv) fragment (scFv);

(b) a dimeric scFv (di-scFv); and

(c) one of (a) or (b) linked to a constant region of an antibody, a constant fragment (Fc) or a heavy chain constant domain (CH) 2 and/or CH3; or

(ii) the VH and the VL are in separate polypeptide chains and the protein is selected from the group consisting of:

(a) a diabody;

(b) a triabody;

(c) a tetrabody;

(d) a fragment antigen binding (Fab);

(e) a F(ab’)2;

(f) a Fv;

(g) one of (a) to (f) linked to a constant region of an antibody, a constant fragment (Fc) or a heavy chain constant domain (CH) 2 and/or CH3; and

(h) an antibody.

For example, the humanized Fn14-binding protein of the present disclosure comprises a VH and a VL wherein the VH and the VL are in a single polypeptide chain and the protein is selected from the group consisting of:

(a) a scFv;

(b) a di-scFv; and

(c) one of (a) or (b) linked to a constant region of an antibody, a constant fragment (Fc) or a CH2 and/or CH3

In another example, the humanized Fn14-binding protein of the present disclosure comprises a V _H and a VL, wherein the V _H and the V _L are in separate polypeptide chains and the protein is selected from the group consisting of:

(a) a diabody;

(b) a triabody;

(c) a tetrabody;

(d) a Fab;

(e) a F(ab’)2;

(f) a Fv;

(g) one of (a) to (f) linked to a constant region of an antibody, a constant fragment (Fc) or a CH2 and/or CH3; and

(h) an antibody.

In one example, the Fn14-binding protein of the present disclosure is an antibody. Exemplary antibodies are full length and/or naked antibodies. For example, the Fn14- binding protein is an anti-Fn14 antibody. In one example, the anti-Fn14 antibody is a monoclonal anti-Fn14 antibody.

The present disclosure provides a humanized anti-Fn14 antibody, wherein the antibody comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16 or 18.

The present disclosure also provides a humanized anti-Fn14 antibody, wherein the antibody comprises a V _H comprising an amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

The present disclosure further provides a humanized anti-Fn14 antibody, wherein the antibody comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 13. The present disclosure also provides a humanized anti-Fn14 antibody, wherein the antibody comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 14.

The present disclosure further provides a humanized anti-Fn14 antibody, wherein the antibody comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in SEQ ID NO: 16.

The present disclosure provides a humanized anti-Fn14 antibody, wherein the antibody comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 8 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 18.

The present disclosure also provides a Fn14-binding protein comprising a V _H and a VL of the present disclosure, wherein the VH is linked to a human heavy chain constant region and the VL is linked to a human light chain constant region. For example, the Fn14- binding protein comprises a heavy chain constant region of isotype IgGi or lgG4 and a light chain constant region of isotype kappa. In one example, the Fn14-binding protein comprises an IgGi or lgG ₄ heavy chain and a kappa light chain. In one example, the Fn14-binding protein comprises a heavy chain constant region of isotype IgGi and a light chain constant region of isotype kappa. For example, the Fn14-binding protein comprises an IgGi heavy chain and a kappa light chain. In another example, Fn14-binding protein comprises a heavy chain constant region of isotype lgG ₄ and a light chain constant region of isotype kappa. For example, the Fn14-binding protein comprises an lgG ₄ heavy chain and a kappa light chain. In another example, the lgG ₄ heavy chain comprises a stabilized lgG ₄ constant region.

The present disclosure provides a humanized anti-Fn14 antibody, wherein the antibody comprises:

(i) an IgGi heavy chain comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 8; and

(ii) a kappa light chain comprising a V _L comprising an amino acid sequence set forth in SEQ ID NO: 18.

The present disclosure further provides a humanized anti-Fn14 antibody, wherein the antibody comprises:

(i) an lgG ₄ heavy chain comprising a V _H comprising an amino acid sequence set forth in SEQ ID NO: 8; and

(ii) a kappa light chain comprising a VL comprising an amino acid sequence set forth in SEQ ID NO: 18.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid sequence comprising SEQ ID NO: 7 and a VL comprising a sequence expressed from or encoded by a nucleic acid comprising any one of SEQ ID NOs: 9-11 , 15 or 17.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 7 and a V _L comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 9.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 9 and a V _L comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 10.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic comprising SEQ ID NO: 7 and a V _L comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 1 1. In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 8 and a VL comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 15.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 7 and a VL comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 17.

The present disclosure provides a humanized anti-Fn14 antibody, wherein the antibody comprises:

(i) an IgGi heavy chain comprising a VH comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 7; and

(ii) a kappa light chain comprising a V _L comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 17.

The present disclosure further provides a humanized anti-Fn14 antibody, wherein the antibody comprises:

(i) an lgG ₄ heavy chain comprising a V _H comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 7;

(ii) a kappa light chain comprising a VL comprising a sequence expressed from or encoded by a nucleic acid comprising SEQ ID NO: 17.

In one example, the Fn14-binding protein or antibody of the present disclosure is conjugated to a compound. For example, the compound is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the Fn14- binding protein in a subject and mixtures thereof.

In one example, the Fn14-binding protein or antibody of the present disclosure is conjugated to a radioisotope. For example, the radioisotope is zirconium-89 ( ⁸⁹ Zr).

In one example, the Fn14-binding protein or antibody of the present disclosure is conjugated to the compound via a metal ion chelator. For example, the metal ion chelator is p-lsothiocyanatobenzyldesferrioxamine (Df).

In one example, the Fn14-binding protein or antibody of the present disclosure is conjugated to ⁸⁹ Zr via Df.

In one example, the polynucleotide is codon optimized.

In one example, the polynucleotide of the disclosure is operably linked to a heterologous promoter.

The present disclosure additionally provides an expression construct comprising the nucleic acid of the disclosure operably linked to a promoter. Such an expression construct can be in a vector, e.g., a plasmid.

In examples of the disclosure directed to single polypeptide Fn14-binding proteins, the expression construct may comprise a promoter linked to a nucleic acid encoding that polypeptide chain.

In examples directed to multiple polypeptides that form a Fn14-binding protein, an expression construct of the disclosure comprises a nucleic acid encoding one of the polypeptides (e.g., comprising a V _H ) operably linked to a promoter and a nucleic acid encoding another of the polypeptides (e.g., comprising a V _L ) operably linked to another promoter.

In another example, the expression construct is a bicistronic expression construct, e.g., comprising the following operably linked components in 5’ to 3’ order:

(i) a promoter

(ii) a nucleic acid encoding a first polypeptide;

(iii) an internal ribosome entry site; and

(iv) a nucleic acid encoding a second polypeptide. For example, the first polypeptide comprises a VH and the second polypeptide comprises a VL, or the first polypeptide comprises a VL and the second polypeptide comprises a V _H .

The present disclosure also contemplates separate expression constructs one of which encodes a first polypeptide (e.g., comprising a VH) and another of which encodes a second polypeptide (e.g., comprising a V _L ). For example, the present disclosure also provides a composition comprising:

(i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH operably linked to a promoter); and

(ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a V _L operably linked to a promoter), wherein the first and second polypeptides associate to form a Fn14-binding protein of the present disclosure.

The present disclosure additionally provides an isolated cell expressing the Fn14- binding protein or antibody of the present disclosure or a recombinant cell genetically- modified to express a Fn14-binding protein or antibody of the disclosure. For example, the disclosure provides use of an isolated cell for preparing the humanized Fn14-binding protein or antibody of the disclosure. In one example, the cell is an isolated hybridoma. In another example, the cell comprises the nucleic acid of the disclosure or the expression construct of the disclosure or:

(i) a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a V _H ) operably linked to a promoter; and

(ii) a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a V _L ) operably linked to a promoter, wherein the first and second polypeptides associate to form a Fn14-binding protein or antibody of the present disclosure.

The present disclosure additionally provides a pharmaceutical composition comprising the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell of the present disclosure and a suitable carrier. In one example, the pharmaceutical composition comprises the Fn14-binding protein or the antibody of the present disclosure.

In one example, the carrier is pharmaceutically acceptable.

The present disclosure additionally provides the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure for use as a medicament.

The present disclosure additionally provides the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure for use in treating, preventing and/or delaying progression of a disease or condition in a subject. In one example, the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure is for use in treating a disease or condition in a subject. In another example, the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure is for use in preventing a disease or condition in a subject. In a further example, the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure is for use in delaying progression of a disease or condition in a subject.

The present disclosure also provides a method of treating, preventing and/or delaying progression of a disease or condition in a subject, the method comprising administering to the subject the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure to the subject. For example, the disclosure provides a method of treating a disease or condition in a subject. In another example, the disclosure provides a method of preventing a disease or condition in a subject. In a further example, the disclosure provides a method of delaying progression of a disease or condition in a subject. The present disclosure additionally provides use of the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure in the manufacture of a medicament for treating, preventing and/or delaying progression of a disease or condition in a subject in need thereof. For example, the disclosure provides for use of the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure in the manufacture of a medicament for treating a disease or condition in a subject. In another example, the disclosure provides for use of the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure in the manufacture of a medicament for preventing a disease or condition in a subject. In a further example, the disclosure provides for use of the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure in the manufacture of a medicament for delaying progression of a disease or condition in a subject.

In one example, the subject is suffering from the disease or condition (i.e. , the subject is in need of treatment).

In one example, the disease and/or condition is a Fn14-mediated condition. For example, the Fn14-mediated condition is cancer, metastasis, tissue invasion by a cancer, excessive vascularization or angiogenesis, an autoimmune disease, an inflammatory disease, a neurodegenerative diseases, a wasting disorder, keloid scarring, graft versus host disease, graft rejection or ischemia.

In one example, the Fn14-mediated condition is cancer.

In one example, the Fn14-mediated condition is metastasis.

In one example, the Fn14-mediated condition is tissue invasion by a cancer.

In one example, the Fn14-mediated condition is excessive vascularization or angiogenesis.

In one example, the Fn14-mediated condition is an autoimmune disease or an inflammatory disease. For example, the condition is a connective tissue disease (including inflammatory arthritis, such as rheumatoid arthritis, psoriatic arthritis, reactive arthritis or gout), lupus (including systemic lupus erythematosus), type 1 diabetes, multiple sclerosis, vasculitis (including Wegener’s granulomatosis and Henoch Schonlein Syndrome), nephritis (including glomerulonephritis and pneumonitis), atherosclerosis or inflammation of the eye (including uveitis).

In one example, the Fn14-mediated condition is a neurodegenerative disease.

In one example, the Fn14-mediated condition is keloid scarring.

In one example, the Fn14-mediated condition is graft versus host disease.

In one example, the Fn14-mediated condition is graft rejection.

In one example, the Fn14-mediated condition is ischemia.

In one example, the Fn14-mediated condition is a wasting disorder. In one example, the wasting disorder is selected from the group consisting of unintended body weight loss, cachexia, muscle wasting and fat wasting. In one example, the wasting disorder is unintended body weight loss. In another example, the wasting disorder is cachexia. In a further example, the wasting disorder is muscle wasting. In an additional example, the wasting disorder is fat wasting.

In one example, the wasting disorder is associated with a condition. For example, the wasting disorder is associated with a condition selected from the group consisting of cancer, metabolic acidosis, infectious diseases, diabetes, autoimmune immune deficiency syndrome (AIDS), autoimmune disorders, addiction to drugs, cirrhosis of the liver, chronic inflammatory disorders, anorexia, chronic heart failure, chronic kidney disease, osteoporosis, skeletal muscle disease, motor neuron disease, multiple sclerosis, muscle atrophy and neurodegenerative disease.

In one example, the wasting disorder is cachexia or sarcopenia (e.g., muscle wasting associated with aging or muscle wasting associated with extended confinement to bed or other restrictions to muscle use). In one example, the wasting disorder is cachexia. For example, the cachexia is precachexia. In another example, the cachexia is overt cachexia. In a further example, the cachexia is refractory cachexia.

In one example, the wasting disorder is sarcopenia (e.g., muscle wasting associated with aging or muscle wasting associated with extended confinement to bed or other restrictions to muscle use).

In one example, the subject is suffering from cachexia (i.e., the subject is in need of treatment).

In one example, the cachexia is associated with cancer, infectious disease (e.g., tuberculosis or leprosy), AIDS, autoimmune disease (including rheumatoid arthritis or type 1 diabetes), cystic fibrosis, drug addiction, alcoholism or liver cirrhosis.

In one example, the cachexia is associated with a condition selected from rheumatoid arthritis, diabetes, cardiac disease, chronic kidney disease, chronic pulmonary inflammation, intestinal inflammation, inflammatory bowel disease, age, sepsis or AIDS.

In one exemplary form of the present disclosure the wasting disorder is cachexia associated with cancer (i.e., cancer cachexia). For example, the cachexia is cancer cachexia (i.e., the subject has or is suffering from cancer). Numerous types of cancer are associated with cachexia, including but not limited to, solid tumors, carcinoma, neuroma, melanoma, leukemia, lymphoma, sarcoma, fibroma, thyroid cancer, bladder cancer, lung cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, bronchial tumor, cervical cancer, colon cancer, colorectal cancer, neuroepithelial tumor, endometrial cancer, endometrial uterine cancer, fallopian tube cancer, kidney cancer, oral cancer, myeloma, neoplasm, neurinoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer or renal cell carcinoma. Additional cancers are described herein.

