WO2011084714A2

WO2011084714A2 - STABILIZED ANTI-TNF-ALPHA scFv MOLECULES OR ANTI-TWEAK scFv MOLECULES AND USES THEREOF

Info

Publication number: WO2011084714A2
Application number: PCT/US2010/061164
Authority: WO
Inventors: Brian Robert Miller; Jennifer Michaelson; Stephen Demarest; Dikran Aivazian; Alexey Alexandrovich Lugovskoy
Original assignee: Biogen Idec Ma Inc.
Priority date: 2009-12-17
Filing date: 2010-12-17
Publication date: 2011-07-14
Also published as: WO2011084714A3

Abstract

The instant invention is based, at least in part on the finding that binding molecules which simultaneously bind to multiple epitopes within TNF-alpha result in improved TNFR-1 and/or TNFR-2 blocking capabilities when compared to binding molecules that bind to a single TNF-alpha epitope. The instant invention provides compositions that bind to multiple epitopes of TNF-alpha, for example, combinations of monospecific binding molecules or multispecific binding molecules (e.g., bispecific molecules). Methods of making the subject binding molecules and methods of using the binding molecules of the invention to antagonize TNF-alpha signaling are also provided.

Description

STABILIZED ANTI-TNF-ALPHA scFv MOLECULES OR ANTI-TWEAK scFv

MOLECULES AND USES THEREOF

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

VIA EFS-WEB

[0001] The content of the electronically submitted sequence listing (Name:

2159.2370001 New Sequence Listing.TXT; Size: 103,362 bytes; and Date of Creation: December 17. 2010) filed herewith with the application is incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

[0002] Tumor necrosis factor-alpha (TNFa), also called cachectin, is a pleiotropic cytokine with a broad range of biological activities including cytotoxicity, immune cell proliferationn, inflammation, tumorigenesis, and viral replication. Kim et al, J. Mol. Biol. 374. 1374 (2007). TNF-alpha is first produced as a transmembrane protein (tmTNF- alpha), which is then cleaved by a metal loproteinase to a soluble form (sTNFa). Wall is. Lancet Infect. Dis. 8(10): 601 (2008). TNF-alpha (-17 kDa) exists as a rigid homotrimeric molecule, which binds to cell-surface TNF Receptor 1 or TNF Receptor 2, inducing receptor oligomerization and signal transduction. See id.

[00031 TNF-rclated weak inducer of apoptosis (TWEAK), a TNF super family cytokine, stimulates the secretion of proinflammatory cytokines and chemokines from a variety of cell types. TWEAK is also reported to promote apoptosis and/or necrosis or stimulate angiogenesis. Chicheportiche et al., J Biol Chem. 272(51 ):32401 ( 1997): Ho et al, Cancer Res 64: 8968 (2004).

[0004] TNF-alpha blocking agents have been used to treat inflammatory disease or autoimmune disease such as inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, polyarticular course juvenile chronic arthritis (JCA), plaque psoriasis, or Crohn's disease. Examples of commercially available TNF-alpha blocking agents include etanercept, infliximab, certolizumab pegol, golimumab. and adalimumab. [00051 However. TNF-alpha blocking agents are not effective in all patients. About 30% of patients treated with a TNF inhibitor failed to achieve an improvement of 20% in American College of Rheumatology criteria (ACR20; primary failure or inefficacy). Rubbert-Roth and Finckh, Arthritis. Res. 1 1 (Suppl 1): SI (2009). In addition, more patients lose efficacy during therapy (secondary failure or acquired therapeutic resistance). Allez et ah, alimen. Pharm. Therap. 31 : 92-101 (2006). Furthermore, some patients using a TNF-alpha blocker suffer from unwanted side effects. The main adverse events are infections and malignancies such as serious and fatal blood disorders, tuberculosis reactivation, lymphoma, solid tissue cancers, liver injury, demyelinating CNS disorder, congestive heart failure, or chronic infection such as tuberculosis.

[0006] Therefore, there is a need in the art for additional or improved anti-TNF agents that can more effectively and/or safely block TNF-alpha mediated inflammatory responses.

[0007] One promising class of biologies that may improve treatment potency and efficacy are multivalent and bispecific binding molecules. These molecules, however, may suffer from instability and/or sub-optimal yields of dimerized antibody. Moreover, instability in the variable regions of some multivalent or bispecific antibodies may result in a variety of production problems, including one or more of: unsuitability for scale-up production in bioreactors (e.g. , because of low yield, significant levels of unwanted byproducts such as unassembled product, and/or aggregated material), difficulties in protein purification, and unsuitability for pharmaceutical preparation and use (e.g., owing to significant levels of breakdown product, poor product quality, and/or unfavorable pharmacokinetic properties).

SUMMARY OF THE INVENTION

[00081 The present invention is directed to a binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein said scFv polypeptide comprises a heavy chain variable domain (VH) at least 90% identical to SEQ ID NO: 9 and a light chain variable domain (VL) at least 90% identical to SEQ ID NO: 11, wherein said scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of: (1) Glycine (G) at Kabat position 16 in the VH, (2) Serine (S) at Kabat position 30 in the VI I. (3) Glycine (G) at Kabat position 49 in the VH, (4) Alanine (A) at Kabat position 49 in the VH, (5) Serine (S) at Kabat position 52 in the VH, (6) Threonine (T) at Kabat position 57 in the VH, (7) Proline (P) at Kabat position 61 in the VH, (8) Lysine (K) at Kabat position 64 in the VH, (9) Glycine (G) at Kabat position 70 in the VH, (10) Glutamine (Q) at Kabat position 77 in the VH, (11) Threonine (T) at Kabat position 77 in the VH, (12) Lysine (K) at Kabat position 77 in the VH, (13) Asparagine (N) at Kabat position 82b in the VH. (14) Proline (P) at Kabat position 84 in the VI I, (15) Proline (P) at Kabat position 3 in the VL, (16) Arginine (R) at Kabat position 54 in the VL. (17) Glutamic acid (E) at Kabat position 83 in the VL. and any combinations thereof.

[0009] The present invention is also directed to a binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein the scFv polypeptide comprises a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 1 1 ; except for one or more stabilizing mutations. In one embodiment, the stabilized anti-^'l NF-alpha scFv polypeptide further comprises a linker connecting the VH and the VL. In another embodiment, the linker comprises (Gly₄Ser)4 (SEQ ID NO: 32).

[0010] In another embodiment, a binding molecule of the present invention comprises a

VH having the amino acid sequence of SEQ ID NO: 9 and a VL hav ing the amino acid sequence f SEQ ID NO: 1 1 with one or more stabilizing mutations comprising amino acid substitutions in the VH at any combination of Kabat positions 16, 30, 49. 52, 57. 61. 64, 70, 77. 82b, and 84 or in the VL at any combination of Kabat positions 3, 54, and 83. For example, the amino acid substitutions in the VI I are selected from the group consisting of R 16G. D30S, S49G. S49A, T52S, I57T. D61 P. E64K, S70G, S77Q, S77T, S77K, S82bN, Λ84Ρ. and any combinations thereof, and the amino acid substitutions in the VL are selected from the group consisting of Q3P, I.54R, V83E, and any combinations thereof.

[0011 ] Also included is a binding molecule of the present invention comprising a VI I having the amino acid sequence of SEQ ID NO: 9 and a VL having the amino acid sequence of SEQ ID NO: 1 1 , with amino acid substitutions R16G and D30S in VI I and L54R and V83E in VL or alternatively with amino acid substitutions R16G, D30S, and T52S in VI I and L54R and V83E in VI, [0012] In one embodiment, a binding molecule of the present invention comprising at least one stabilized scFv polypeptide that specifically binds to TNF-alpha is characterized by an improved biophysical property compared to a scFv polypeptide without the one or more stabilizing mutations (i. e., conventional scFv polypeptide). The biophysical property is selected from the group consisting of thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, ligand binding stoichiometry, ligand binding affinity, and a combination of two or more of the biophysical properties. For example, the thermal stability of the stabilized anti-TNF-alpha scFv polypeptide is characterized by a melting temperature (Tm) o at least 60 °C, 65 °C, or 67°C as measured by differential scanning calorimetry. In another embodiment, a stabilized anti-TNF-alpha scFv polypeptide binds to human TNF-alpha with a dissociation constant of 150 pM or less or, at equilibrium, the binding stoichiometry of the binding molecule to a TNF-alpha trimer is 1 : 1 . In other embodiments, the binding molecule is incapable of forming complexes comprising two or more TNF-alpha trimers. In certain embodiments, the binding molecule binds rabbit TNF-alpha with a binding affinity greater than that of adalimumab.

10013] The invention also provides a binding molecule having multivalent binding specificity for TNF-alpha. In one embodiment, the binding molecule is multispecific or bispecific. For example, a binding molecule o the invention comprises a first binding region comprising the stabilized TNF-alpha speci fic scFv molecule and a second binding region which specifically binds to a molecule such as, but not limited to. TNF-alpha, TWEAK, and IL-6. In another embodiment, the second binding region that binds to TWEAK is an anti-TWEAK antibody which binds to the same epitope as hP2D10 or P5G9 or competitively inhibits binding of hP2D 10 or P5G9 to TWEAK. In certain embodiments. TWEAK binding regions are connected to the N-terminus or C-terminus of the heavy chain polypeptide or the light chain polypeptide of one or more of the stabilized anti-TNF-alpha scFv polypeptides. In certain embodiments, multispecific or bispecific binding molecules of the present invention inhibit TWEAK- or TNF-alpha induced IT- 8 release in the IL-8 release assay. In other embodiments, multispecific or bispecific binding molecules of the present invention simultaneously inhibit TWEAK and TNF- alpha activity in the WiDr MTT combo assay.

[0014] The binding molecule o the invention comprising a first binding region, which comprises the stabilized TNF-alpha specific scFv molecule, and a second binding region, which specifically binds to IL-6, can comprise MJH258 (SEQ ID NO: 49) in the heavy chain and MJH259 (SEQ ID NO: 5 1 ) in the light chain. A binding molecule specifically binds to IL-6 can be an IL-6 antagonist, e.g., IL-6 antibody.

[0015] The invention is also directed to a binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the scFv polypeptide comprises a heavy chain variable domain (VH) at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ I D NO: 13 (P2D10 VI I) and a light chain variable domain (VL) at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 5 ( P2D 10 VL). wherein said scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of: (i) Asparagine (N) at Kabat position 60 in the VI I, (ii) Lysine (K) at Kabat position 62 in the VH, (iii) Glutamic Acid (E) at Kabat position 12 in the VL. and any combinations thereof.

[00161 In one embodiment, a binding molecule comprises a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the scFv polypeptide comprises a heavy chain variable domain (VH). which comprises three complementarity domain regions (CDRs) o SEQ ID NO: 13, and a light chain variable domain (VL), which comprises three CDRs of SEQ ID NO: 1 5. except for one or more stabilizing mutations. For example, the amino acid substitutions in the VI 1 can comprise one or more amino acid substitution of P60N, T62K, or both. The stabilizing mutation in VL can comprise an amino acid substitution of P12E.

[0017] In certain embodiments, the present invention is directed to a nucleic acid molecule comprising a polynucleotide encoding the binding molecule of the present invention or a binding site or variable region thereof. In a particular embodiment, a nucleic acid molecule of the invention further comprises a promoter operably associated with the polynucleotide. The present invention is also directed to a vector comprising the nucleic acid molecule or a host cell comprising the vector.

[0018] In other embodiments, the present invention is directed to a pharmaceutical composition comprising (1) the binding molecule of the present invention. (2) the nucleic acid encoding the binding molecule. (3) the vector comprising the nucleic acid, and (4) the host cell comprising the vector, or a combination thereof. In certain embodiments, a pharmaceutical composition of the invention further comprises a heterologous binding molecule, e.g. , an i mm unomodu 1 at i ng agent. For example, the heterologous binding molecule for a binding molecule comprising the stabilized TNF-alpha scFv is an antibody specifically binding to e.g., TWEAK or 11.-6. The heterologous binding molecule comprising the stabilized TWEAK scFv is an antibody specifically binding to, e.g., TNF- alpha or II . -6. In one embodiment, the composition is administered once a week, once in two weeks, once in three weeks, once in four weeks, once in five weeks, once in 10 days, once in 20 days or once in 30 days. In another embodiment, the composition is administered subcutaneously, intravenously, intraarterially. intraperitoneal ly. intramuscularly, rectal ly. or vaginally.

[001 ] In certain embodiments, the composition of the present invention is administered to a subject who does not respond to or has developed resistance to one or more disease- modifying antirheumatic drugs (DMARDs), e.g. , methotrexate, or to at least one TNF- alpha antagonist. In other embodiments, the composition of the present invention is administered with one or more DMARDs. e.g. , methotrexate. In further embodiments, the pharmaceutical composition of the present invention is administered to a subject in need thereof to prevent, ameliorate, or treat a disease or disorder associated with an inflammatory response or autoimmune response. Non-limiting examples of diseases or disorders associated with an inflammatory response or autoimmune response include rheumatoid arthritis, juvenile ideopathic arthritis, psoriatic arthritis, ankylosing spondylitis. Crohn's disease, psoriasis, ulcerative colitis, or a combination of the diseases or disorders.

100201 Also provided is a method of producing the binding molecule comprising: (i) culturing the host cell comprising the nucleic acid encoding the binding molecule such that the binding molecule is secreted in host cell culture media and (ii) isolating the binding molecule from the media.

[0021] In certain embodiments, the present invention is directed to a method of reducing or inhibiting an activity of TNF-alpha or binding o TNF-alpha to a TNF-alpha receptor or an activity of TWEAK or binding of TWEAK to a TWEAK receptor in a subject in need thereof comprising administering to the subject (1) the binding molecule of the instant invention. (2) the nucleic acid encoding the binding molecule. (3) the vector comprising the nucleic acid. (4) the host cell comprising the nucleic acid, and (5) the composition comprising the binding molecule. The present invention is also directed to a method of preventing, ameliorating, or treating a disease or disorder associated with an inflammatory response or autoimmune response, e.g., rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, Sjogren's syndrome, inflammatory myositis, Langerhans-cell histiocytosis, adult respiratory distress s v ndro me/bro nc hi o 1 i t i s obliterans. Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, noninfectious sclcritis. chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, and a combination of two or more of said diseases or disorders. The method reduces one or more side effects compared to the side effects induced by administration of adalimumab or P2D10.

BRIEF DESCRIPTION OF THE DRAWINGS

10022] Figure 1 A-B: A Differential Scanning Calorimetry profiles of adalimumab

Fab fragment and purified pIEH-254 wild-type adalimumab scFv ("Adalimumab WT scFv"). B. Thermal challenge assay graph o adalimumab Fab fragment and pIHI 1-254 wild-type adalimumab scFv.

100231 Figure 2: Differential Scanning Calorimetry profiles o selected adalimumab scFv mutants, including the final quintuple mutant (VH R16G D30S T52S; VL L54R V83E).

100241 Figure 3. Solution binding data of TNF-alpha to the conventional adalimumab scFv and a stabilized anti-TNF-alpha scFv molecule expressed by pIEH298. The straight lines represent either the conventional or stabilized anti-TNF-alpha scFvs without h TNF- alpha. and the curved lines represent binding of h I^'NF-alpha to either the conventional or stabilized scFvs.

100251 Figure 4: Schematic diagram of the structure of an anti-TNF-alpha-TWEAK bispecific anti body of the invention.

|0()26| Figure 5A-D: Solution binding of XWU 198 TNF-TWEAK Bispecific Antibody

(A) and adalimumab (B) to 0.8 nM human TNF-alpha, as measured by solution affinity biacore analysis. Standard curve: straight line (triangle), TNF-alpha solution binding: curved lower line (square). Binding of XWU198 TNF-TWEA Bispecific Antibody (C) and hP2D10 anti-TWEAK antibody (D) to 0.8 nM human TWEAK, as measured by solution affinity biacore analysis. Standard curve: straight line (triangle), TWEAK solution binding: curved lower line (square).

100271 Figure 6A-B: Kinetic biacore analysis of sequential binding o 33 nM TNF- alpha and TWEAK to biacore chip-captured XWU 198 TNF-TWEAK BsAb, adahmumab and hP2D 10 anti-TWEAK antibody. TNF-alpha is bound first in A, while TWEAK is bound first in B.

[00281 Figure 7A-D: A. Kinetic biacore analysis of TNF-alpha binding, at varying concentrations (0, 1 .2. 3.7. 1 1 , 33. 100 nM) to chip-captured XWU198 TNF-TWEAK. B. Kinetic biacore analysis of TNF-alpha binding, at varying concentrations (0, 1.2. 3.7. 1 1. 33. 100 nM) to chip-captured Adalinuimab. C. Kinetic biacore analysis o TWEAK binding, at varying concentrations (0, 1 .2, 3.7. 1 1. 33, 100 nM) to chip-captured TNF- TWEAK. D. Kinetic biacore analysis of TWEAK binding, at varying concentrations (0, 1 .2. 3.7. 1 1 , 33, 100 nM) to chip-captured hP2D10 anti-TWEAK antibody.