In one exemplary form of the present disclosure the wasting disorder is cachexia associated with diabetes. For example, the cachexia is diabetic cachexia (i.e., the subject has or is suffering from diabetes). In one example, the cachexia is associated with or caused by type 1 diabetes. In another example, the cachexia is associated with or caused by type 2 diabetes.

In one example, the method comprises administering the Fn14-binding protein to a subject suffering from a condition associated with a wasting disorder, wherein the condition is associated with or caused by a cell expressing Fn14. For example, the method comprises administering the Fn14-binding protein or antibody of the disclosure to a subject suffering from cancer cachexia, wherein the subject suffers from a cancer expressing Fn14.

In one example, the method additionally comprises selecting a subject suffering from a wasting disorder associated with a condition associated with or caused by a cell expressing Fn14. For example, the method additionally comprises selecting a subject suffering from cancer cachexia, wherein the subject suffers from a cancer expressing Fn14.

Methods of treatment described herein can additionally comprise administering a further treatment for a wasting disorder (e.g., cachexia).

Methods of treatment of a wasting disorder (e.g., cancer cachexia or diabetic cachexia) described herein can additionally comprise administering a further compound to treat or prevent (or delay progression of) cancer and/or diabetes. Exemplary compounds are described herein.

The present disclosure also provides a kit comprising the Fn14-binding protein or the antibody or the nucleic acid or the expression construct or the cell or the composition of the present disclosure packaged with instructions for use in the treatment or prevention or delaying the progression of a wasting disorder (e.g., cachexia).

In one example, the subject is a mammal, for example a primate, such as a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the clustal alignment of murine and humanized first generation (A) light chain variable region protein sequences and (B) heavy chain variable region protein sequences. “*’ indicates fully conserved residue, indicates strongly conserved residue, 7 Indicates weakly conserved residue and indicates a gap in the sequence.

Figure 2 is a graphical representation of size exclusion chromatography analysis of (C, D) first and (A, B) second generation hu002 IgGi and lgG4 constructs. Each panel includes chromatograms with detection at A214nm (upper panels) and A280nm (lower panels).

Figure 3A is a graphical representation showing body weight curves expressed as percentage of body weight at therapy commencement (% of initial); Arrows indicate time points of therapy (10 mg/kg, IP).

Figure 3B is a graphical representation showing individual mouse body weights at end point (17 days post therapy commencement) expressed as percent of body weight at therapy commencement (% of initial). Data are presented as mean ± SEM, n=6 (exception pair-fed control n=8); *, P<0.05.

Figure 4 is a graphical representation showing the anti-cachetic therapeutic efficacy of anti-Fn14 002 antibodies in C26 tumor bearing CD2F1 mice. Individual mouse body weights at end point (17 days post therapy commencement) expressed as percent of body weight at therapy commencement (% of initial); Data are presented as mean ± SEM, n=6. At study endpoint significant preservation of body weight (P<0.005) was observed with all anti-Fn14 antibody therapy arms compared to hulgG1 isotype control or PBS.

Figure 5 is a graphical representation of X-ray crystallography structural data showing the interaction of Fn14 with (A, C) mu002 and (B, D) ITEM-1 .

Figure 6 is a series of graphical representations showing NF-kB reporter assay upon the treatments of humanized constructs 002-hlgG1 or 002-hlgG4 or PDL192 or P484 at indicated concentrations with addition of TWEAK-Fc.

Figure 7 is a graphical representation showing solid phase in vitro binding properties of non-optimised ⁸⁹ Zr-Df-mu002, (20 ng) alone or in the presence of excess 20 pg mu002 binding to increasing concentrations of Fn14 -Ni-NTA agarose beads (1 pg/ pL).

Figure 8 is a series of graphical representations showing biodistribution (%ID/g) properties of radiolabeled anti-Fn14 ⁸⁹ Zr-Df-antibodies (mu002 (A), hulgG1 002 (B), hulgG4 002 (C)) over 7 days in BALB/c nu/nu mice bearing Fn14-positive PC-3* prostate carcinoma tumors, (bars; mean ± SD; n=5).

Figure 9 is a series of graphical representations showing biodistribution (%ID/g) properties of ⁸⁹ Zr-Df-labelled anti-Fn14 antibodies and a hulgG1 isotype control on day 0 (A), day 2 (B) and day 7 (C) post-injection in BALB/c nu/nu mice bearing Fn14 -positive PC- 3* prostate carcinoma tumors, (bars; mean ± SD; n=5).

Figure 10 is a series of graphical representations showing statistical analysis of tumor, kidney and liver uptake (%l D/g) on day 2 and day 7 post injection of ⁸⁹ Zr-radiolabeled 002 constructs and isotype lgG1 control antibody. (A-B) Tumor uptake (%ID/g) on day 2 (A) and day 7 (B); (C-D) Kidney uptake (%ID/g) on day 2 (C) and day 7 (D); (E-F) Liver uptake (%l D/g) on day 2 (E) and day 7 (F); Mean and individual values are shown, n=5 (day 2) and n=4 (day 7); *, P<0.05; **, P<0.005; ***, P<0.005; P<0.0005

Figure 11 is a series of graphical representations showing blood clearance of ⁸⁹ Zr- labelled anti-Fn14 antibodies constructs in PC-3* tumor xenografted BALB/c nu/nu mice. Clearance curves fitted with one-phase (A) or two-phase (B) decay non-linear fits for pharmacokinetic parameter determination, (bars; mean ± SD; n=5)

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 nucleotide sequence of mu002 V _L

SEQ ID NO: 2 nucleotide sequence of mu002 VH

SEQ ID NO: 3 amino acid sequence of mu002 V _L

SEQ ID NO: 4 amino acid sequence of mu002 VH

SEQ ID NO: 5 nucleotide sequence encoding human Fn14

SEQ ID NO: 6 amino acid sequence of human Fn14

SEQ ID NO: 7 nucleotide sequence of hu002 VH SEQ ID NO: 8 amino acid sequence of hu002 VH

SEQ ID NO: 9 nucleotide sequence of hu002 variant 1 VL

SEQ ID NO: 10 nucleotide sequence of hu002 variant 2 V _L

SEQ ID NO: 11 nucleotide sequence of hu002 variant 3 VL

SEQ ID NO: 12 amino acid sequence of hu002 variant 1 VL

SEQ ID NO: 13 amino acid sequence of hu002 variant 2 V _L

SEQ ID NO: 14 amino acid sequence of hu002 variant 3 V _L

SEQ ID NO: 15 nucleotide sequence of hu002 variant 3 PE VL

SEQ ID NO: 16 amino acid sequence of hu002 variant 3 PE VL

SEQ ID NO: 17 nucleotide sequence of hu002 variant 3 ALLQ V _L

SEQ ID NO: 18 amino acid sequence of hu002 variant 3 ALLQ V _L

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1 -4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 , Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997. Any discussion of a protein or antibody herein will be understood to include any variants of the protein or antibody produced during manufacturing and/or storage. For example, during manufacturing or storage an antibody can be deamidated (e.g., at an asparagine or a glutamine residue) and/or have altered glycosylation and/or have a glutamine residue converted to pyroglutamate and/or have a N-terminal or C-terminal residue removed or “clipped” and/or have part or all of a signal sequence incompletely processed and, as a consequence, remain at the terminus of the antibody. It is understood that a composition comprising a particular amino acid sequence may be a heterogeneous mixture of the stated or encoded sequence and/or variants of that stated or encoded sequence.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Selected Definitions

As used herein, the term “Fn14” collectively refers to Fn14 from all mammals, such as from humans and from rodents. The term “hFn14” or “human Fn14” refers to Fn14 from humans. For the purpose of nomenclature and not limitation, an amino acid sequence of an hFn14 is set forth in SEQ ID NO: 6. For the purpose of nomenclature and not limitation, a nucleotide sequence encoding a hFn14 is set forth in SEQ ID NO: 5.

The term “Fn14-binding protein” shall be taken to include a single polypeptide chain, (i.e. , a series of contiguous amino acids linked by peptide bonds), or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex) capable of binding to Fn14 in the manner described and/or claimed herein. For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions. A non-covalent bond contemplated by the present disclosure is the interaction between a V _H and a V _L , e.g., in some forms of diabody or a triabody or a tetrabody or a Fv.

As the term suggests, “anti-Fn14 antibody” means an antibody that specifically binds to Fn14. Based on the disclosure herein, it will be apparent to the skilled person that anti- Fn14 antibodies suitable for use in the present disclosure are antagonistic (i.e., an anti- Fn14 antibody that blocks the normal ligand from binding and activating the receptor) rather than an agonistic antibody (i.e., an anti-Fn14 antibody that activates receptor signalling).

The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody variable region, this term does not encompass an antibody naturally-occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody variable region. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody variable region. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

The skilled artisan will be aware that an “antibody” is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a polypeptide comprising a VL and a polypeptide comprising a VH. An antibody also generally comprises constant domains, some of which can be arranged into a constant region, which includes a constant fragment or fragment crystallizable (Fc), in the case of a heavy chain. A V _H and a V _L interact to form a Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a K light chain or a A light chain and a heavy chain from mammals is a, 6, s, y, or p. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi , lgG ₂ , lgG ₃ , lgG ₄ , IgAi and lgA ₂ ) or subclass. The term “antibody” encompasses humanized antibodies.

The terms “full-length antibody”, “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

The term “naked antibody” refers to an antibody that is not conjugated to another compound, e.g., a toxic compound or radiolabel.

As used herein, “variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDRI, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1 , FR2, FR3 and optionally FR4) together with three CDRs. In the case of a protein derived from an IgNAR, the protein may lack a CDR2. VH refers to the variable region of the heavy chain. V _L refers to the variable region of the light chain.

As used herein, the term "complementarity determining regions” (syn. CDRs; i.e., CDRI, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDRI, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196 901 -917, 1987; Chothia et al. Nature 342, 877-883, 1989; and/or Al-Lazikani et al., J Mol Biol 273 927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27: 55-77, 2003; or the AHO numbering system of Honnegher and Plukthun J. Mol. Biol., 309: 657-670, 2001 .

"Framework regions" (FRs) are those variable domain residues other than the CDR residues.

As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a V _L and a V _H associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. The V _H and the V _L which form the antigen binding site can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding sites which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the V _H is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fabz” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

As used herein, the term “binds” in reference to the interaction of a protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled “A” bound to the antibody.

As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that a protein of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a protein binds to Fn14 with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold greater affinity) avidity, more readily, and/or with greater duration than it binds to other antigens, e.g., to other TNF superfamily receptors or to antigens commonly recognized by polyreactive natural antibodies (i.e. , by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

As used herein, the terms “disease”, “disorder” or “condition” refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.

The term “Fn14-mediated condition” shall be taken to encompass any disease or disorder caused by or associated with excess numbers of cells expressing Fn14 and/or overexpression of Fn14 by cells and/or excess activity of Fn14 in a subject.

As used herein, the term “wasting disorder” refers to a disorder which involves, results at least in part from, or includes loss of weight, muscle atrophy, fatigue, weakness in someone who is not actively trying to lose weight. Wasting disorders are commonly characterized by inadvertent and/or uncontrolled (in the absence of medical intervention) loss of muscle and/or fat. The term encompasses cachexia or other forms of wasting, e.g., denervation-induced wasting.

The term “wasting disorder associated with another condition” will be understood to mean a wasting that is observed in a subject suffering from a condition, i.e., the wasting may result from changes (e.g., metabolic changes) caused by the condition. In one example, the condition is a Fn14-mediated condition. In one example, the condition is caused by or associated with Fn14 expression in a cell (or a cell expressing Fn14) other than muscle. As used herein, the term “cachexia” will be understood to refer to a complex metabolic condition associated with an underlying (or another) condition, wherein cachexia is characterized by loss of body weight and loss of muscle with or without loss of fat mass. Cachexia is generally associated with increased protein catabolism due to underlying disease(s). As used herein, the term “cachexia” encompasses all stages of cachexia, including “pre-cachexia”, “overt cachexia” (also known as cachexia) and “refractory cachexia”. .

The term “cancer cachexia”, also known as “cancer anorexia cachexia” shall be understood to refer to cachexia that is associated with cancer or occurring in a subject that is suffering from cancer and is characterised by an ongoing loss of muscle mass (with loss of fat mass), leading to progressive functional impairment which cannot be fully reversed by normal nutritional support.

The term “diabetic cachexia” (also known as “diabetic neuropathic cachexia”) shall be understood to refer to cachexia that is associated with diabetes or occurring in a subject that is suffering from diabetes mellitus and is characterised by bilateral, painful neuropathy over the limbs and trunk, with dramatic weight loss.

The term “unintended body weight loss” refers to a condition where the subject is incapable of maintaining a healthy body weight or loses a considerable amount of body weight, without actually attempting to reduce body weight. For example a body mass index (BMI) of less than 18.5 (or any another BMI range defined by a medical specialist) is considered underweight. For the purposes of the present disclosure, the term “body mass index” or “BMI” is calculated by the following formula: mass (kg)Z(height (m) ² ).

The term “total body mass” will be understood to mean a subject’s weight.

As used herein, a subject “at risk” of developing a disease or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment according to the present disclosure. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of the disease or condition, as known in the art and/or described herein.

As used herein, the terms “treating”, “treat” or “treatment” include administering a protein described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition or to slow progression of the disease or condition.

As used herein, the term “preventing”, “prevent” or “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a specified disease or condition in a subject. A subject may be predisposed to or at risk of developing the disease or disease relapse but has not yet been diagnosed with the disease or the relapse.

As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

Humanized Fn14-binding proteins

The present disclosure provides humanized Fn14-binding proteins and humanized anti-Fn14 antibodies.

The term “humanized” shall be understood to refer to a protein comprising a humanlike variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse) grafted onto or inserted into FRs from a human antibody (this type of antibody is also referred to a “CDR-grafted antibody”). Humanized proteins also include proteins in which one or more residues of the human protein are modified by one or more amino acid substitutions and/or one or more FR residues of the human protein are replaced by corresponding non-human residues. Humanized proteins may also comprise residues which are found in neither the human antibody or in the non-human antibody. Any additional regions of the protein (e.g., Fc region) are generally human. Humanization can be performed using a method known in the art, e.g., US5225539, US6054297, US7566771 or US5585089. The term “humanized protein” also encompasses a super-humanized protein, e.g., as described in US7732578.

Antibody-Based Fn14 Binding Proteins

Antibodies

The present disclosure provides humanized anti-Fn14 antibodies, e.g., comprising the variable regions described herein.

In one example, the humanized antibody has been codon optimised.

In one example, the present disclosure provides a humanized anti-Fn14 antibody, wherein the antibody comprises a V _H comprising an amino acid sequence set forth in SEQ ID NO: 8 and a V _L comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16 or 18.

In one example, the disclosure provides a humanized Fn14-binding protein or a humanized anti-Fn14 antibody, wherein the binding protein or antibody comprises a VH comprising a sequence expressed from or encoded by a nucleic acid sequence comprising SEQ ID NO: 7 and a V _L comprising a sequence expressed from or encoded by a nucleic acid comprising any one of SEQ ID NOs: 9-11 , 15 or 17.

In one example, the humanized antibody is a recombinant antibody. Methods for producing humanized antibodies comprising variable regions described herein will be apparent to the skilled artisan based on the disclosure herein and/or documents referred to herein.

Monoclonal antibodies are exemplary antibodies contemplated by the present disclosure. The term “monoclonal antibody" or TnAb” or “MAb” refers to a homogeneous antibody population capable of binding to the same antigen(s) and, for example, to the same epitope within the antigen. This term is not intended to be limited with respect to the source of the antibody or the manner in which it is made.

Antibody Fragments

Single Chain Fv (scFv) Fragments and dimeric-scFv (di-scFv)

The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain. The polypeptide chain further comprises a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e. , for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly ₄ Ser) ₃ being one of the more favoured linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv (see, for example, Brinkmann et al., 1993).

Alternatively, or in addition, the present disclosure provides a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun) (see, for example, Kruif and Logtenberg, 1996). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

For a review of scFv, see Pluckthun (1994).

Diabodies, Triabodies, Tetrabodies

Exemplary humanized Fn14-binding proteins comprising an antibody antigen binding domain are diabodies, triabodies, tetrabodies and higher order protein complexes such as those described in WQ98/044001 and WQ94/007921 .

For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure V _L -X-V _H or V _H -X-V _L , wherein V _L is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a V _L of the other polypeptide chain to form an antigen binding site, i.e., to form an Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

Minibodies

The skilled artisan will be aware that a minibody comprises the VH and VL domains of an antibody fused to the CH2 and/or CH3 domain of an antibody. Optionally, the minibody comprises a hinge region between the VH and a VL, sometimes this conformation is referred to as a Flex Minibody. A minibody does not comprise a CH1 or a CL. In one example, the V _H and VL domains are fused to the hinge region and the CH3 domain of an antibody. At least one of the variable regions of said minibody binds to Fn14 in the manner of the disclosure. Exemplary minibodies and methods for their production are described, for example, in WO94/09817.

Constant Regions

The present disclosure encompasses humanized Fn14-binding proteins comprising a variable region and a constant region or a domain(s) thereof, e.g., Fc, CH2 and/or CH3 domain. The skilled artisan will be aware of the meaning of the terms constant region and constant domain based on the disclosure herein and references discussed herein.

Constant region sequences useful for producing the humanized Fn14-binding proteins of the present disclosure may be obtained from a number of different sources. In some examples, the constant region or portion thereof of the humanized Fn14-binding protein is derived from a human antibody. Moreover, the constant domain or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGi, lgG ₂ , lgG ₃ and lgG ₄ . In one example, the human isotype IgGi is used. In another example, the human isotype lgG ₄ is used.

A variety of constant region gene sequences are available in the form of publicly accessible deposits or the sequence thereof is available from publicly available databases. Constant regions can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity.

Effector Function

In one example, a humanized protein and/or humanized antibody of the present disclosure has or displays an effector function that facilitates or enables at least partial depletion, substantial depletion or elimination of cells expressing Fn14. Such an effector function may be enhanced binding affinity to Fc receptors, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cell mediated phagocytosis (ADCP) and/or complement dependent cytotoxicity (CDC).

In one example, the humanized Fn14-binding protein or humanized anti-Fn14 antibody of the disclosure is capable of inducing an enhanced level of effector function.

In one example, the level of effector function induced by the constant region is enhanced relative to a wild-type Fc region of an IgGi antibody or a wild-type Fc region of an lgG3 antibody.

In another example, the constant region is modified to increase the level of effector function it is capable of inducing compared to the constant region without the modification. Such modifications can be at the amino acid level and/or the secondary structural level and/or the tertiary structural level and/or to the glycosylation of the Fc region.

The skilled addressee will appreciate that greater effector function may be manifested in any of a number of ways, for example as a greater level of effect, a more sustained effect or a faster rate of effect. Exemplary constant region modifications include amino acid substitutions, such as, S239D/I332E, numbered according to the EU index of Kabat or S239D/A330L/I332E, numbered according to the EU index of Kabat.

Additional amino acid substitutions that increase ability of an Fc region to induce effector function are known in the art and/or described, for example, in US6737056 or US7317091.

In one example, the glycosylation of the constant region is altered to increase its ability to induce enhanced effector function. In some examples, Fc regions according to the present disclosure comprise a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region, i.e., the Fc region is “afucosylated”. Such variants may have an improved ability to induce ADCC. Methods for producing afucosylated antibodies include, expressing the Fn14-binding protein in a cell line incapable of expressing a-1 ,6- fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et al., 2004). Other methods include the use of cell lines which inherently produce antibodies capable of inducing enhanced effector function (e.g. duck embryonic derived stem cells for the production of viral vaccines, W02008/129058; Recombinant protein production in avian EBX® cells, WO 2008/142124).

Fn14-binding proteins can also comprise an Fc region capable of inducing enhanced levels of CDC. For example, hybrids of lgG1 and lgG3 produce antibodies having enhanced CDC activity (Natsume et a/., 2008).

Methods for determining the ability of an antibody or antigen binding fragment thereof to induce effector function and known in the art and/or described herein.

The skilled addressee will also appreciate that reduced or absent effector function may also be desirable.

In one example, the humanized Fn14-binding protein or humanized anti-Fn14 antibody of the disclosure lacks effector functions, such as ADCC and/or CDC, e.g., as described in Offringa and Glennie, Cancer Cell. 28:273-8, 2015.

In another example, the humanized protein comprises one or more amino acid substitutions that increase the half-life of the humanized Fn14-binding protein. For example, the humanized Fn14-binding protein comprises a constant region comprising one or more amino acid substitutions that increase the affinity of the constant region for the neonatal Fc region (FcRn). For example, the constant region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an endosome. In one example, the constant region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which facilitates the re-release of Fc into blood following cellular recycling. These amino acid substitutions are useful for extending the half life of a protein, by reducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.

Stabilized Proteins

Humanized Fn-14 binding proteins of the present disclosure can comprise an lgG4 constant region or a stabilized lgG4 constant region. The term “stabilized lgG4 constant region” will be understood to mean an lgG ₄ constant region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a halfantibody or a propensity to form a half antibody. “Fab arm exchange” refers to a type of protein modification for human lgG4, in which an lgG4 heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another lgG ₄ molecule. Thus, lgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A “half antibody” forms when an lgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

In one example, a stabilized lgG ₄ constant region comprises a proline at position 241 of the hinge region according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and/or 1991 ). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human lgG ₄ , this residue is generally a serine. Following substitution of the serine for proline, the lgG ₄ hinge region comprises a sequence CPPC. In this regard, the skilled person will be aware that the “hinge region” is a proline-rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgGi according to the numbering system of Kabat. Hinge regions of other IgG isotypes may be aligned with the IgGi sequence by placing the first and last cysteine residues forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for example WO2010/080538).

Protein Production

In one example, a humanized Fn14-binding protein or humanized anti-Fn14 antibody of the disclosure is produced by culturing a cell line under conditions sufficient to produce the protein, e.g., as described herein and/or as is known in the art.

Recombinant Expression

In the case of a recombinant protein, nucleic acid encoding same is placed into one or more expression construct, e.g., expression vector(s), which is/are then transfected into host cells, such as cells that can produce a disulphide bridge or bond, such as E. coli cells, yeast cells, insect cells, or mammalian cells. Exemplary mammalian cells include simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel or Sambrook. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art. See US4816567; US7923221and US7022500.

Following isolation, the nucleic acid encoding a humanized protein of the disclosure is inserted into an expression construct or replicable vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. For example, the nucleic acid is operably linked to a promoter,

As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term “operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter. Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a humanized Fn14-binding protein of the present disclosure (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of a protein. For example, exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters include those active in prokaryotes (e.g., phoA promoter, p- lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter).

Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EF1 ), small nuclear RNA promoters (U1 a and U1b), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, p-actin promoter; hybrid regulatory element comprising a CMV enhancer/ p-actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, AUSTRALIAN CELL BANK CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, AUSTRALIAN CELL BANK CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL 1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

Means for introducing the isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, viral transduction (e.g., using a lentivirus) and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., Wl, USA) amongst others.

The host cells used to produce the humanized Fn14-binding protein of this disclosure may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

Isolation of Proteins

A humanized Fn14-binding protein or humanized anti-Fn14 antibody of the present disclosure can be isolated or purified.

Methods for purifying a humanized Fn14-binding protein or humanized anti-Fn14 antibody of the disclosure are known in the art and/or described herein.

When using recombinant techniques, the humanized Fn14-binding protein of the disclosure can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the protein is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The protein prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in WO99/57134 or Zola (1997).

The skilled artisan will also be aware that a humanized Fn14-binding protein of the disclosure can be modified to include a tag to facilitate purification or detection, e.g., a polyhistidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. For example, the tag is a hexa-his tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexahis tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

Conjugates

The present disclosure also provides conjugates of humanized Fn14-binding proteins described herein according to any example. Examples of compounds to which a humanized protein can be conjugated are selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the protein in a subject and mixtures thereof. Exemplary therapeutic agents include, but are not limited to an anti-angiogenic agent, an anti-neovascularization and/or other vascularization agent, an anti-proliferative agent, a pro-apoptotic agent, a chemotherapeutic agent or a therapeutic nucleic acid.

A toxin includes any agent that is detrimental to (e.g., kills) cells. For a description of these classes of drugs which are known in the art, and their mechanisms of action, see Goodman et al., (1990). Additional techniques relevant to the preparation of immunoglobulin-immunotoxin conjugates are provided in for instance in US5194594. Exemplary toxins include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO93/21232.

Suitable chemotherapeutic agents for forming immunoconjugates of the present disclosure include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)). In one example, a humanized Fn14-binding protein as described herein according to any example is conjugated or linked to another protein, including another humanized Fn14- binding protein of the disclosure or a protein comprising an antibody variable region, such as an antibody or a protein derived therefrom, e.g., as described herein. Other proteins are not excluded. Additional proteins will be apparent to the skilled artisan and include, for example, an immunomodulator or a half-life extending protein or a peptide or other protein that binds to serum albumin amongst others.

Exemplary serum albumin binding peptides or protein are described in US20060228364 or US20080260757.

A variety of radionuclides are available for the production of radioconjugated proteins. Examples include, but are not limited to, low energy radioactive nuclei (e.g., suitable for diagnostic purposes), such as ¹³ C, ¹⁵ N, ² H, ¹²⁵ l, ¹²³ l, "Tc, ⁴³ K, ⁵² Fe, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Zr, ¹¹¹ ln and the like. For example, the radionuclide is a gamma, photon, or positron-emitting radionuclide with a half-life suitable to permit activity or detection after the elapsed time between administration and localization to the imaging site. The present disclosure also encompasses high energy radioactive nuclei (e.g., for therapeutic purposes), such as ¹²⁵ l, ¹³¹ l, ¹²³ l, ¹¹¹ ln, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re and ¹⁸⁸ Re. These isotopes typically produce high energy a- or p-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on nonlocalized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes may be generated by thermal irradiation of an otherwise stable isotope, for example as in boron neutron-capture therapy (Guan et al., 1998).

In another example, the humanized Fn14-binding protein is conjugated to a "receptor" (such as streptavidin) for utilization in cell pretargeting wherein the conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is conjugated to a therapeutic agent (e.g., a radionucleotide).