[0029] Figure 8A-B. TNF-TWEAK and adahmumab binding stoichiometry determination by size exclusion chromatography/static light scattering (SEC/LS) analysis.

A. SEC/LS profile of 5.0 μΜ TNF-TWEAK in the absence (black, dotted line) or presence (black, solid line) of 10 μΜ TNF-alpha. 10 μΜ TNF-alpha alone is in grey. A model for the proposed mechanism of TNF-alpha binding to TNF-TWEAK is shown on the right. B. SEC/LS profile of 5.0 μΜ adahmumab in the absence (black, dotted line) or presence (black, solid line) of 10 μΜ TNF-alpha. 10 μΜ TNF-alpha alone is in grey. A model for the proposed mechanism of TNF-alpha binding to adahmumab is shown on the right.

[0030] Figure 9A-B. TNF-alpha binding stoichiometry determination of TNF-

TWEAK and adahmumab by biacore analysis. A. Graph showing moles of TNF-alpha trimer per mol XWU 198 TNF-TWEAK (squares) or adahmumab (triangles) versus amount of TNF-TWEAK or adahmumab captured on chip, in arbitrary resonance units.

B. Model for TNF-alpha binding to chip-captured TNF-TWEAK and adahmumab.

[0031] Figure 1 0Λ-Β. A. Schematic diagram of the binding stoichiometry of adahmumab to TNF-alpha in equilibrium, excess TNF-alpha state, and excess antibody state. B. Schematic diagram of binding stoichiometry of the anti-TNF-alpha/TWEAK bispecifie antibody binding to TNF-alpha trimer. [0032J Figure 1 1 : Sequence Alignment of TNF-alpha from mouse (Mus musculus) (SEQ

ID NO: 1), rat (Rattus norvegicus) (SEQ ID NO: 2), rabbit-short sequence (Oryctolagus cuniculus) (SEQ ID NO: 3), rabbit-long sequence {Oryctolagus cuniculus) (SEQ ID NO: 4), Cynomolgus monkey (Macaca fasciculari )( EQ ID NO: 5), and human (Homo sapiens) (SEQ ID NO: 6). The conserved primate TACE recognition site is highlighted. Cleavage occurs between the AV residues in bold.

[0033] Figure 12: Biacore solution affinity analysis of 0.8 nM rabbit and human TNF- alpha binding to XWU198 TNF-TWEAK BsAb (left) and adalimumab (right). The XWU198 and adalimumab standard curves are the straight lines (triangles), the rabbit TNF-alpha binding curves are indicated with circles, and the human TNF-alpha curves with squares.

[0034] Figure 13A-C. A. Test for XWU198 TNF-TWEAK. BsAb and adalimumab neutralization of murine TNF-alpha in WEHI cytostatic assay. Varying concentrations of TNF inhibitor (XWU198 TNF-TWEAK BsAb, adalimumab, control Ig, murine TNFR- Fc) were incubated with 5 pM murine TNF prior to incubation with WEHI cells. Percent survival was measured. B. Test for XWU 198 TNF-TWEAK BsAb and adalimumab neutralization of rabbit TNF-alpha in WiDr cytotoxicity assay. Varying concentrations of TNF inhibitor (XWU198 TNF-TWEAK BsAb. adalimumab. control Ig, murine TNFR- Fc) were incubated with 5 pM rabbit TNF prior to incubation with WiDr cells. Percent survival was measured. C. Test for XWU198 TNF-TWEAK BsAb and adalimumab neutralization of human TNF-alpha in WiDr cytotoxicity assay. Varying concentrations of TNF inhibitor (XWU198 TNF-TWEAK BsAb. adalimumab, control Ig. murine TNFR- Fc) were incubated with 5 pM human TNF prior to incubation with WiDr cells. Percent survival was measured.

1 0351 Figure 14Λ-Β: A. A graph showing inhibition of TNF-alpha (InM) induced II. -8 release in A375 cells by various treatments. Key: anti-TNF-alpha/TWEAK bispecific antibody (XWU198, diamond), adalimumab (big square), anti-TWEAK antibody (P2D10, triangle), adalimumab and anti-TWEAK antibody (P2D10) (X), control immunoglobulin (asterisk), liTNF-alpha (circle), and cells (small squares). B. A graph showing inhibition of TWEAK (InM) induced IL-8 release in A375 cells by anti-TNF-alpha/TWEAK bispecific antibody (XWU198, diamond), adalimumab (big square), anti-TWEAK antibody (P2D10, triangle), adalimumab and anti-TWEAK antibody (P2D10) (X), control immunoglobulin (asterisk), hTNF-alpha (circle), and cells (small squares).

[0036] Figure 15. A graph showing the effect of anti-TNF/TWEAK antibody treatment on TNF-alpha plus TWEAK-induced WiDr cell death. Key: anti-TWEAK antibody (diamond), adalimumab (big square), adalimumab and anti-TWEAK antibody (P2D10) (triangle), anti-TNF-alpha/TWEAK antibody (XWU198, X), control immunoglobulin (asterisk), hTNF-alpha and TWEAK (circle), TNF-alpha (triangle), and TWEAK (small square).

[0037] Figure 16. A. A. graph showing inhibition of TNF-alpha plus mFcTWEAK

(murine TWEAK Fc fusion protein) induced NF-κΒ activation in 293 cells stably expressing an NF-kB reporter gene by administration of various treatments. Key: anti- TNF-TWEAK bispecific antibody (XWU198, diamond), adalimumab (big square), anti- TWEAK antibody (triangle), adalimumab and p2D10 (X), and control immunoglobulin (asterisk).

[0038] Figure 17A-B. A: ADCC activity of an anti-TNF-alpha/TWEAK bispecific antibody using NK cells from donor 22. The lines represent (1) adalimumab, (2) an anti- TNF-alpha/TWEAK bispecific antibody (XWU198), (3) etanercept, (4) human IgGl, and (5) adalimumab Fab. B. ADCC activity of an anti-TNF-alpha/TWEAK bispecific antibody using NK cells from donor 88.

[0039] Figure 18. CDC activity of adalimumab, etanercept and an anti-TNF- alpha/TWEAK bispecific antibody (XWU198). The data is representative of three independent experiments. The lines represent (1) adalimumab, (2) an anti-TNF- alpha/TWEAK bispecific antibody (XWU198), (3) etanercept, (4) hlgGl, and (5) hP2D10.

[0040] Figure 19. Activity of a TNF-TWEAK bispecific antibody in Balb Oslo NF-KB- luciferease transgenic mice. The lines represent (1) TNF and FcTWEAK (diamond), (2) TNF, FcTWEAK. and an anti-TWEAK antibody (hP2D10)(square), (3) TNF, FcTWEAK, and adalimumab (triangle). (4) TNF, FcTWEAK, and XWU 198 (X).

[0041 ] Figure 20 A -F. TNF-alpha binding stoichiometry determination by SEC/LS analysis. A. SEC/LS profile of 5.0 μΜ adalimumab with 10 μΜ TNF-alpha. B. SEC/LS profile o 5.0 μΜ MJF 149 with 10 μΜ TNF-alpha. C. SEC/LS profile of 5.0 μΜ with 10μΜ TNF-alpha. D. SEC/LS profile of 5.0 μΜ MJF193 with 10μΜ TNF- alpha. E. SEC/LS profile of 5.0 μΜ XWU242 with ΙΟμΜ TNF-alpha. F. SEC/LS profile of 5.0 μΜ XWU243 with Ι ΟμΜ TNF-alpha.

[0042] Figure 21A-C. TWEAK binding stoichiometry determination by SEC/LS analysis. A. SEC/LS profile of 5.0 μΜ hP2D10 with 10μΜ TWEAK. B. SEC/LS profile of 5.0 μΜ SWU 198 with 10μΜ TWEAK. C. SEC/LS profile of 5.0 μΜ MJF149 with 10μΜ TWEAK.

[0043] Figure 22. Biacore Binding Assay showing binding stoichiometries of TNF and

TWEAK to various constructs.

[0044] Figure 23. Biacore Binding Assay showing TNF binding stoichiometry determination of TWEAK-TNF (circle). TNF-TWEAK (diamond), and HUMIRA® anti- TNF antibody (square).

[0045] Figure 24. Biacore Binding Analysis showing TWEAK binding stoichiometry determination of TWEAK-TNF (diamond), TNF-TWEAK (circle), and hP2D10 anti- TWEAK antibody (square).

[0046] Figure 25. Biacore Binding Assay showing biniding stoichimetries of TNF and

TWEAK to alternative anti-TNF scFv containing bispecific antibodies (MJF258 and MFJ260).