The humanized Fn14-binding proteins of the present disclosure can be modified to contain additional non-proteinaceous moieties that are known in the art and readily available. For example, the moieties suitable for derivatization of the protein are physiologically acceptable polymer, e.g., a water soluble polymer. Such polymers are useful for increasing stability and/or reducing clearance (e.g., by the kidney) and/or for reducing immunogenicity of a Fn14-binding protein of the disclosure. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propropylene glycol (PPG).

In one example, a humanized Fn14-binding protein as described herein according to any example comprises one or more detectable markers to facilitate detection and/or isolation. For example, the compound comprises a fluorescent label such as, for example, fluorescein (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3- diazol-4- yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein- N-hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine). The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm).

Alternatively, or in addition, the humanized Fn14-binding protein as described herein according to any example is labeled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US6,306,610).

Alternatively, or in addition, the humanized Fn14-binding protein is labeled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobaltplatinum, or strontium ferrite. Assaying Activity

Binding Assays

One form of such an assay is an antigen binding assay, e.g., as described in Scopes (1994). Such a method generally involves labeling the humanized Fn14-binding protein and contacting it with immobilized antigen or a fragment thereof, e.g., a protein comprising an extracellular domain of Fn14 fused to an Fc region of an antibody. Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound protein is detected. Of course, the humanized Fn14-binding protein can be immobilized and the antigen labeled. Panning-type assays, e.g., as described or exemplified herein can also be used.

In Vivo Therapeutic Efficacy Assays

A humanized Fn14-binding protein of the disclosure can also be tested in vivo.

For example, a humanized Fn14-binding protein can be tested in an animal model of a wasting disorder as described herein, e.g., in which a non-human mammal is administered a tumor cell expressing Fn14 under conditions for a wasting disorder to develop. A humanized Fn14-binding protein of the disclosure is then administered and the effect on the wasting disorder is assessed, e.g., by monitoring body weight changes. A humanized Fn14- binding protein that reduces or prevents loss of body weight or induces a gain in body weight is selected as a potential therapeutic agent.

A humanized Fn14-binding protein of the disclosure can also be selected on the basis of its ability to reduce or prevent invasiveness of a tumor cell. For example, a tumor cell is implanted into a subject, e.g., into a muscle, and the subject is administered a test humanized Fn14-binding protein (or for controls, no Fn14-binding protein is administered). A reduction in invasion of tissue surrounding the tumor cell (e.g., as assessed using histopathology) in the presence of the humanized Fn14-binding protein compared to in the absence of the humanized Fn14-binding protein indicates that the humanized Fn14-binding protein reduces or prevent invasiveness of a tumor cell.

A humanized Fn14-binding protein of the disclosure can also be assessed for therapeutic efficacy by determining its ability to slow or prevent development of a tumor in a xenograft model.

A humanized Fn14-binding protein of the disclosure can also be assessed for therapeutic efficacy by determining it ability to reduce the amount of angiogenesis or vasculogenesis in a tumor in a xenograft model.

Therapeutic efficacy can also be assessed in animal models of rheumatoid arthritis e.g., a SKG strain of mouse (Sakaguchi et al.), rat type II collagen arthritis model, mouse type II collagen arthritis model; a mouse model of GVHD (e.g., as described in Trenado (2002)) or a model of ischemic stroke, e.g., aorta/vena cava occlusion, external neck torniquet or cuff, hemorrhage or hypotension, intracranial hypertension or common carotid artery occlusion, two-vessel occlusion and hypotension, four-vessel occlusion, unilateral common carotid artery occlusion (in some species only), endothelin-1 -induced constriction of arteries and veins, middle cerebral artery occlusion, spontaneous brain infarction (in spontaneously hypertensive rats), macrosphere embolization, blood clot embolization or microsphere embolization.

Therapeutic efficacy can also be determined by administration of a humanized Fn14- binding protein to a model of diabetes, e.g., type 1 diabetes. For example, the test subject is a non-obese diabetic (NOD) mouse (a model of Type I diabetes) or a mouse or rat to which streptozotocin has been administered (models of Type I and/or Type II diabetes).

Affinity Assays

Optionally, the dissociation constant (Kd) or association constant (Ka) or binding constant (K _D , i.e., Ka/Kd) of a humanized Fn14-binding protein for Fn14 or an epitope containing peptide thereof is determined. These constants for a humanized Fn14-binding protein is in one example measured by a radiolabeled or fluorescently-labeled Fn14 binding assay. This assay equilibrates the humanized Fn14-binding protein with a minimal concentration of labeled Fn14 in the presence of a titration series of unlabeled Fn14. Following washing to remove unbound Fn14, the amount of label is determined. According to another example the constants are measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized Fn14 or a region thereof.

Methods of Treatment or Prevention

The present disclosure encompasses the use of a humanized Fn14-binding protein or humanized anti-Fn14 antibody or composition described herein to treat a Fn14-mediated condition. Exemplary conditions include cancer, metastasis, excessive vascularization or angiogenesis, an autoimmune disease, an inflammatory disease, a neurodegenerative diseases, keloid scarring, graft versus host disease, graft rejection or ischemia.

In one example, the Fn 14- mediated condition is an inflammatory disease or an autoimmune disease. In one example, the condition is a connective tissue disease (including inflammatory arthritis, such as rheumatoid arthritis, psoriatic arthritis, reactive arthritis or gout), lupus (including systemic lupus erythematosus), type 1 diabetes, multiple sclerosis, vasculitis (including Wegener’s granulomatosis and Henoch Schonlein Syndrome), nephritis (including glomerulonephritis and pneumonitis), atherosclerosis or inflammation of the eye (including uveitis).

In one example, the autoimmune condition is multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Sjogren's syndrome, arthritis (such as rheumatoid arthritis), Type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, celiac disease, glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroid diseases, Hashimoto's thyroiditis, Graves disease, myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, or systemic lupus erythematosis (SLE). In one example, the condition is rheumatoid arthritis or SLE.

In one example, the condition is a connective tissue disease, such as rheumatoid arthritis. In this regard, Dharmapatni et al., (2011 ) have shown that Tweak/Fn14 play a role in rheumatoid arthritis.

In one example, the condition is scleroderma (including systemic scleroderma).

In another example, the condition is graft rejection (e.g., allograft rejection) or graft versus host disease (including weight loss associated with graft versus host disease). In this regard, Tweak/Fn14 have been show to play a role in pathogenesis of graft versus host disease, e.g., by Zhao et al., (2007).

In another example, the condition is cardiac allograft vasculopathy.

In one example, the condition is graft rejection associated intimal thickening.

In another example the condition is intramyocardial infarction or ischemic repurfusion injury. In this regard, Tweak/Fn14 has been shown to play a role in ischemia by, e.g., Frauenknecht et al., (2010) and Inta et al., (2008).

In another example, the condition is associated with excessive angiogenesis and/or neovascularization, e.g., cancer (including solid tumors, leukemias, lymphoma, melanoma, glioma, breast cancer, colonic cancer, gastric cancer, esophageal cancer, renal cell cancer, ovarian cancer, cervical cancer, carcinoid cancer, testicular cancer, prostate cancer, head and neck cancer and hepatocellular carcinoma), cancer metastasis, cancer neovascularization, autoimmune disease (including psoriasis), nephropathy, retinopathy, preeclampsia, hepatitis, sepsis and macular degeneration.

In one example, the condition is cancer or a metastasis thereof. The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

An adenocarcinoma is a cancer of an epithelium that originates in glandular tissue. Exemplary adenocarcinomas include forms of colorectal cancer, lung cancer, cervical cancer, prostate cancer, urachus cancer, vulval cancer, breast cancer, esophageal cancer, pancreatic cancer and gastric cancer.

Digestive/gastrointestinal cancers include anal cancer; bile duct cancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumor, gastrointestinal cancer; colon cancer; colorectal cancer including childhood colorectal cancer; esophageal cancer including childhood esophageal cancer; gallbladder cancer; gastric (stomach) cancer including childhood gastric (stomach) cancer; hepatocellular (liver) cancer including childhood hepatocellular (liver) cancer; pancreatic cancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma; rectal cancer; and small intestine cancer.

Endocrine cancers include islet cell carcinoma (endocrine pancreas); adrenocortical carcinoma including childhood adrenocortical carcinoma; gastrointestinal carcinoid tumor; parathyroid cancer; pheochromocytoma; pituitary tumor; thyroid cancer including childhood thyroid cancer; childhood multiple endocrine neoplasia syndrome; and childhood carcinoid tumor.

Eye cancers include intraocular melanoma; and retinoblastoma.

Musculoskeletal cancers include Ewing's family of tumors; osteosarcoma/malignant fibrous histiocytoma of the bone; rhabdomyosarcoma including childhood rhabdomyosarcoma; soft tissue sarcoma including childhood soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and uterine sarcoma.

Neurologic cancers include childhood brain stem glioma; brain tumor; childhood cerebellar astrocytoma; childhood cerebral astrocytoma/malignant glioma; childhood ependymoma; childhood medulloblastoma; childhood pineal and supratentorial primitive neuroectodermal tumors; childhood visual pathway and hypothalamic glioma; other childhood brain cancers; adrenocortical carcinoma; central nervous system lymphoma, primary; childhood cerebellar astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumors; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; and supratentorial primitive neuroectodermal tumors including childhood and pituitary tumor.

Genitourinary cancers include bladder cancer including childhood bladder cancer; renal cell (kidney) cancer; ovarian cancer including childhood ovarian cancer; ovarian epithelial cancer; ovarian low malignant potential tumor; penile cancer; prostate cancer; renal cell cancer including childhood renal cell cancer; renal pelvis and ureter, transitional cell cancer; testicular cancer; urethral cancer; vaginal cancer; vulvar cancer; cervical cancer; Wilms tumor and other childhood kidney tumors; endometrial cancer; and gestational trophoblastic tumor;

Germ cell cancers include childhood extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; and testicular cancer.

Head and neck cancers include lip and oral cavity cancer; childhood oral cancer; hypopharyngeal cancer; laryngeal cancer including childhood laryngeal cancer; metastatic squamous neck cancer with occult primary; mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer including childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer; pharyngeal cancer; salivary gland cancer including childhood salivary gland cancer; throat cancer; and thyroid cancer.

Hematologic/blood cell cancers include leukemia (e.g., acute lymphoblastic leukemia in adults and children; acute myeloid leukemia, e.g., in adults and children; chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g., AIDS-related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphoma including Hodgkin's lymphoma in adults and children; Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including non-Hodgkin's lymphoma in adults and children; non-Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom's macroglobulinemia; and primary central nervous system lymphoma); and other hematologic cancers (e.g., chronic myeloproliferative disorders; multiple myeloma/plasma cell neoplasm; myelodysplastic syndromes; and myelodysplastic/myeloproliferative disorders).

Respiratory cancers include non-small cell lung cancer; small cell lung cancer; malignant mesothelioma including malignant mesothelioma in adults and children; malignant thymoma; childhood thymoma; thymic carcinoma; bronchial adenomas/carcinoids including childhood bronchial adenomas/carcinoids; pleuropulmonary blastoma.

Skin cancers include Kaposi's sarcoma; Merkel cell carcinoma; melanoma; basal cell carcinoma and childhood skin cancer.

In a further example, the condition is a wasting disorder, such as cachexia as described in more detail herein. In one example, the wasting disorder is associated with a condition, such as, cancer, metabolic acidosis, infectious diseases, diabetes, HIV infection, autoimmune immune deficiency syndrome (AIDS), autoimmune disorders, addiction to drugs, cirrhosis of the liver, chronic inflammatory disorders, anorexia, chronic heart failure, chronic kidney disease, osteoporosis, skeletal muscle disease, motor neuron disease, multiple sclerosis, muscle atrophy and neurodegenerative disease.

In one example, the wasting disorder is cachexia or sarcopenia (e.g., wasting associated with aging or muscle wasting associated with extended confinement to bed or other restrictions to muscle use).

In one example, the wasting disorder is cachexia. For example, the cachexia can be pre-cachexia, overt cachexia (or cachexia) or refractory cachexia.

In one example, the cachexia is associated with cancer, infectious disease (e.g., tuberculosis or leprosy), AIDS, autoimmune disease (including rheumatoid arthritis or type 1 diabetes), cystic fibrosis, drug addiction, alcoholism or liver cirrhosis.

In one example, the cachexia is associated with an autoimmune disease. In one example, the cachexia is associated with rheumatoid arthritis. In one example, the cachexia is associated with type 1 diabetes. For example, the cachexia is diabetic cachexia. For example, the subject is suffering from diabetic cachexia. For example, a subject suffering from diabetic cachexia has a clinically accepted marker of diabetes, such as:

• Fasting plasma glucose of greater than or equal to 7nmol/L or 126mg/dl;

• Casual plasma glucose (taken at any time of the day) of greater than or equal to

11 .1 nmol/L or 200 mg/dl with the symptoms of diabetes.

• Oral glucose tolerance test (OGTT) value of greater than or equal to 1 1 .1 nmol/L or 200 mg/dl measured at a two-hour interval. The OGTT is given over a two or three-hour time span.

In one example, the subject suffers from type 1 diabetes. For example, the subject suffers from cachexia associated with type 1 diabetes.