[0047] Figure 26. 293/NFkB luciferase assay comparing the anti-TNF inhibitory activity of anti-TWEAK-TNF bispecific antibody (TNF-mP5G9 (square)), anti-TNF/IL-6 bispecific antibody (triangle). BIIB040 (diamond) (i.e., XWU 198 TNF-TWEAK), adalimumab (Humira®) (cross) and other constructs. Antibodies at varying concentrations were incubated with 5pM TNF.

10048] Figure 27. 293/NFkB luciferase assay comparing the anti-TWEAK inhibitory activity of anti-TWEAK-TNF bispecific antibody (TNF-mP5G9 (square)). anti-TNF/IL-6 bispecific antibody (triangle). BIIB040 (diamond) (i.e., XWU 198 TNF-TWEAK), adalimumab (Humira®) (cross) and other constructs. Antibodies at varying concentrations were incubated with 133pM human Fc-TWEAK.

[0049] Figure 28. 293/NFkB luciferase assay comparing simultaneously the anti-

TWEAK and anti-TNF inhibitory activity of bispecific antibody TNF-mP5G9 (square), BIIB040 (diamond) (i.e., XWU198 TNF-TWEAK), adalimumab (Humira®) (cross), and other constructs. Antibodies at varying concentrations were incubated w ith 133pM human Fc-TWEAK and 5 pM TNF. 100501 Figure 29. Graph showing the effect of bispecific antibody constructs TNF- mP5G9 (square) and TNF/IL-6 (triangle) on TNF-alpha induced WiDr cell death. Cells were incubated with 20 pM TNF.

(00511 Figure 30. Graph showing the effect of bispecific antibody constructs TNF- mP5G9(square) on TWEAK-induced WiDr cell death. Cells were incubated with 200 pM hisTWEAK.

[00521 Figure 3 1 . Graph showing the effect of bispecific antibody construct TNF-mP5G9

(square) on WiDr cell induced by simultaneous incubation with 15 pM TNF and 150 pM

TWEAK.

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

[0053] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a scFv polypeptide," is understood to represent one or more scFv polypeptides. As such, the terms "a" (or "an"), "one or more." and "at least one" can be used interchangeably herein.

1 05 1 As used herein, the term "binding molecule^" refers to a molecule which binds

(e.g. , specifically binds or preferentially binds) to a target molecule of interest, e.g., an antigen. For example, in certain embodiments, a binding molecule of the invention is a polypeptide which speci fically or preferentially binds to at least one epitope of TNF-alpha or TWEAK. Binding molecules within the scope of the invention also include small molecules, nucleic acids, peptides, peptidomimetics, dendrimers, and other molecules within binding specificity for an epitope of TNF-alpha or TWEAK described herein.

[0055] As used herein, the term "polypeptide^" is intended to encompass a singular

"polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

100561 A polypeptide of the invention can be o a size of about 3 or more. 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1 ,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded, or linear. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g. , a serine residue or an asparagine residue.

[0057] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially puri fied by any suitable technique.

[0058] As used herein the term "derived from" a designated protein refers to the origin of the polypeptide. In one embodiment, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide is a variable region sequence (e.g. a VH or VL) or sequence related thereto (e.g. a CDR or framework region) derived from an immunoglobulin molecule or antibody. In one embodiment, the amino acid sequence which is derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from a starting antibody. In one embodiment, the polypeptide or amino acid sequence that is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence or a portion thereof, wherein the portion consists of at least 3-5 amino acids, 5- 10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.

[0059] Also included as binding molecules of the present invention are fragments or variants of polypeptides, and any combination thereof. The term "fragment" or "variant" when referring to polypeptide binding molecules f the present invention include any polypeptides which retain at least some of the binding properties of the corresponding molecule. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants occur naturally or are non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0060] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue in a polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in another embodiment, mutations are introduced randomly along all or part of the polypeptide. [0061] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix. Genetics Computer Group. University Research Park, 575 Science Drive. Madison, WI 5371 1). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence described herein, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0062] As used herein the term "valency" refers to the number of potential binding sites in a binding molecule. A binding molecule can be "monovalent" and have a single binding site or a binding molecule can be "multivalent" (e.g. , bivalent, trivalent, tetravalent, or greater valency). Each binding site specifically binds one target molecule or specific site on a target molecule (e.g. , an epitope). When a binding molecule comprises more than one target binding site (i.e. a multivalent binding molecule), each target binding site specifically binds the same or different molecules (e.g. , binds to different TNF-alpha molecules or to different epitopes on the same TNF-alpha molecule).

[0063] As used herein, the phrase "binding moiety", "binding site", "binding region," or

"binding domain" refers to the portion of a binding molecule that specifically binds to a target molecule of interest (e.g. , TNF-alpha or TWEAK). Exemplary binding domains include an antibody variable domain, a receptor binding domain of a ligand, a ligand binding domain of a receptor or an active site of an enzyme. For example, the binding region is an antibody or antigen binding fragments including, but not limited to, Fab, scFv, F(ab')2, or a polypeptide comprising at least one CDR from an antibody. The polypeptide comprising at least one CDR retains the ability to bind to the target that the antibody from which the CDR is derived binds. In one embodiment, the binding regions have at least one binding site specific for TNF-alpha or TWEAK. In another embodiment, a binding site has a single TNF-alpha or TWEAK binding moiety. In other embodiments, a binding site has two or more binding specificities. For example, a binding molecule has a single binding site having dual specificity.

'4] The term "binding specificity" or "specificity" refers to the ability of a binding molecule to specifically bind (e.g., immunoreact with) a given target molecule or epitope. In certain embodiments, the binding molecules of the invention comprise two or more binding specificities (i.e. , they bind two or more different epitopes present on one or more different antigens at the same time). In one embodiment, a binding molecule is "monospecific" and has a single binding specificity. In another embodiment, a binding molecule is "multispecific" (e.g. , bispecific or trispecific or of greater multispecificity) and has two or more binding specificities. In exemplary embodiments, the binding molecules of the invention are "bispecific" and comprise two binding specificities. Thus, whether a T NF-alpha or TWEAK binding molecule is "monospecific^" or "multispecific," e.g. , "bispecific," refers to the number of different epitopes with which a binding molecule reacts. In exemplary embodiments, multispecific binding molecules of the invention are specific for a TNF-alpha epitope and a TWEAK epitope.

5] In one embodiment, a binding molecule comprises a dual binding specificity. As used herein the phrase "dual binding specificity" or "dual specificity" refers to the ability of a binding molecule to specifically bind to one or more different epitopes. For example, in certain embodiments, a binding molecule comprises a binding specificity having at least one binding site which specifically binds two or more different epitopes (e.g. , two or more non-overlapping or discontinuous epitopes) on a target molecule. In other embodiments, a binding molecule specifically binds to two or more different targets. Accordingly, a binding molecule having a dual binding specificity is said to cross-react with two or more epitopes.

6] A given binding molecule of the invention can be monovalent or multivalent for a particular binding specificity. For example, when a TNF-alpha or TWEAK binding molecule is monospecific, the binding specificity comprises a single binding site which specifically binds an epitope (i.e. , a "monovalent monospecific" binding molecule) and such a binding molecule can be used in combination with a second binding molecule having at least one binding specificity for a different epitope of TNF-alpha or TWEAK, respectively. In one embodiment, the monospecific TNF-alpha or TWEAK binding molecule comprises two binding domains which specifically bind the same epitope. Such a binding molecule is bivalent and monospecific. In other embodiments, where a binding molecule is multispecific, one or more of its binding specificities comprise two or more binding domains which specifically bind the same epitope (i.e. , a "multivalent binding specificity"). For example, a bispecific molecule comprises a first binding specificity that is bivalent (i.e., two binding sites which bind a first epitope) and a second binding specificity which is bivalent (i.e. , two binding sites which bind a second, different epitope). In another embodiment, a bispecific molecule comprises a first binding specificity that is monovalent (i.e., one binding site which binds a first epitope) and a second binding specificity which is bivalent or monovalent. In certain embodiments, a binding molecule is multispecific and binds to two or more different targets.

[0067] Binding molecules disclosed herein are described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide (e.g. , TNF-alpha or TWEAK) that they recognize or specifically bind. The portion of a target polypeptide which specifically interacts with the binding site or moiety of a binding molecule is an "epitope," or an "antigenic determinant." In certain embodiments, a target polypeptide comprises a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

10068] By "specifically binds." it is generally meant that a binding molecule binds to an epitope via a binding site of the binding molecule (e.g. , antigen binding domain), and that the binding entails some complementarity between that binding site and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope, via the binding site, more readily than it would bind to an unrelated epitope. Where a binding molecule is multispecific, the binding molecule can specifically bind to a second epitope (i.e. , unrelated to the first epitope) via another binding site (e.g., antigen binding domain) of the binding molecule.

[0069] By "preferentially binds," it is meant that the binding molecule specifically binds to an epitope via a binding site more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such a binding molecule may cross-react with the related epitope. [0070] As used herein, the term "cross-reactivity" refers to the ability of a binding molecule specific for one epitope, to react with a second, related epitope; a measure of relatedness between two different antigenic substances. The term "cross-reactivity" also means the ability of a binding molecule that is specific for an epitope of an antigen derived from one species (e.g., human) to react with an epitope of the corresponding antigen from another species (e.g. , rabbit) when the antigens from the two different species are not identical. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.

[0071] For example, certain binding molecules have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g. , epitopes with at least 95%, at least 90%), at least 85%, at least 80%, at least 75%>, at least 70%, at least 65%, at least 60%, at least 55%), and at least 50%o identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%>, less than 90%, less than 85%), less than 80%o, less than 75%>, less than 70%o, less than 65%, less than 60%, less than 55%, and less than 50%> identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be deemed "highly specific" for a certain antigen or epitope, if it does not bind any other analog, ortholog, or homolog of that antigen or epitope.

[0072] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the binding site of a binding molecule. See, e.g., Harlow et al , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. "Affinity" can be expressed as a dissociation constant, i.e., a specific type of equilibrium constant that measures the propensity of an object (e.g. , ligand) to separate (dissociate) reversibly from another object (e.g. , receptor) in solution. Binding affinities include those with a dissociation constant or Kd less than 5 x 10^" M. 10^"2 M, 5 x 10^"3 M, 10^"3 M, 5 x 10^"4 M, 10^"4 M, 5 x 10^"5 M, 10^"5 M, 5 x 10^"6 M, 10^"6 M, 5 x 10^"7 M, lO^"7 M, 5 x 10^"8 M, 10^"8 M, 5 x 10^"9 M, 10^"9 M, 5 x 10^"10 M, 10^"10 M, 5 x 10^"" M, 10^"" M. 5 x 10^"12 M, 10^"12 M, 5 x 10^"13 M, 10^"13 M, 5 x 10^"14 M, 10^"14 M, 5 x 10^"15 M, or 10^" ¹⁵ M. [0073] As used herein, the term "avidity" refers to the overall stability of the complex between a population of binding molecules (e.g. , stabilized scFv polypeptides) and an antigen, that is, the functional combining strength of a binding molecule mixture with the antigen. See, e.g. , Harlow at pages 29-34. Avidity is related to both the affinity of individual binding molecules in the population with specific epitopes, and also the valencies of the binding molecules and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.

1 0741 In certain embodiments, the binding site of a binding molecule of the invention is an antigen binding site. An antigen binding site is formed by variable regions that vary from one polypeptide to another. In one embodiment, the polypeptides of the invention comprise at least two antigen binding sites. As used herein, the term "antigen binding site" includes a site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble form of an antigen). The antigen binding site includes an immunoglobulin heavy chain and light chain variable region and the binding site formed by these variable regions determines the specificity of the antibody. In one embodiment, an antigen binding site of the invention comprises at least one heavy or light chain CDR of an antibody molecule (e.g. , the sequence of which is known in the art or described herein). In another embodiment, an antigen binding site of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding site of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding site of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding site of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding site of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary binding sites comprising at least one CDR (e.g. , CDRs 1 -6 ) that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.

[0075] Binding molecules o the invention comprise framework and constant region amino acid sequences derived from a human amino acid sequence. However, in certain embodiments, binding polypeptides comprise framework and/or constant region sequences derived from another mammalian species. For example, binding molecules comprising murine sequences can be appropriate for certain applications. In one embodiment, a primate framework region (e.g., non-human primate), heavy chain portion, and/or hinge portion can be included in the subject binding molecules. In one embodiment, one or more murine amino acids are present in the framework region of a binding polypeptide, e.g. , a human or non-human primate framework amino acid sequence comprises one or more amino acid back mutations in which the corresponding murine amino acid residue is present and/or can comprise one or mutations to a different amino acid residue not found in the starting murine antibody.

[0076] A "fusion" or chimeric protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0077] The term "heterologous" as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a gcnotypically distinct entity from that of the entity to which it is being compared. For instance, a heterologous polynucleotide or antigen can be derived from a different species, different cell type of an individual, or the same or different type of cell of distinct individuals.

[0078] The term "receptor binding domain" or "receptor binding portion" as used herein refers to any native ligand or any region or derivative thereof retaining at least a qualitative receptor binding ability, and/or the biological activity of a corresponding native ligand.

[0079] The terms "antibody" and "immunoglobulin" are used interchangeably herein. An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). [0080] As will be discussed in more detail below, the term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g. , γ1 -γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g. , IgGl , IgG2. IgG3, IgG4, IgAl , etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

100811 Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be paired with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other via disulfide linkages, and the "tail" portions of the two heavy chains are bonded to each other by c ova lent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0082] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VI I) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI , CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and the C- terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0083] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody (e.g. , in some instances a CH3 domain) combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. In one embodiment, the antigen binding site is defined by three CDRs on each f the VI I and VL chains. In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule consists of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0084] As used herein the term "variable region CDR amino acid residues" includes amino acids in a CDR or complementarity determining region as identified using sequence or structure based methods. As used herein, the term "CDR" or "complementarity determining region" refers to the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions hav been described by Kabat et: al., J. Biol. Chem. 252, 6609-6616 ( 1 77) and Kabat et al., Sequences o protein of immunological interest. (1991), and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum et al, J. Mol. Biol. 262:732-745 ( 1996) where the definitions include overlapping or subsets f amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in Table 1 for comparison. In certain embodiments, the term "CDR" is a CDR as defined by Kabat based on sequence comparisons.

Table 1 : CDR Definitions

CDR Definitions

Kabat¹ Chothia² MacCallum³

VH CDRI 31 -35 26-32 30-35

V„ CDR2 50-65 53-55 47-58 V_H CDR3 95- 102 96- 1 01 93- 101

VL CDRI 24-34 26-32 30-36

V, CDR2 50-56 50-52 46-55

V_L CDR3 89-97 91 -96 89-96

Residue numbering follows the nomenclature of Kabat et al., supra

2Residue numbering follows the nomenclature of Chothia et al., supra

3Residue numbering follows the nomenclature of MacCallum et al, supra

[0085] As used herein the term "variable region framework (FR) amino acid residues" refers to those amino acids in the framework region of an Ig chain. The term "framework region" or "FR region^" as used herein, includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g. , using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs.

[0086] As used herein, the term "Fc domain" refers to the portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 1 14) and ending at the C-terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0087] As used herein, the term "Fc domain portion" includes amino acid sequences derived from an Fc domain. A polypeptide comprising a Fc domain portion comprises at least one of: a hinge (e.g. , upper, middle, and/or lower hinge region) domain, a CH2 domain, a CI 13 domain, a CH4 domain, or a variant or fragment thereof. In one embodiment, a polypeptide of the invention comprises at least one Fc domain comprising at least a portion of a hinge domain, and a CH2 domain. In another embodiment, a polypeptide of the invention comprises at least one Fc domain comprising a CH I domain and a CH3 domain. In another embodiment, a polypeptide of the invention comprises at least one Fc domain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain. In another embodiment, a polypeptide of the invention comprises at least one Fc domain comprising a CH3 domain. In one embodiment, a polypeptide of the invention comprises at least one Fc domain which lacks at least a portion of a CH2 domain (e.g. , all or part of a CH2 domain). As set forth herein, it will be understood by one of ordinary skill in the art that any Fc domain can be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule.

[0088] The Fc domains of a polypeptide of the invention can be derived from different immunoglobulin molecules. In one example, an Fc domain of a polypeptide comprises a CHI domain derived from an IgGl molecule and a chimeric hinge region derived from an IgG3 molecule. In another example, an Fc domain can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an lgG4 molecule.

[0089] Exemplary binding molecules include or comprise, for example, polyclonal, monoclonal, multispecitic. human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')₂, Fd, Fvs, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g. , anti-Id antibodies to TNF-alpha antibodies or TWEAK antibodies disclosed herein). ScFv molecules are known in the art and are described, e.g. , in US patent 5,892,019. Binding molecules of the invention which comprise an Ig heavy chain constant region can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG 3. IgG 4. IgAl and IgA2) or subclass of immunoglobulin molecule.