In one example, the subject suffers from chronic obstructive pulmonary disease (COPD).

In one example, the cachexia is associated with cardiac disease.

In one example, the cachexia is associated with chronic kidney disease.

In one example, the cachexia is associated with chronic pulmonary inflammation.

In one example, the cachexia is associated with intestinal inflammation.

In one example, the cachexia is associated with inflammatory bowel disease.

In one example, the cachexia is associated with aging (i.e., sarcopenia).

In one example, the cachexia is associated with extended confinement to bed or other restrictions to muscle use.

In one example, the cachexia is associated with sepsis.

In one example, the cachexia is associated with AIDS.

In one exemplary form of the present disclosure the wasting disorder is cachexia associated with cancer. Exemplary cancers are described supra. In one example, the method additionally comprises identifying a subject suffering from cachexia. Such a subject can be identified, for example, based on detection of unintentional weight loss following diagnosis of another condition (e.g., cancer). In one example, the different stages of cachexia can be diagnosed based on the following clinically acceptable criteria:

Pre-cachexia

• Weight loss < 5%; and/or

• Anorexia and metabolic changes. Cachexia or overt cachexia

• Weight loss >5% over a period of six months (in the absence of starvation);

• A BMI <20 together with weight loss;

• Weight loss >2% together with an appendicular skeletal muscle index consistent with sarcopenia (males <7.26kg/m2; females <5.45kg/m2); and/or

• Reduced food intake with or without systemic inflammation. Refractory cachexia

• Weight loss non-responsive to treatment;

• <3 months expected survival.

In one example, performing a method described herein according to any example of the disclosure results in enhancement of a clinical response and/or delayed disease progression.

By "clinical response" is meant an improvement in the symptoms of disease. The clinical response may be achieved within a certain time frame, for example, within or at about 8 weeks from the start of treatment with, or from the initial administration. Clinical response may also be sustained for a period of time, such as for >24 weeks, or >48 weeks.

Pharmaceutical Compositions

Humanized Fn14-binding proteins and humanized anti-Fn14 antibodies of the disclosure (syn. active ingredients) are useful for formulations into a pharmaceutical composition for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration, for prophylactic or for therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges.

The pharmaceutical compositions of this disclosure are useful for parenteral administration, such as intravenous administration or subcutaneous administration or administration into a body cavity or lumen of an organ or joint. The compositions for administration will commonly comprise a solution of the humanized Fn14-binding protein of the disclosure dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable carriers 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 of humanized Fn14-binding proteins of the present disclosure 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 patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

The humanized Fn14-binding protein of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, transdermal, or other such routes, including peristaltic administration and direct instillation into a tumor or disease site (intracavity administration). The preparation of an aqueous composition that contains the compounds of the present disclosure as an active ingredient will be known to those of skill in the art.

Suitable pharmaceutical compositions in accordance with the disclosure will generally include an amount of the humanized Fn14-binding protein of the present disclosure admixed with an acceptable pharmaceutical carrier, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980.

Dosage and Administration

Upon formulation, humanized proteins and humanized antibodies of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically/prophylactically effective.

As used herein, the term “therapeutically effective amount” shall be taken to mean a sufficient quantity of humanized Fn14-binding protein of the disclosure to reduce or inhibit one or more symptoms of a disorder being treated.

As used herein, the term “prophylactically effective amount” shall be taken to mean a sufficient quantity of humanized Fn14-binding protein of the disclosure to prevent or inhibit or delay the onset of one or more detectable symptoms of a disorder being treated.

The dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

Dosage can vary from about 0.1 mg/kg to about 300 mg/kg, e.g., from about 0.2 mg/kg to about 200 mg/kg, such as, from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

In some examples, the humanized Fn14-binding protein is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses). For example, the humanized Fn14-binding protein is administered at an initial dose of between about 1 mg/kg to about 30mg/kg. The humanized Fn14-binding protein is then administered at a maintenance dose of between about 0.0001 mg/kg to about 1 mg/kg. The maintenance doses may be administered every 7-35 days, such as, every 14 or 21 or 28 days.

In some examples, a dose escalation regime is used, in which a humanized Fn14- binding protein is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject’s initially suffering adverse events

In the case of a subject that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.

A subject may be retreated with the humanized Fn14-binding protein, by being given more than one exposure or set of doses, such as at least about two exposures of the humanized Fn14-binding protein, for example, from about 2 to 60 exposures, and more particularly about 2 to 40 exposures, most particularly, about 2 to 20 exposures.

In another example, any retreatment may be given at defined intervals. For example, subsequent exposures may be administered at various intervals, such as, for example, about 24-28 weeks or 48-56 weeks or longer. For example, such exposures are administered at intervals each of about 24-26 weeks or about 38-42 weeks, or about 50-54 weeks.

A method of the present disclosure may also include co-administration of a humanized Fn14-binding protein of the disclosure together with another therapeutically effective agent for the treatment of a wasting disorder and/or an associated condition (e.g., cancer and/or diabetes).

In one example, the humanized Fn14-binding protein of the disclosure is used in combination with at least one additional known compound which is currently being used or is in development for preventing or treating a wasting disorder. Exemplary compounds include orexigenic agents (i.e., appetite stimulants, such as L-carnitine, megestrol acetate, and melatonin), anabolic steroids (e.g., selective androgen receptor modulators (SARMs) such as enobosarm, espindolol and testosterone) and/or anti-inflammatory drugs (e.g., thalidomide, pentoxyphylline, a monoclonal antibody against interleukin-1 a, ghrelin and the ghrelin agonist anamorelin).

In one example, the humanized Fn14-binding protein of the disclosure is used in combination with at least one additional known compound which is currently being used or is in development for preventing or treating a cancer.

In one example, the additional therapeutic agent for preventing or treating a cancer is a chemotherapeutic agent. Exemplary chemotherapy agents include, for example, caboplatin, cytarabine, chlorambucil, cisplatin, cyclophosphamide, danorubicin, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, fludarabine, idarubicin, irinotecan, methotrexate, mitoxantrone, paclitaxel, topotecan, vincristine and vinblastine.

In one example, the additional therapeutic agent for preventing or treating a cancer is a therapeutic antibody. Exemplary therapeutic antibodies are known to the skilled person and include, but are not limited to, Abagovomab; Abciximab; Abituzumab; Abrilumab; Actoxumab; Adalimumab; Adecatumumab; Aducanumab; Afelimomab; Afutuzumab; Alacizumab pegol; Alemtuzumab; Alirocumab; Altumomab pentetate; Amatuximab; Anatumomab mafenatox; Anetumab ravtansine; Anifrolumab; Anrukinzumab; Apolizumab; Arcitumomab; Ascrinvacumab; Aselizumab; Atezolizumab; Atinumab; Atlizumab (tocilizumab); Atorolimumab; Bapineuzumab; Basiliximab; Bavituximab; Bectumomab; Begelomab; Belimumab; Benralizumab; Bertilimumab; Besilesomab; Bevacizumab; Bezlotoxumab; Biciromab; Bimagrumab; Bimekizumab; Bivatuzumab mertansine; Blinatumomab; Blosozumab; Bococizumab; Brentuxim abvedotin; Briakinumab; Brodalumab; Brolucizumab; Brontictuzumab; Canakinumab; Cantuzumab mertansine; Cantuzumab ravtansine; Caplacizumab; Capromab pendetide; Carlumab; Catumaxomab; cBR96-doxorubicin immunoconjugate; Cedelizumab; Certolizumab pegol; Cetuximab; Citatuzumab bogatox; Cixutumumab; Clazakizumab; Clenoliximab; Clivatuzumab tetraxetan; Codrituzumab; Coltuximab ravtansine; Conatumumab; Concizumab; Crenezumab; Dacetuzumab; Daclizumab; Dalotuzumab; Dapirolizumab pegol; Daratumumab; Dectrekumab; Demcizumab; Denintuzumab mafodotin; Denosumab; Derlotuximab biotin; Detumomab; Dinutuximab; Diridavumab; Dorlimomab aritox; Drozitumab; Duligotumab; Dupilumab; Durvalumab; Dusigitumab; Ecromeximab; Eculizumab; Edobacomab; Edrecolomab; Efalizumab; Efungumab; Eldelumab; Elgemtumab; Elotuzumab; Elsilimomab; Emactuzumab; Emibetuzumab; Enavatuzumab; Enfortumab vedotin; Enlimomab pegol; Enoblituzumab; Enokizumab; Enoticumab; Ensituximab; Epitumomab cituxetan; Epratuzumab; Erlizumab; Ertumaxomab; Etanercept; Etaracizumab; Etrolizumab; Evinacumab; Evolocumab; Exbivirumab; Fanolesomab; Faralimomab; Farletuzumab; Fasinumab; Felvizumab; Fezakinumab; Ficlatuzumab; Figitumumab; Firivumab; Flanvotumab; Fletikumab; Fontolizumab; Foralumab; Foravirumab; Fresolimumab; Fulranumab; Futuximab; Galiximab; Ganitumab; Gantenerumab; Gavilimomab; Gemtuzumab ozogamicin; Gevokizumab; Girentuximab; Glembatumumab vedotin; Golimumab; Gomiliximab; Guselkumab; Ibalizumab; Ibritumomab tiuxetan; Icrucumab; Idarucizumab; Igovomab; Imalumab; Imciromab; Imgatuzumab; Inclacumab; Indatuximab ravtansine; Indusatumab vedotin; Infliximab; Inolimomab; Inotuzumab ozogamicin; Intetumumab; Ipilimumab; Iratumumab; Isatuximab; Itolizumab; Ixekizumab; Keliximab; Labetuzumab; Lambrolizumab; Lampalizumab; Lebrikizumab; Lemalesomab; Lenzilumab; Lerdelimumab; Lexatumumab; Libivirumab; Lifastuzumab vedotin; Ligelizumab; Lilotomab satetraxetan; Lintuzumab; Lirilumab; Lodelcizumab; Lokivetmab; Lorvotuzumab mertansine; Lucatumumab; Lulizumab pegol; Lumiliximab; Lumretuzumab; Mapatumumab; Margetuximab; Maslimomab; Matuzumab; Mavrilimumab; Mepolizumab; Metelimumab; Milatuzumab; Minretumomab; Mirvetuximab soravtansine; Mitumomab; Mogamulizumab; Morolimumab; Motavizumab; Moxetumomab pasudotox; Muromonab-CD3; Nacolomab tafenatox; Namilumab; Naptumomab estafenatox; Narnatumab; Natalizumab; Nebacumab; Necitumumab; Nemolizumab; Nerelimomab; Nesvacumab; Nimotuzumab; Nivolumab; Nofetumomab merpentan; Obiltoxaximab; Obinutuzumab; Ocaratuzumab; Ocrelizumab; Odulimomab; Ofatumumab; Olaratumab; Olokizumab; Omalizumab; Onartuzumab; Ontuxizumab; Opicinumab; Oportuzumab monatox; Oregovomab; Orticumab; Otelixizumab; Otlertuzumab; Oxelumab; Ozanezumab; Ozoralizumab; Pagibaximab; Palivizumab; Panitumumab; Pankomab; Panobacumab; Parsatuzumab; Pascolizumab; Pasotuxizumab; Pateclizumab; Patritumab; Pembrolizumab; Pemtumomab; Perakizumab; Pertuzumab; Pexelizumab; Pidilizumab; Pinatuzumab vedotin; Pintumomab; Placulumab; Polatuzumab vedotin; Ponezumab; Priliximab; Pritoxaximab; Pritumumab; Quilizumab; Racotumomab; Radretumab; Rafivirumab; Ralpancizumab; Ramucirumab; Ranibizumab; Raxibacumab; Refanezumab; Regavirumab; Reslizumab; Rilotumumab; Rinucumab; Rituximab; Robatumumab; Roledumab; Romosozumab; Rontalizumab; Rovelizumab; Ruplizumab; Sacituzumab govitecan; Samalizumab; Sarilumab; Satumomab pendetide; Secukinumab; Seribantumab; Setoxaximab; Sevirumab; Sibrotuzumab; Sifalimumab; Siltuximab; Simtuzumab; Siplizumab; Sirukumab; Sofituzumab vedotin; Solanezumab; Solitomab; Sonepcizumab; Sontuzumab; Stamulumab; Sulesomab; Suvizumab; Tabalumab; Tacatuzumab tetraxetan; Tadocizumab; Talizumab; Tanezumab; Taplitumomab paptox; Tarextumab; Tefibazumab; Telimomab aritox; Tenatumomab; Teneliximab; Teplizumab; Teprotumumab; Tesidolumab; Tetulomab; Ticilimumab; Tigatuzumab; Tildrakizumab; Tocilizumab; Toralizumab; Tosatoxumab; Tositumomab; Tovetumab; Tralokinumab; Trastuzumab; Tregalizumab; Tremelimumab; Trevogrumab; Tucotuzumab celmoleukin; Tuvirumab; Ublituximab; Ulocuplumab; Urelumab; Urtoxazumab; Ustekinumab; Vandortuzumab vedotin; Vantictumab; Vanucizumab; Vapaliximab; Varlilumab; Vatelizumab; Vedolizumab; Veltuzumab; Vepalimomab; Vesencumab; Visilizumab; Volociximab; Vorsetuzumab mafodotin; Votumumab; Zalutumumab; Zanolimumab; Zatuximab; Ziralimumab; Zolimomab aritox.

In one example, the humanized Fn14-binding protein of the disclosure is used in combination with at least one additional known compound which is currently being used or is in development for preventing or treating a chronic kidney disease. Examples of such compounds include but are not limited to: ACE inhibitor drugs (e.g. captopril (Capoten™), enalapril (Innovace™), fosinopril (Staril™), lisinopril (Zestril™), perindopril (Coversyl™), quinapril (Accupro™), trandanalopril (Gopten™), lotensin, moexipril, ramipril); RAS blockers; angiotensin receptor blockers (ARBs) (e.g. Olmesartan, Irbesartan, Losartan, Valsartan, candesartan, eprosartan, telmisartan, etc); protein kinase C (PKC) inhibitors (e.g. ruboxistaurin); inhibitors of AGE-dependent pathways (e.g. aminoguanidine, ALT-946, pyrodoxamine (pyrododorin), OPB-9295, alagebrium); anti-inflammatory agents (e.g. clyclooxigenase-2 inhibitors, mycophenolate mophetil, mizoribine, pentoxifylline), GAGs (e.g. sulodexide (U.S. Pat. No. 5,496,807)); pyridoxamine (U.S. Pat. No. 7,030,146); endothelin antagonists (e.g. SPP 301), COX-2 inhibitors, PPAR-gamma antagonists and other compounds like amifostine (used for cisplatin nephropathy), captopril (used for diabetic nephropathy), cyclophosphamide (used for idiopathic membranous nephropathy), sodium thiosulfate (used for cisplatin nephropathy).

In one example, the humanized Fn14-binding protein of the disclosure is used in combination with at least one additional known compound which is currently being used or is in development for preventing or treating diabetes. Examples of such known compounds include but are not limited to common anti-diabetic drugs such as sulphonylureas (e.g. glicazide, glipizide), metformin, glitazones (e.g. rosiglitazone, pioglitazone), prandial glucose releasing agents (e.g. repaglinide, nateglinide), acarbose and insulin (including all naturally-occurring, synthetic and modified forms of insulin, such as insulin of human, bovine or porcine origin; insulin suspended in, for example, isophane or zinc and derivatives such as insulin glulisine, insulin lispro, insulin lispro protamine, insulin glargine, insulin detemir or insulin aspart). As will be apparent from the foregoing, the present disclosure provides methods of concomitant therapeutic treatment of a subject, comprising administering to a subject in need thereof an effective amount of a first compound and a second compound, wherein said first compound is a humanized binding protein of the disclosure (i.e., humanized Fn14- binding protein or humanized anti-Fn14 antibody), and the second agent is for the prevention or treatment of cancer and/or diabetes.

As used herein, the term "concomitant" as in the phrase "concomitant treatment" includes administering a first agent in the presence of a second agent. A concomitant therapeutic treatment method includes methods in which the first, second, third or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer to a subject a first agent and as a second actor may administer to the subject a second agent and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and/or additional agents) are after administration in the presence of the second agent (and/or additional agents). The actor and the subject may be the same entity (e.g., a human).

In one example, the disclosure also provides a method for treating a wasting disorder in a subject, the method comprising administering to the subject a first pharmaceutical composition comprising at least one humanized Fn14-binding protein of the disclosure and a second pharmaceutical composition comprising one or more additional compounds.

In one example, a method of the disclosure comprises administering a humanized Fn14-binding protein to a subject suffering from cachexia (e.g., cancer cachexia or diabetic cachexia) and receiving another treatment (e.g., for cancer or diabetes).

Kits

Another example of the disclosure provides kits containing humanized Fn14-binding proteins or humanized Fn14-antibodies of the disclosure for the treatment or prevention of a disease or condition as described above.

In one example, the kit comprises (a) a container comprising a humanized Fn14- binding protein as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a disease or condition (e.g., a wasting disorder) in a subject.

In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the humanized Fn14-binding protein. The label or package insert indicates that the composition is used for treating a subject eligible for treatment, e.g., one having or predisposed to a wasting disorder, with specific guidance regarding dosing amounts and intervals of compound and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit optionally further comprises a container comprises a second medicament, wherein the humanized Fn14-binding protein is a first medicament, and which article further comprises instructions on the package insert for treating the subject with the second medicament, in an effective amount. The second medicament may be any of those set forth above.

The present disclosure includes the following non-limiting Examples.

EXAMPLES

Example 1 : Identification of human heavy and light chain sequence homologous to mu002 heavy and light chain sequences

The heavy and light chain variable region sequences of murine antibody 002 are shown in SEQ ID NOs: 1 to 4. The heavy and light chain murine sequences of 002 were entered into the V-Quest/IMGT database (IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org (founder and director: Marie-Paule Lefranc, Montpellier, France) and VBASE2/ DNAPIot (Brochet, 2008, Better, et al., 2005; Mollova et al., 2007) and a homology search was performed against the human germline sequences contained in the databases. Highly homologous germline sequences were chosen to use as templates for the humanization process.

The human kappa light chain sequences chosen were IMGT/VBASE2 IGKV3-11 *01 germline sequence (clone name L6, Huber et al., 1993; Cox et al., 1994), NCBI ref X01668.1 and the human kappa consensus sequence subgroup III from Padlan, EA (1991 ). The light chain kappa Fn14 murine sequence shares 73.2% homology with the IGKV3-11 *01 human germline sequence.

The human heavy chain sequences chosen were IMGT/VBASE2 IGHV3-72*01/DP29 germline sequence, NCBI reference X9226.1 and the human heavy chain consensus sequence subgroup III from Padlan, EA (1991 ). The heavy chain Fn14 murine sequence shares 75.17% homology with the DP29 human germline sequence (Almagro et al., 1997).

Identification of Invariant, Similar Residue and Residue Class Sites

Chothian numbering was applied to the sequences to assign framework and CDR regions. Residues in both the heavy and light chain of mu002 were assigned a certain designation (as defined in Chothia et al., 1998), as follows:

1 ) Invariant residue class (IR): Nine amino acid positions were identified which are occupied by a single particular residue in almost all sequences,

2) Similar residue class (SR): At 17 sites, in almost all sequences, there resides only one of a small number of very similar residues. Similar residues are defined as those that have the same chemical character and whose volumes differ by no more than the equivalent of one methylene group, and

3) Residue class (RC): two thirds of the sites are occupied by residues that have a wider range in chemical character and/ or volume than that found at SR sites. In nearly all cases, these sites do not contain all types of residues but are limited to certain classes of residues. Different types of RC sites can be defined by the particular class of residues that they conserve:

Surface (s): R, K, E, D, Q and N

Neutral (n): P, H, Y, G, A, S and T

Buried (b): C, V, L, I, M, F and W

Identification of the structural role of amino acids in the mu002 heavy and light chain sequences

Using the classification described above (Chothia et al., 1998), the structural role of amino acids in the heavy and light chains of mu002 were identified, p-sheet assignment, variable light-variable heavy CDR (VL-VH) and variable region CDR-constant region (V-C) interface residues, central hydrophobic core, buried polar residues, sites around the central hydrophobic core, sites on the corners of the p-sheets, and interstrand loop residues were identified. Some residues had neither a residue class designation nor a structural role identified. First generation humanization constructs: rationale and criteria for changing amino acid residues from murine to human

CDR amino acids of the heavy and light chains were excluded from the humanization process. This was to preserve the integrity and affinity of the antibody binding to the target Fn14.

The following rationale and criteria were used for selection of amino acids to change from mouse to human:

1 ) buried residues were changed if the amino acid residues were considered conservative for example Leucine to Methionine or Valine,

2) the amino acid residues were surface residues and the human germline candidate and consensus sequences had the same amino acid at that residue,

3) if the murine Fn14 amino acid residue was the same in both of the human germline candidate sequence and the human germline consensus sequence, the murine amino acid was left unchanged,

4) if the murine amino acid differed from both human germline candidate and consensus sequences or only shared similarity with one of the human candidate sequences, change to the most commonly occurring human amino acid among the human candidate sequences was considered (according to Chothia, 1998),

5) amino acids that were not identified by Chothia et al. (1992) as being important structurally, and were different from all human candidate sequences, were considered for change to the most common sequence shared among the human candidate sequences,

6) residues that were identified as being part of the variable light-variable heavy CDR interfaces (V _L /V _H ) and variable region CDR-constant region interfaces (V-C) were considered for conservative changes based on germline sequences, and

7) where the germline amino acids in both the template and the human consensus sequences proved to be destabilizing when considered for substitution at a particular murine residue, identification of alternate less destabilizing germline amino acid for substitution was made within the confines of the published known germline sequences at that particular residue.

Accelrys modelling software for determination of effect of amino acid changes on structure of mu002 and CDR presentation

The Accelrys protein modelling software (Biovia, San Diego, CA) was used to perform a comparison of the mu002 antibody variable region structure with the versions of heavy and light chains designed during the first generation of humanization. The antibody modelling software by Accelrys was used to create 3D models of the mu002 antibody followed by the build mutants and calculate mutational energy programs to analyse the effect of the humanization process on the stability of the antibody.

Homology modelling allowed investigation of how amino acid residue changes would affect the presentation of the CDRs as well as any alterations that might result in the framework regions.

Example 2: First generation hu002 lgG4 constructs

Three versions of the hu002 light chain and one version of the hu002 heavy chain were prepared containing a small number of amino acids differences designed to determine which amino acid combination provided the most suitable candidate.

Clustal alignment of murine and humanized 002 first generation light and heavy chain variable region protein sequences are shown in Figure 1.

Codon optimization for expression of hu002 antibody variants in CHO SV cells

Based on the Accelrys models, the sequences were submitted to GenScript for codon optimization of product expression in the mammalian cell line CHO SV. Codon optimization can improve the expression and production of heterologous proteins. By changing the codons of a protein at the DNA level to ones that are most commonly recognized by the mammalian cells to be used for expression, this can improve the expression and secretion of the heterologous protein.

Cloning strategy for expression of humanized 002 variable light and heavy chains in light and heavy chain mammalian expression vectors

Synthesized, sequence verified humanized variable light chain (LC) and heavy chain (HC) domains were excised from GeneArt pcDNA3.1 mammalian expression vectors using EcoRI and HinDIII restriction enzymes and cloned into heavy chain and light chain mammalian expression vectors.

Heavy and light chain constructs were sequence verified prior to construction of the final expression vector that contained both the heavy and light chain sequences. The final construct was then sequence verified (Mircromon Sequencing, Monash University, Australia). Subsequently the humanized heavy chain and light chain variable region combinations were evaluated for binding. The following constructs were created:

Variant 1 :hu002 HC LC.V1 ;

Variant 2:hu002 HC LC.V2;

Variant 3:hu002 HC LC.V3.

The DNA and protein sequences for heavy chain variable regions are disclosed in SEQ ID NOs: 7 and 8 respectively.

The DNA and protein sequences for the variable regions of light chain variants 1 -3 are disclosed in SEQ ID NOs: 9 to 14.

First generation hu002 variants expression and characterization

The pCDNA 3.1 vectors containing the heavy and light chain hu002 antibody sequences were co-transfected in to Freestyle 293T cells (Invitrogen) and transiently expressed following manufacturer’s protocol. Analysis of the binding activity indicated that all three variants performed equally well.

Variant 3: hu002 HC LC. V3 containing the highest number of humanized amino acids was chosen for further analysis.

Following protein A purification of Freestyle 293T transiently expressed antibody variants, SDS-PAGE gel analysis of murine and first generation humanized antibody constructs under reduced and non-reduced conditions was performed. Although the reduced conditions demonstrated the typical profile of an immunoglobulin light and heavy chain bands, a multiple banding pattern was observed in all lgG ₄ LC variants in nonreducing conditions.

First generation hu002 lgG _t construct generation and characterization

A human lgG1 version of the hu002 heavy chain antibody was created to enable investigation of the possibility that the constant region of the hu002 lgG4 antibody construct was causing the apparent intact IgG instability. The lgG1 constant region sequence from another successfully produced humanized monoclonal antibody within the laboratory was used to create the hu002 heavy chain lgG1 construct. Restriction enzyme Pmel and Drain digests were performed on i) the heavy chain mammalian expression vector containing the lgG1 constant region and ii) the hu002 heavy chain mammalian expression vector. The hu002 heavy chain variable region and the heavy chain IgG 1 mammalian expression vector were agarose gel separated, excised and purified. The hu002 heavy chain variable region was then ligated into the heavy chain lgG1 mammalian expression vector to create the hu002 heavy chain lgG1 construct.

The mammalian expression vector containing both the hu002 heavy chain lgG1 and light chain V3 variable regions was then created (phu002 HC lgG1 -LCV3). Transient expression of the sequence verified final construct was successfully performed. Following protein-A purification of the antibody protein analyses were performed by SDS PAGE gel and size exclusion chromatography (SEC)- HPLC. Investigation of control lgG4 protein characteristics in two mammalian expression systems To investigate the possibility that the expression vectors were contributing to the apparent instability, a previously humanized, lgG4 antibody was subcloned into the heavy and light chain mammalian expression vectors used to create the hu002 constructs. The lgG4 constant region sequence of this antibody was identical to that used for hu002, so only the vector sequence was the variable being assessed. Subcloning of the variable regions of the heavy and light chains of the test antibody was performed using Pmel and Drain (HC) or Rsrll (LC) restriction enzymes and digesting the hu002 heavy chain and light chain mammalian expression vectors. The variable regions were then subcloned into the respective light and heavy chain constant region containing vectors followed by sequence verification (Micromon). A final mammalian expression vector containing both the heavy and light chains of the test antibody was constructed. Transient expression in the Freestyle 293T cells followed by protein purification and SDS PAGE analysis.