[0090] As used herein the term "scFv polypeptide" or "scFv molecule" includes binding molecules which consist of one light chain variable domain (VL) or portion thereof, and one heavy chain variable domain (VI I) or portion thereof, wherein each variable domain (or portion thereof) is derived from the same or different antibodies. scFv molecules optionally comprise an scFv linker interposed between the VI I domain and the VL domain. scFv molecules are known in the art and are described, e.g., in US patent 5,892,019, Flo et al. 1989. Gene 77:51 ; Bird et al, 1988 Science 242:423; Pantoliano et al. 1991. Biochemistry 30: 101 17; Milenic et al. 1991. Cancer Research 51 :6363; Takkinen et al. 1991. Protein Engineering 4:837. The VL and VH domains of an scFv molecule are derived from one or more antibody molecules. It will also be understood by one of ordinary skill in the art that the variable regions of the scFv molecules of the invention can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived. For example, in one embodiment, nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues). Alternatively or in addition, mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. The binding molecules of the invention maintain the ability to bind to a given antigen, e.g., TNF-alpha or TWEAK.

[00 11 A "scFv linker" as used herein refers to a moiety interposed between the VL and

VH domains of the scFv. ScFv linkers may maintain the scFv molecule in a antigen binding conformation. In one embodiment, an scFv linker comprises or consists of an scFv linker peptide. In certain embodiments, an scFv linker peptide comprises or consists of a Gly-Ser connecting peptide. In other embodiments, an scFv linker comprises a disulfide bond.

10092] As used herein, the phrase "Gly-Ser connecting peptide^" refers to a peptide that consists of Glycine and Serine residues. An exemplary Gly/Ser connecting peptide comprises the amino acid sequence (Gly₄ Ser)„ (SEQ ID NO: 28) In various embodiments, n can be any integer, e.g., 1 , 2, 3, 4, 5, or 6. Another exemplary Gly/Ser connecting peptide comprises the amino acid sequence Ser(Gly₄Ser)_n (SEQ ID NO: 29), also indicated as S(G₄S)_n.

1 0931 As used herein the term "disulfide bond^" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CHI and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229. EU numbering system).

[0094] As used herein the term "conventional scFv molecule" refers to a scFv molecule which is not stabilized. In one embodiment, a conventional scFv molecule lacks stabilizing mutations described herein as compared to a non-conventional or stabilized scFv molecule. Examples of conventional scFv molecules used herein include, but are not limited to, a scFv polypeptide comprising the adalimumab VH, the adalimumab VL, and a (G₄S)₃ linker interposed between the VH and VL.

[0095] A "stabilized scFv polypeptide" of the invention is an scFv molecule or polypeptide comprising at least one change or alteration as compared to a conventional scFv molecule which results in stabilization of the scFv molecule. As used herein, the term "stabilizing mutation" includes a mutation which confers enhanced protein stability (e.g. thermal stability) to the scFv molecule and/or to a larger protein comprising the scFv molecule. In one embodiment, the stabilizing mutation comprises the substitution of a destabilizing amino acid with a replacement amino acid that confers enhanced protein stability (herein a "stabilizing amino acid"). In one embodiment, a stabilized scFv molecule of the invention comprises one or more amino acid substitutions. For example, in one embodiment, a stabilizing mutation comprises a substitution of at least one amino acid residue which substitution results in an increase in stability o the VH and VI , interface of an scFv molecule. In one embodiment, the amino acid is within the interface. In another embodiment, the amino acid is one which scaffolds the interaction between VFI and VL. In another embodiment, a stabilizing mutation comprises substituting at least one amino acid in the VII domain or VL domain that covaries with two or more amino acids at the interface between the VH and VL domains. In another embodiment, the stabilizing mutation is one in which at least one cysteine residue is introduced (i.e. , is engineered into one or more of the VH or VL domain) such that the VI I and VL domains are linked by at least one disulfide bond between an amino acid in the VH and an amino acid in the VL domain. In certain embodiments, a stabilized scFv molecule of the invention is one in which both the length of the scFv linker is optimized and at least one amino acid residue is substituted and/or the VH and VL domains are linked by a disulfide bond between an amino acid in the VI I and an amino acid in the VL domain. In one embodiment, one or more stabilizing mutations made to an scFv molecule simultaneously improve the thermal stability of both the VH and VL domains of the scFv molecule as compared to a conventional scFv molecule. In another embodiment, the stabilized scFv molecules of the population comprise the same stabilizing mutation or a combination of stabilizing mutations. In other embodiments, the individual stabilized scFv molecules of the population comprise different stabilizing mutations. Exemplary methods of preparing stabilized scFv molecules are described in U.S. Patent Application Publication No. 2008/0050370, which is incorporated herein by reference in its entirety.

As used herein the term "protein stability" refers to an art-recognized measure of the maintenance of one or more physical properties of a protein in response to an environmental condition (e.g. an elevated or lowered temperature). In one embodiment, the physical property is the maintenance of the covalent structure of the protein (e.g. , the absence of proteolytic cleavage, denaturation, unwanted oxidation or deamidation). In another embodiment, the physical property is the presence of the protein in a properly folded state (e.g., the absence of soluble or insoluble aggregates or precipitates). In one embodiment, stability of a protein is measured by assaying a biophysical property of the protein, for example thermal stability, pH unfolding profile, stable removal of glycosylation, solubility, biochemical function (e.g. , ability to bind to a protein (e.g., a ligand, a receptor, an antigen, etc.) or chemical moiety, etc.), and/or combinations thereof. In another embodiment, biochemical function is demonstrated by the binding affinity of an interaction. In one embodiment, a measure of protein stability is thermal stability, i.e. , resistance to thermal challenge. Stability can be measured using methods known in the art and/or described herein.

[0097] As used herein the term "properly folded polypeptide" includes polypeptides (e.g., anti-TNF-alpha or TWEAK scFv molecules) in which all of the functional domains comprising the polypeptide are distinctly active. As used herein, the term "improperly folded polypeptide" includes polypeptides in which at least one of the functional domains of the polypeptide is not active. In one embodiment, a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.

[00981 The term "polynucleotide" or "nucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g. , messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide comprises a conventional phosphodiester bond or a non- conventional bond (e.g. , an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g. , DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding an TNF-alpha or TWEAK binding molecule contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0099] As used herein, a "coding region" is a portion of nucleic acid molecule which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA. or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g. , on a single vector, or in separate polynucleotide constructs, e.g. , on separate (different) vectors. Furthermore, a vector can contain a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an TNF- alpha or TWEAK binding molecule or fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

1 010 1 In certain embodiments, the polynucleotide or nucleic acid molecule is a DNA molecule. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of niRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[00101] A variety of transcription control regions arc known to those skilled in the art.

These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin. heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as iymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

1001021 Similarly, a variety of translation control elements are known to those of ordinary- skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons. and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[001031 In other embodiments, a polynucleotide of the present invention is an RNA molecule, for example, in the form of messenger RN A (mRNA).

[00104] Polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion o a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. In one embodiment, a wild-type leader sequence is substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse β-glucuronidase.

[00105] As used herein the term "engineered^" with reference to nucleic acid or polypeptide molecules refers to such molecules manipulated by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).

[00106] As used herein, the term "linked," "fused" or "fusion" is used interchangeably.

These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co- translated as part o a continuous polypeptide.

[00107] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. [00108] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g. , polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0 10 1 As used herein, the terms "treat" or "treatment" refer to therapeutic treatment, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of inflammatory synovitis. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need o treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. Other uses include prophylactic and preventative measures.

[00110] By "subject" or "individual" or "animal" or "patient" or "mammal." is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, e.g. , humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on.

[00111] As used herein, phrases such as "a subject that would benefit from administration of a binding molecule" and "an animal in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g. , for a diagnostic procedure) and/or from treatment, i.e., palliation or prevention of a disease such as rheumatoid arthritis, with a binding molecule which specifically binds a given target protein. As described in more detail herein, the binding molecule can be used in unconjugated form or can be conjugated, e.g. , to a drug, prodrug, or an isotope.

[00112] The phrase "inflammatory disease or disorder" means a disease or condition caused by or manifested as biological responses of tissues to harmful stimuli such as pathogens, damaged cells, or irritants. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissues. Prolonged inflammation, known as chronic inflammation, leads to a progressive shi t in the type of cells which are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

[00113] The phrase "autoimmune disease or disorder" as used herein means an overactive immune response of a body against substances and tissues normally present in the body.

[00114] Examples of inflammatory or autoimmune diseases, disorders, conditions and/or responses include, but are not limited to. rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, Sjogren's syndrome, inflammatory myositis, Langerhans-cell histiocytosis, adult respiratory distress syndrome/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, noninfectious scleritis. chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, and a combination of two or more of said diseases or disorders.

II. ANTI- TNF-ALPHA ANTIBODY OR ANTI-TWEAK ANTIBODY

(A) ANTI-TNF-ALPHA ANTIBODY [0100] Human TNF-alpha (abbreviated herein as hTNF-alpha, or simply hTNF) is a human cytokine that exists as a 17 kD soluble form (sTNF-alpha) and a 26 kD membrane associated form (tmTNF-alpha), the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNF-alpha is described for example, in Pennica, D., et al. (1984) Nature 572:724-729: Davis, J. M., et al. (1987) Biochemistry 26: 1322-1326; and Jones, E. Y.. et al. (1989) Nature 338:225- 228, which are incorporated herein by reference in their entireties. TNF-alpha may bind to TNF-receptor type 1 (TNFR- 1 ) or TNF-receptor ty pe 2 (TNFR-2 ) and is involved in regulating immune cells, inducing apoptosis or inflammation, or inhibiting tumorigenesis or viral replication. The cell signaling cascades produced by TNF/TNFR binding are described, e.g. , in Wajant, I I.. et al. (2003) Cell Death Differ, 10(1 ): 45-65 or Chen, G., et al. (2002) Science 296: 1634-5. The term human TNF-alpha is intended to include recombinant human TNF-alpha (rhTNF-alpha), which can be prepared by standard recombinant expression methods or purchased commercially (e.g. , R & D Systems, Catalog No. 210-TA, Minneapolis, Minn.). The amino acid sequence and accession number in Genbank of TNF-alpha from human, rat, murine, and rabbit are given in the examples and an alignment of the sequences is in Figure 1 1.

[0101 j The sequence alignment of T F-alpha from human, mouse, rat. rabbit, and cynomolgus monkey is shown in Figure 1 1. The TNF-alpha binding molecule of the present invention comprises a binding region which specifically binds to TNF-alpha. in particular human TNF-alpha, and is derived from adalimumab, which is described in U.S. Patent Nos. 6,090,382; 6,258,562; 6,509,015, and 7,223,394 and in U.S. Application Publication Nos. and 2003/0219438 A l and 2007/0249813 Al , all of which are incorporated herein by reference in their entireties.

[ 1021 Adalimumab is a fully-human anti-TNF-alpha antibody (also known as D2E7 or

HU M IRA ^H ) containing Immunoglobulin gamma- 1 constant region. Adalimumab has 1330 amino acids with molecule weight of 148 kDa. The amino acid and nucleotide sequences of the heavy and light chain variable domains of adalimumab are shown below: TABLE 2: REFERENCE VH-CDRl, VH-CDR2, AND VH-CDR3 SEQUENCES OF

ADALIMUMAB *

* The nucleotide sequence encoding the adalimumab VH is listed as SEQ ID NO: 8.

** The nucleotide sequence encoding the adalimumab VL is listed as SEQ ID NO: 10.

(B) ANTI-TWEAK ANTIBODY

[0103] TWEAK is a member of TNF superfamily, which also includes TNF-alpha.

TWEAK was isolated in a screen for RNA that hybridized to an erythropoietin probe. Chicheportiche et al, J. Biol. ( hem. 272:32401 -32410 ( 1 97). The mouse and human peptides have an unusually high degree of conservation, including 93% amino acid identity in the receptor binding domain. TWEAK has been shown to be efficiently secreted from cells and it is abundantly expressed in many tissues, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, lymph nodes, thymus, appendix, and peripheral blood lymphocytes. [0104J One known TWEAK receptor is Fnl4, which is known to be encoded by a growth factor-regulated immediate-early response gene that decreases cellular adhesion to the extracellular matrix and reduces serum-stimulated growth and migration (Meighan- Mantha et al. , J. Biol. Chem. 274:33166-33176 (1999)).

[0105J TWEAK has been implicated in many biological processes. In some cases, blocking TWEAK using a TWEAK antagonist can treat inflammatory disorders, e.g., an arthritic disorder, e.g., rheumatoid arthritis, psoriatic arthritis, or Sjogren's Syndrome. See US 2008/0187544 Al, which is incorporated herein by reference in its entirety. In other cases, targeting the TWEAK pathway may treat cancer, e.g., a carcinoma, e.g., an adenocarcinoma such as a pancreatic adenocarcinoma. See US 2008/0279853A1 , which is incorporated herein by reference in its entirety.

[0106] An example of TWEAK antagonists is an anti-TWEAK antibody or antigen binding fragment thereof such as P2D10 or P5G9. The amino acid sequence and nucleotide sequence of P2D10 is shown in US 2008/0279853 Al, which is incorporated herein by reference in its entirety. The P2D10 amino acid sequence is reproduced in Table 3.

TABLE 3

REFERENCE CDR1, CDR2, AND CDR3 SEQUENCES OF THE

VH AND VL DOMAINS OF hP2D10

* The nucleotide sequence encoding the hP2D10 VI I is listed as SEQ ID NO: 12.

** The nucleotide sequence encoding the hP2D10 VL is listed as SEQ ID NO: 14. III. STABILIZED ANTI-TNF- ALPHA scFv MOLECULES OR ANTI-

TWEAK scFv MOLECULES

(A) STABILIZED AN TI-TNF-ALPHA scFv MOLECULE

[0107] The present invention is directed to a binding molecule comprising a binding region, which specifically binds to human I F -alpha, wherein the binding region comprises a VH domain and/or a VL domain derived from an anti-TNF-alpha antibody, i.e., adalimumab. In one embodiment, a binding molecule of the present invention comprises a binding region comprising the adalimumab VH domain except one or more stabilizing mutations. In another embodiment, a binding molecule of the invention comprises a binding region comprising the adalimumab VL domain except one or more stabilizing mutations. In other embodiments, a binding molecule comprising the adalimumab VH domain except one or more stabilizing mutations or the adalimumab VL domain except one or more stabilizing mutations further comprises a VL domain or a VH domain, respectively, wherein the binding molecule specifically or preferentially binds to TNF-alpha.

[0108] In some embodiments, the binding molecule of the present invention acts to antagonize TNF-alpha activity. In certain embodiments, for example, a binding molecule of the present invention as administered to a host has at least one of the following activities: inhibits binding of TNF-alpha to TNF-alpha receptor I (TNF receptor type 1, also known as CD 120a, p55/60). TNF-alpha receptor II (TNF receptor type 2, also known as CD 120b. p75/80), r both: inhibits activation of the NF-κΒ pathway and/or MAPK pathway, and/or inhibits TNF-alpha induced cell death.

[0109] In one embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein said scFv polypeptide comprises a heavy chain variable domain (VH) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to adalimumab VH (SEQ ID NO: 9) and a light chain variable domain (VL) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to adalimumab VL (SEQ I NO: 11), wherein the scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of: (1) Glycine (G) at Kabat position 16 in the VH, (2) Serine (S) at Kabat position 30 in the VH, (3) Glycine (G) at Kabat position 49 in the VH, (4) Alanine (A) at Kabat position 49 in the VH. (5) Serine (S) at Kabat position 52 in the VH. (6) Threonine (T) at Kabat position 57 in the VH, (7) Proline (P) at Kabat position 61 in the VI I. (8) Lysine (K) at Kabat position 64 in the VH, (9) Glycine (G) at Kabat position 70 in the VH, (10) Glutamine (Q) at Kabat position 77 in the VH, (11) Threonine (T) at Kabat position 77 in the VH, (12) Lysine (K) at Kabat position 77 in the VH, (13) Asparagine (N) at Kabat position 82b in the VH, (14) Proline (P) at Kabat position 84 in the VH, (15) Proline (P) at Kabat position 3 in the VL, (16) Arginine (R) at Kabat position 54 in the VL, (17) Glutamic acid (E) at Kabat position 83 in the VL, and any combinations thereof. In one embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein said scFv polypeptide comprises a VH at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 9 and a VL at least 95% identical to VL, wherein said scFv polypeptide comprises stabilizing amino acid substitutions Glycine (G) at Kabat position 16 in the VH, Serine (S) at Kabat position 30 in the VI 1, Arginine (R) at Kabat position 54 in the VL, and Glutamic acid (E) at Kabat position 83 in the VL. In another embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein said scFv polypeptide comprises a VH at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 9 and a VL at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to VL, wherein said scFv polypeptide comprises stabilizing amino acid substitutions Glycine (G) at Kabat position 16 in the VH, Serine (S) at Kabat position 30 in the VH. Serine at Kabat position 52 in the VH, Arginine (R) at Kabat position 54 in the VL, and Glutamic acid (E) at Kabat position 83 in the VL.