Protein expression analysis demonstrated no difference in lgG4 product between the original expression vectors and those previously used. Therefore the mammalian expression vectors were not the source of the apparent instability.

SDS-PAGE analysis of purified hu002 lgG1 LCv3 protein under non-reduced conditions demonstrated less pronounced small molecular weight fragments, but similar multiple banding pattern to the hu002 lgG4 construct. Furthermore, the multiple banding was more marked in the first generation hu002 lgG1 and lgG4 constructs compared to control hulgG4 expressed in the same vector and control hulgG1 suggesting protein instability.

Example 3: Second generation hu002 constructs

Second generation constructs were created due to the apparent instability in the humanized constructs when analyzed by SDS PAGE under non-reducing conditions. The investigation of the apparent instability focused on three areas: 1 ) amino acid residues, 2) the heavy chain constant region, and 3) the mammalian expression system.

Review of hu002 amino acid residue substitution

Review of the amino acid changes implemented during the initial humanization process led to further alteration of the sequences. The previous rationale and criteria for the first generation of humanization were applied with some amendments. Additional amino acids were considered for change in the second generation based on these further criteria:

1 ) the amino acid structure, side chain interactions and charge, were investigated where the amino acids were not considered conservative in their changes.

2) The amino acids initially substituted from murine to human were reconsidered even though these were conservative changes.

Second generation light chain variable region constructs were synthesized using the Agilent Technologies Quick Change Mutagenesis kit. The mammalian expression vector containing the light chain variable region variant 3 was used as the template and the mutations were introduced following the manufacturers protocol. After each combination of mutations was completed, the vectors were sent for sequence verification (Micromon, Monash University, Australia). Final mammalian expression vector constructs containing both the heavy and light chain variable region sequences were prepared, sequence verified (Micromon) and tested for apparent binding affinity following transient expression in Freestyle 293T cells (Thermofisher Scientific).

Back mutation of light chain residues 77 and 80

Light chain variable region amino acid residues 77 and 80 were considered for back mutation to the parental murine residues: Hu77 Serine —> Mu77 Proline and Hu80 Proline Mu80 Glutamic acid. The amino acid proline can introduce turns in the antibody super structure (tertiary) that can impact on the binding affinity. Both amino acid 77 and 80 are in the interstrand loops according to Chothia et al. (1998). Changes to the amino acids in the interstrand loops are unlikely to impact the binding affinity as this is an unordered and unstructured area between the CDR2 and CDR3. Amino acid 77 was considered for substitution in the first round of humanization due the fact that there is no proline in the human germline sequences reviewed at this residue and the human consensus sequence subgroup III indicates arginine at this residue. Chothia et al. (1998) indicates that serine is the most common amino acid found at this residue in germline sequences and therefore the amino acid was changed to serine. Amino acid 80 was considered for substitution in the first round of humanization due to the fact that glutamic acid was present in only one germline sequence whereas there were 26 germline sequences with proline at this residue. Additionally, L6, the germline sequence used for the template in the humanization of muFn14 light chain variable region sequence, has a proline at this residue.

Accordingly light chain residues 77 and 80 were back mutated to the respective parental murine residues proline (P) and glutamic acid (E).

The DNA and protein sequences of the Variant PE variable light chain regions are shown in SEQ ID NOs: 15 and 16 respectively.

Back mutation of light chain residues 100, 104, 106 and 107

Light chain variable region amino acids 100, 104, 106 and 107: the amino acid substitutions were considered to be conservative. Mu100 Alanine —> Hu100 Glycine, Mu104 Leucine —> Hu 104 Valine, Mu106 Leucine —> Hu106 Isoleucine, Mu107 Glutamine — > Hu107 Lysine. Residue 100 is part of the VL/VH interface however the substitution was considered conservative and G was the most common residue at that site in germline sequences (Chothia et al., 1998). Residues 104 and 106 are part of the central hydrophobic core however the residues Valine, Leucine, Isoleucine and Methionine are all hydrophobic and part of the same closely related group of residues defined by Chothia et al., 1998. Residue 107 is part of the variable/ constant region interface.

Hsu et al., 2014 found from extensive sequencing of the antibody variable regions that the C-terminal p-strands could accoSEQ mmodate flexible sequence features while remaining optimized in thermal stability. Their work also found that “the residue side chains involving the lower hydrophobic packing peripheral to the central core of variable domains are interchangeable in hydrophobic amino acids and moreover, the optimization to the peripheral hydrophobic packing can improve the thermal stability of the variable domains”. The lower hydrophobic core in the light chain variable region includes amino acid residue 104. This analysis concurs with Chothia et al., 1998 and their analysis of the residues, their conservation and importance in determining the folding and binding affinity of the CDR regions.

Accordingly, amino acid residues 100, 104, 106 and 107 substituted in the initial humanization process were back mutated to the parental murine residues of Alanine (A), Leucine (L), Leucine (L) and Glutamine (Q), respectively.

The DNA and protein sequences of the Variant ALLQ variable light chain regions are shown in SEQ ID NOs: 17 and 18 respectively.

Second generation construct characterisation

Light chain variant 3 PE back mutations

Additional mammalian expression constructs using the light chain variable region variant 3 PE mutant and both the heavy chain variable region lgG4 and lgG1 constant region sequences were prepared (phu002 HC lgG4-LCV3 PE back mutation, phu002 HC lgG1 -LCV3 PE back mutation) and transiently expressed in Freestyle 293T cells. SDS PAGE and anti-human Fc western blot analysis demonstrated that the back mutation effected a decrease in the presence of high molecular weight bands around 180kDa in the original lgG4 heavy chain construct and no remarkable change in the expression of the heavy chain lgG1 containing constructs. The multiple banding pattern under non-reduced conditions was more apparent in the 002 constructs compared to control lgG1 and not improved by PE back mutations. The PE back mutation was not further pursued.

Light chain variant 3 ALLQ back mutations

The mammalian expression constructs were created with the hu002 heavy chain variable region lgG1 and lgG4 sequences combined with the light chain variable region variant 3 ALLQ mutant sequence (phu002 HC lgG1 -LCV3 ALLQ and phu002 HC lgG4- LCV3 ALLQ). These were transiently expressed in Freestyle 293 cells and protein was purified and analysed in vitro for stability and Fn 14 binding.

1. SDS PAGE analysis

An experiment was designed to determine if heating the protein samples that did not contain reducing agent for 10 minutes at 70°C prior to loading was causing the apparent instability in addition to evaluating the expression profile of the ALLQ mutants. SDS PAGE protein gel analysis was performed on samples that were heated (70°C, 10 mins) or not heated (retained at room temperature) prior to gel loading. Results demonstrated that 1 ) the ALLQ mutant lgG4 and lgG1 antibody proteins did not show any difference to the first generation hu002 lgG4 and lgG1 antibody protein profiles 2) heating the non-reduced samples resulted in multiple protein bands suggesting antibody fragmentation and instability.

2. SEC-HPLC analysis

Size exclusion chromatography was performed on the first generation hu002 LCV3 lgG4 and lgG1 constructs and second generation hu002 LCV3 ALLQ mutants. The results demonstrated that the back mutation resolved the aggregation that was observed in the first generation hu002 lgG4 antibody (Figure 2). Single peak chromatograms for the lgG1 constructs and second generation hu002 lgG4 were observed corresponding to the molecular weight of an intact immunoglobulin.

Collectively the results demonstrated that the second generation hu002 constructs bound Fn14 with no loss of affinity compared to the parental murine 002 antibody (Table 2), did not aggregate (Figure 2), and without sample heating for SDS-PAGE analyses the expected profile of an intact immunoglobulin was evident under reduced and non-reduced conditions.

Table 2: Apparent binding affinities of parental murine 002 and humanized 002 antibodies for different species Fn14 using surface plasmon resonance analysis.

(abbreviations: h, human; WT, wildtype; cyno, cynomolgus; r, rat; m, mouse) Example 4: Anti Cachectic efficacy of humanized anti-Fn14 antibodies in animal models of cancer cachexia

In vivo analysis of anti-cachectic properties of hu002 lqG1 ALLQ, hu002 lqG4 ALLQ compared to mAb 002 (also referred to as mu002) and hu lqG1 controls (Human Prostate Cancer model)

Human prostate carcinoma PC-3* cells, a variant of the PC-3 cell line causing cachexia, were grown in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 100 units/mL penicillin/streptomycin (Thermo Fisher Scientific) and 2mM L-alanyl-L-glutamine dipeptide (Thermo Fisher Scientific) until cells were between 80% and 90% confluent, incubated at 37°C in 5% CO2. Cells were harvested or passaged using 2.5% Trypsin/0.05% EDTA solution in phosphate-buffered saline (PBS, Gibco) for detachment.

Therapeutic study

PC3* prostate carcinoma xenografts were established in male NOD-SCID-IL2R-/- (NSG) mice age 5-6 weeks (ARC, or Bioresources Facility, Austin Health) . PC-3* cells (5 x 10 ⁶ ) were resuspended in RPMI 1640 medium supplemented with 50% (v/v) BD Matrigel Basement membrane Matrix, then injected subcutaneously in left lower flank using a 27G insulin needle. Tumors were measured via a caliper using formula: TV = (L x W ² )/2 [where TV, tumor volume; L, length; W, width]. Once tumors reached an average size of 100mm ³ , mice were randomized into four groups of n = 6 to achieve similar group average tumor size and body weights. The study was performed blinded. Groups of 6 mice received 002 therapy (10mg/kg of mu002 mouse lgG1 , or hu002 lgG1 or hu002 lgG4) or vehicle control (PBS) twice per week via intraperitoneal injections, for a total 5 doses over 14 days. A pair fed non-tumor bearing control arm (n=8) was included for comparison with the PBS control arm for body weight assessments to preclude the impact of animal handling on weight loss in the treated mice. Body weight was monitored three times per week until body weight loss was identified (5%) after which body weight was monitored daily until study end point (body weight loss of 20% or tumor size of 1000mm ³ ).

Study endpoint was Day 17 post commencement of therapy due to control arm tumor burden. At study endpoint significant retention of body weight (P<0.05) was observed for all three antibody therapy groups compared to PBS vehicle control arm (Figure 3 A and B). The anti-cachectic effect was apparent following the third antibody dose (Figure 3 A). The three anti-Fn14 antibodies tested demonstrated equivalent anti-cachectic properties compared to PBS control (P< 0.005).

In vivo analysis of anti-cachectic properties of hu002 ALLQ lqG1 , hu002 ALLQ lqG4 compared to mAb 002 and hu lqG1 controls (C26 model in CD2F1 mice)

Male CD2F1 mice (10-1 1 weeks old) bearing Colon 26 (C26) Fn 14 expressing tumors were used to analyse the anti-cachectic properties of humanized antibodies hu002 lgG1 , hu002 lgG4 in comparison with parental murine antibody mu002 and a humanized lgG1 isotype control. Briefly, 1 x 10 ⁶ C26 cells were subcutaneously injected into the flank of CD2F1 male mice (day one of experiment) as previously described (Johnston et al Cell 162: 1365-1378, 2015). Mice were monitored daily (body weight and condition). On day 6, when tumors were average size of 100 mm ³ (caliper measurements), groups of six mice were randomized into groups of n = 6 to achieve similar group average tumor size and body weights. Mice received single dose 20 mg/kg intraperitoneal administration of hu002 lgG1 , hu002 lgG4, mu002, hulgG1 isotype or PBS vehicle control. Study endpoint was Day 17 post commencement of therapy and mice were weighed and euthanised. At study endpoint significant preservation of body weight (P<0.005) was observed with 002 antibody therapy arms compared to hulgG1 control, and PBS. There was no significant difference in anti- cachectic efficacy between hu002 lgG1 and hu002 lgG4 or mu002 (P>0.2) (Figure 4). Statistical analysis

Statistical analysis of blinded raw data of the study results for individual mouse body weight and tumor sizes were performed using GraphPad Prism (GraphPad) software. A Brown- Forsythe and Welch ANOVA (equal standard deviations not assumed) with a post hoc Dunnett T3 (to correct for multiple comparisons) test between groups was performed.

Example 5: Anti-cachectic properties of hu002 ALLQ IgGI, hu002 ALLQ lgG4 compared to commercially available antibodies

To demonstrate that mAb 002, hu002 ALLQ lgG1 and lgG4 are antagonistic in nature and are functionally distinct (i.e., anti-cachectic) from commercial antibodies PDL192 (Enavatuzumab - a humanized lgG1), ITEM1 (mouse lgG1 ) and P4A8 that are not anti- cachectic, analysis of the binding properties and interaction of the antibodies to Fn14 was performed.

Crystallography of Fn14 and ITEM1 in complex with Fab

Crystallography was used to determine the structure of Fn14 and ITEM1 in complex with Fab.

As shown in Figure 5 and Table 3, the interaction of Fn14 and mu002 is asymmetric, in that a large area of Fn14 is in contact with the heavy chain (HC) (15.6%; A) compared to the light chain (LC) (10.7%; C). While the interaction of Fn14 and ITEM1 is more evenly shared by HC (B) and LC (D), 15.7% and 13.4% respectively.

Tweak induced NFKB blocking

Anti-Fn14 IgG monoclonal antibodies mouse 002, humanized ALLQ 002 lgG1 , humanized 002 ALLQ lgG4, PDL192 and P4A8 were assessed for functional antagonistic activity in vitro. Briefly, 293T NF-KB reporter cells were treated with mu002 or 002-hlgG1 or 002-hlgG4 or PDL192 or P4A8 at 100ng/mL or 1 p with addition of increasing concentrations of TWEAK-Fc (i.e., Ong/mL, 50ng/mL, 100ng/mL and 200ng/mL).

As shown in Figure 6, all 002 antibodies exhibited strong antagonistic activity and efficiently blocked NFKB activation by 50ng/mL of Tweak-Fc when added at a concentration of 1 pg/mL. At the lower antibody concentration of 100 ng/mL, blocking was also observed.

Example 6: In vivo Biodistribution in tumor bearing nude mice

Antibody conjugates were assessed for their suitability for use in bioimaging by exploring chelation with deferoxamine (Df-) and radiolabeling with zirconium-89 ( ⁸⁹ Zr-) of mu002 and hu002 anti-Fn14 monoclonal antibody constructs. An isotype hulgG1 control antibody was included as a negative control.

Materials and Methods

Cell Culture

PC-3* cells, a Fn14 expressing variant of the PC-3 prostate carcinoma cell line (ATCC), were grown in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 100 units/mL penicillin/streptomycin (Thermo Fisher Scientific) and 2mM L- alanyl-L-glutamine dipeptide (Thermo Fisher Scientific) until cells were between 80% and 90% confluent before passaging or use. The cell line was grown at 37°C in a humidified atmosphere of 5% CO2. Conjugation of deferoxamine for radiolabeling

Affinity purified murine monoclonal antibody mu002, or recombinant humanized constructs hu002 lgG1 and hu002 lgG4 produced by HEK293 transient expression were conjugated with the bifunctional metal ion chelator, deferoxamine (Df; Macrocyclics, TX, USA), at a 4.0-fold (mu002) or 2.3 fold (hu002) molar excess in 0.1 M sodium bicarbonate buffer, pH 9. Prepared conjugates were prepared in a buffer formulation of 50 mM sodium acetate, 0.02% w/v Tween 20, 5% w/v sorbitol, pH 6.0, via spin column, aliquoted and stored at or below -80°C. Optimal conditions for chelation and radiolabeling of hulgG1 isotype control had been previously established.

Flow Cytometry

The Fn14 antigen binding by the anti-Fn14 antibodies, Df-anti-FN-14 antibodies or ‘mock’ radiolabelled ⁸⁹ Zr-Df-antibodies using decayed ⁸⁹ Zr- was assessed by flow cytometry. Anti-Fn14 antibody (20 pg/mL) bound to 1 x10 ⁶ PC-3* cells/mL was probed with a Phycoerythrin conjugated goat anti-human or goat anti-mouse IgG secondary antibody. Flow analysis was performed using FACS Canto II Flow Cytometry.

Radiolabeling and Quality Assurance

Analytical grade reagents, sterile technique and pyrogen-free plasticware were used in all labelling steps. The antibody constructs were trace radiolabeled with ⁸⁹ Zr- (Austin Health, Cyclotron, Heidelberg, Australia) on lysine residues via the bifunctional metal ion chelating agent deferoxamine. Following chelation, the optimized radiolabeling conditions for the supplied antibody constructs were determined. Briefly, a solution containing 1 .0 mCi of ⁸⁹ Zr in 0.5 M ammonium acetate buffer containing 5 mg/ml of genetisic acid, pH 7.2, was mixed with 0.5 mg Df-antibody for 1 hour at 37°C. Resultant radioconjugates were assessed for radiochemical purity and immunoreactivity without further purification using Fn14- agarose beads as described below.

Radiochemical Purity

The amount of free versus bound antibody following radiolabeling was determined by instant thin layer chromatography (ITLC) using silica gel impregnated glass fiber ITLC strips (Gelman Sciences, Inc., An Arbor, Ml, USA) and 20 mM citric acid (pH4.8) as mobile phase. Assays were performed in duplicate. Radioactivity was measured with an automated gamma counter (Wizard, PerkinElmer, Australia).

Immunoreactivity

Recombinant extracellular domain Fn 14 with histidine tag (7.5 kDa; 2mg) was coupled to 1 mL Nickel- NTA agarose beads (Ni-NTA; Qiagen, Australia). Briefly, a 100 pL slurry of Fn14-Ni-NTA beads (~20 pg Fn14) with or without the addition of competing unlabelled 002 antibody (20 pg), or non-specific binding control Ni-NTA beads was mixed with 20 ng of each ⁸⁹ Zr-Df-002 construct in PBS for the binding analyses. The beads were incubated for 45 min at room temperature with continuous mixing throughout to keep the beads in suspension. Beads were harvested by centrifugation and washed thrice with PBS to remove unbound antibody, and pellets were measured in a gamma counter (Wizard, Perkin Elmer). Immunoreactivity was calculated as the percentage of radioactivity bound minus nonspecific binding to beads alone/ total radioactivity added.

Serum Stability

Radioconjugate integrity was assessed by Size Exclusion Chromatography (SEC). SEC analyses were performed with an Agilent 1200 Series HPLC system using a TOSOH TSKgel G3000SWXL column and a solution of 50 mM phosphate buffer (pH 7.2), 0.2 M sodium chloride and 0.02% sodium azide as elution buffer at a flow rate of 0.80 mL/min.. Absorbance at 280 nm was recorded using a diode array UV/VIS detector and the radioactivity was measured using a scintillation detector (Packard Radiomatic FLO-ONE Beta 150TR Flow Scintillation Analyzer).

Animal Model

To establish tumors, PC-3* prostate carcinoma cells (5x10 ⁶ ) were injected subcutaneously in the right flank of 5-6-week-old female BALB/c nu/nu mice (Animal Research Centre, WA, Australia) in 50% Matrigel/50% culture medium. Tumor volume (TV) was calculated by the formula [(length x width ² )/2] where length was the longest axis and width the measurement at right angles to length. All animal studies were approved by the Austin Health Animal Ethics Committee and were conducted in compliance with the Australian Code of Practice for the care and use of animals for scientific purposes.

Biodistribution Studies

BALB/c nu/nu mice (n = 105) with established Fn14 expressing PC-3* tumors (mean TV= 124.7 ± 74.2 mm3) were randomized into groups and received a dose of ⁸⁹ Zr-Df- mu002, ⁸⁹ Zr-Df-hu002 IgG 1 , ⁸⁹ Zr-Df-hu002 lgG4, or ⁸⁹ Zr-Df-hulgG1 Isotype control (5.0 pg, 5.0 pCi) intravenously via the tail vein (total volume = 0.1 mL). At designated time points after injection of radioconjugates, groups of mice (n = 5) were sacrificed by over inhalation of isoflurane anaesthesia and biodistribution was assessed on day 0 (4 hour pi), and days 1 , 2, 3, 5 and 7. Biodistribution of ⁸⁹ Zr-Df-hulgG1 isotype control was performed on day 2 and 7.

At the designated time points groups of 5 mice were exsanguinated by cardiac puncture, and tumors and organs [liver, spleen, kidney, muscle, skin, bone (femur), lungs, heart, stomach, brain, small bowel, tail and colon] were collected immediately. All samples were counted in a dual channel gamma scintillation counter (Wizard, PerkinElmer, Australia). Triplicate standards prepared from the injected material were counted at each time point with tissue and tumor samples enabling calculations to be corrected for the physical decay of the isotopes. The tissue distribution data were calculated as the mean ± SD (n=5) percent injected dose per gram tissue (%l D/g) for the radiolabeled constructs per time point.

Pharmacokinetics

Pharmacokinetic analyses were performed on blood biodistribution data (%ID/g) of the ⁸⁹ Zr-radiolabeled antibodies dosed at 5 pg/ 5 pCi using Prism Version 8.2 (Graphpad) Software, San Diego, CA, USA). Estimates were determined for the half lives of the initial and terminal phases of disposition, T ¹ /za and T ¹ /2p.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism Version 8 Software (San Diego, CA, USA). A Brown-Forsythe and Welch ANOVA (equal SD cannot be assumed) with a post hoc Dunnett T3 (to correct for multiple comparisons) test was used to determine significant differences between uptake in tumor, kidney and liver (%ID/g) results. P values were reported as ****P<0.0001 , ***P<0.0005, **P<0.005 and *P<0.05. Data are presented as the Mean ± SD, unless stated differently.

Results

Radiolabeled anti-Fn14 antibody properties

The optimal conditions for conjugation of bifunctional chelate deferoxamine to anti- Fn14 antibodies (mu002, hu002 lgG1 , and hu002 lgG4) and for radiolabeling Df-anti-Fn14 antibody constructs with the PET isotope ⁸⁹ Zr were determined. Radioconjugates were characterised in vitro. ⁸⁹ Zr-radiolabeling produced stable mu002, hu002 lgG1 and hu002 lgG4 radioconjugates in vitro with high radiochemical purity (-99%) and retention of immunoreactivity with ⁸⁹ Zr-Df-002 antibody constructs demonstrating specific binding to Fn14-coated beads (Figure 7). The immunoreactivity of optimized constructs at end of synthesis on day 0 was: ⁸⁹ Zr-Df-mu002, 66.6 ± 7.7%; ⁸⁹ Zr-Df-hu002 lgG1 , 59.9 ± 13.6%; ⁸⁹ Zr-Df-hu002 lgG4, 66.0 ± 5.9%), with low non-specific binding to Fn14-negative beads (immunoreactivity at day 0, 4.2 ± 2.6%). Specific activity was respectively 1.99 ± 0.15, 2.17 ± 0.29, and 2.18 ± 0.31 mCi/mg antibody.

FACS analysis

The Df-conjugated antibodies demonstrated no change in binding properties compared to the original antibody proteins when assessed by FACS analysis with Fn 14- positive PC-3* prostate carcinoma cells. Furthermore, FACS assessment of Df-mu002 radiolabelled with decayed ⁸⁹ Zr (“mock” radiolabelled) showed no change in FACS binding profile for Fn14 positive PC3* cells compared to the original mu002 antibody (data not shown).

SEC Analysis

HPLC SEC analyses assessed the protein integrity of the anti-Fn14 antibodies following conjugation with the bifunctional metal ion chelate Df. Results demonstrated no change in the protein integrity following the conjugation process.

SEC analyses of the ⁸⁹ Zr-radiolabeled anti-Fn14 antibodies at end of synthesis demonstrated by the A280nm traces and the corresponding radioconstruct profile shown by the radioactivity detector trace. The ⁸⁹ Zr-Df-hu002 lgG1 demonstrated a very similar profile to that of the ⁸⁹ Zr-Df-hulgG1 isotype control.

Biodistribution in tumor bearing nude mice

In vivo biodistribution studies showed specific tumor localization and uptake of ⁸⁹ Zr- Df-anti-Fn14 antibody constructs in Fn-14 positive PC-3* prostate cancer tumor models (Figures 8-10). In most normal, healthy tissues Fn14 expression is relatively low. Clearance of blood pool activity with time and pattern of uptake was consistent with low Fn14 expressing organs and the pharmacokinetics and catabolic pathways of radiolabeled intact antibodies. Specific and prolonged tumor retention of the murine and human lgG1 anti-Fn14 radioconjugates over 7 days was observed. ⁸⁹ Zr-Df-mu002 (14.4 ± 1 .1%ID/g) and ⁸⁹ Zr-Df- hu002 lgG1 (12.5 ± 1.4%ID/g) showed highest PC-3* tumor uptake on Day 7 compared to ⁸⁹ Zr-Df-hulgG4 (7.4 ± 1 .1 %ID/g) and isotype control (7.0 ± 1.8 %ID/g) (Figure 8). A significantly increased kidney uptake was observed with radiolabeled hulgG4 002 antibody (day 2, 12.84 ± 0.90 %ID/g) compared to ⁸⁹ Zr-Df-mu002 lgG1 (day 2, 5.17 ± 0.93 %ID/g; P < 0.0001 ) and ⁸⁹ Zr-Df-hu002 lgG1 (day 2, 4.95 ± 0.26 %ID/g; P < 0.0001 ) (Figure 10) suggesting this lgG4 construct is not suitable for in vivo applications.

Pharmacokinetics

The blood clearance of the radiolabeled antibodies enabled determination of the pharmacokinetic parameters of T ¹ /za and T ¹ /2p. Clearance curves for one and two phase exponential decay fits are presented in Figure 11 and results are presented in Table 5. In mice, as anticipated the murine antibody exhibited a slightly longer half-life than the humanized constructs which demonstrated serum clearance patterns typical of humanized constructs in preclinical models.

Table 5: Serum Pharmacokinetics ⁸⁹ Zr-labeled anti-Fn14 antibodies in BALB/c nu/nu mice bearing PC-3* tumors