In certain embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein the scFv polypeptide comprises a heavy chain variable domain (VH), which comprises at least one, two or three complementarity domain regions (CDRs) of adalimumab VH (SEQ ID NO: 9), and/or a light chain variable domain ( VL). which comprises at least one. two, or three CDRs of adalimumab VL (SEQ ID NO: 1 1 ), except for one or more stabilizing mutations in the CDRs selected from the group consisting of T52S in the VH. 157 f in the VH. D61 P in the VH. E64K in the VH. and L54R in the VL. In other embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the scFv polypeptide comprises a VH having three CDRs of adalimumab VFI and/or the scFv polypeptide comprises a VL having three CDRs of adalimumab VL, except one or more stabilizing mutations in the CDRs selected from the group consisting of T52S in the VH, I57T in the VH, D61P in the VH, E64K in the VH, and L54R in the VL. In other embodiments, a binding molecule of the present invention further comprises stabilizing amino acid substitutions in the framework regions, wherein the amino acid substitutions are selected from the group consisting of amino acid Glycine (G) at Kabat position 16 in the VH, amino acid Serine (S) at Kabat position 30 in the VH, amino acid Glycine (G) at Kabat position 49 in the VH, amino acid Alanine (A) at Kabat position 49 in the VH, amino acid Glycine (G) at Kabat position 70 in the VH, amino acid Glutamine (Q) at Kabat position 77 in the VI I. amino acid Threonine (T) at Kabat position 77 in the VH, amino acid Lysine (K) at Kabat position 77 in the VH, amino acid Asparagine (N) at Kabat position 82b in the VH, amino acid Proline (P) at Kabat position 84 in the VH, amino acid Proline (P) at Kabat position 3 in the VL. amino acid Glutamic acid (E) at Kabat position 83 in the VI, and any combinations thereof.

[0111] In certain embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the scFv polypeptide comprises a VH comprising CDR1 , CDR2, and CDR3 of adalimumab VH and a VL comprising CDR1 , CDR2, and CDR3 of adalimumab VL, except that amino acid Leucine (L) at Kabat position 54 is replaced with Arginine (R). In other embodiments, the binding molecule further comprises a stabilizing mutation in CDR2 of the VH domain, wherein amino acid Threonine (T) at Kabat position 52 is replaced with Serine (S). In still other embodiments, the binding molecule further comprises stabilizing amino acids in the framework region, e.g.. Glycine (G) at Kabat position 16 in the VH, Serine at Kabat position 30 in the VH, and Glutamic acid (E) at Kabat position 83 in the VL.

[0112] In one embodiment, a binding molecule of the present invention comprises a stabilized anti-TNF-alpha scFv molecule having the adalimumab VH with one or more stabilizing mutations and the adalimumab VL. In another embodiment, a binding molecule of the present invention comprises a stabilized anti-TNF-alpha scFv molecule having the adalimumab VL with one or more stabilizing mutations and the adalimumab VI I. The binding molecule specifically or preferentially binds to TNF-alpha. In other embodiments, the invention provides a binding molecule comprising the adalimumab VH domain with one or more stabilizing mutations and the adalimumab VL domain with one or more stabilizing mutations, wherein the binding molecule specifically or preferentially binds to TNF-alpha. In certain embodiments, the binding molecule further comprises a scFv linker.

[0113] In one embodiment, a binding molecule of the invention is a single chain TNF- alpha binding molecule (e.g. , a scFv). Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al, Nature 334:544- 554 (1989)) can be adapted to produce single chain binding molecules. The DNA sequence and amino acid sequence f a scFv molecule constructed from 1)2 H 7 (adalimumab) are presented as SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

[0114] In another embodiment, a binding molecule of the present invention is a stabilized anti-TNF-alpha scFv molecule. In other embodiments, the stabilized anti-TNF-alpha scFv molecules of the invention comprise a VH or VL domain having at least one stabilizing amino acid mutation(s) or substitution(s).

[01 15 j In certain embodiments, a binding molecule of the invention is a stabilized anti-

TNF-alpha scFv molecule comprising the adalimumab VH domain with one or more stabilizing mutations and/or the adalimumab VL domain with one or more stabilizing mutations, wherein the stabilized scFv molecule specifically or preferentially binds to TNF-alpha.

[0116] A stabilized anti-TNF-alpha scFv molecule o the invention comprises a VI I or

VL domain with an optional scFv linker and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 1 1 , or at least 12 stabilizing amino acid mutation(s) or substitution(s) in either the VH or VL domain or both. In one embodiment, a stabilized anti-TNF-alpha scFv molecule comprises the adalimumab VH or VL domain with a single mutation (mutant) or double, triple, quadruple, quintuple (or pentuplc). sextuple (or hextuple), septuple, octuple, nonuple, decuple, hendecuple (or undecuple), or duodecuple mutations (or mutants). In one embodiment, the stabilizing mutations or substitutions in the VH domain of the scFv molecule of the present invention are at amino acid 16, amino acid 30, amino acid 49, amino acid 52, amino acid 57, amino acid 61 , amino acid 64, amino acid 70, amino acid 77, amino acid 82b, amino acid 84, or any combinations thereof, according to the Kabat numbering system. In other embodiments, the stabilizing mutations or substitutions in the VL domain of the scFv molecule of the present invention are at amino acid 3, amino acid 54, amino acid 83 or any combination thereof, according to the Kabat numbering system.

[0117] In one embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the anti-TNF -alpha scFv polypeptide comprises an adalimumab VH and an adalimumab VL except for one or more stabilizing substitutions or mutations at (i) amino acid substitution at Kabat position 16 from Arginine(R) to Glycine (G) of VH (R16G), (ii) amino acid substitution at Kabat position 30 from Aspartic acid (D) to Serine (S) of VH (D30S), (iii) amino acid substitution at Kabat position 49 from Serine (S) to Glycine (G) of VH (S49G), (iv) amino acid substitution at Kabat position 49 from Serine (S) to Alanine (A) of VH (S49A), (v) amino acid substitution at Kabat position 52 from Threonine (T) to Serine (S) of VI I (T52S), (vi) amino acid substitution at Kabat position 57 from Isoleucine (I) to Threonine (T) of VH (157T), (vii) amino acid substitution at Kabat position 61 from Aspartic acid (D) to Proline (P) of VH (D61P), (viii) amino acid substitution at Kabat position 64 from Glutamic acid (E) to Lysine (K) of VI 1 (E64K), (ix) amino acid substitution at Kabat position 70 from Serine (S) to Glycine (G) of VH (S70G), (x) amino acid substitution at Kabat position 77 from Serine (S) to Glutamine (Q) of VH (S77Q), (xi) amino acid substitution at Kabat position 77 from Serine (S) to Threonine (T) of VH (S77T), (xii) amino acid substitution at Kabat position 77 from Serine (S) to Lysine (K) of VH (S77K), (xiii) amino acid substitution at Kabat position 82b from Serine (S) to Asparagine (N) of VH (S82bN), (xiv) amino acid substitution at Kabat position 84 from Alanine (A) to Proline (P) of VH (A84P) or any combinations thereof.

[0118] In another embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the anti-TNF-alpha scFv polypeptide comprises an adalimumab VH and an adalimumab VL except for one or more stabilizing substitutions or mutations at (i) amino acid substitution at Kabat position 3 from Glutamine (Q) to Proline (P) of VL (Q3P); (ii) amino acid substitution at Kabat position 54 from Leucine (L) to Arginine (R) of VL (L54R); (iii) amino acid substitution at Kabat position 83 from Valine (V) to Glutamic acid (E) of VL (V83E); or any combinations thereof. ] In certain embodiments, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein the anti-TNF-alpha scFv polypeptide comprises an adalimumab VH and an adalimumab VL except for one or more stabilizing substitutions or mutations at (i) S77Q in VH and I57T in VH; (ii) R16G in VH and S70G in VH; (iii) R16G in VH and D30S in VH; (iv) T52S in VH and V83E in VL; (v) S77T in VH and V83E in VL; (vi) S77K in VH and V83E in VL; (vii) R16G in VI I. S70G in VI I. and V83 in VL; (viii) S49A in VH and V83E in VL: (ix) T52S in VH, L54R in VL. and V83E in VL; (x) R16G in VH. D30S in VH. L54R in VL, V83E in VL; (xi) S49A in VH, L54R in VL, and V83E in VL; (xii) R16G in VH, D30S in VH. S77T in VH. I.54R in VL, and V83E in VL; (xiii) R 16G in VI I. D30S in VH. E64 in VH. L54R in VL. and V83E in VL, (xiv) R16G in VI I. D30S in VI I. A84P in VH. L54R in VL. and V83E in VL. (xv) R16G in VH, 03 OS in VH, T52S in VI I. L54R in VL, and V83E in VL, or any combinations thereof, according to the Kabat numbering system.

] In one embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein the scFv polypeptide comprises an adalimumab VH and an adalimumab VL except for amino acid substitutions R 16G in VH. D30S in VH. L54R in VL. and V83E in VL (also referred to herein as "the quadruple mutant or XWU199). In another embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the scFv polypeptide comprises an adalimumab VI I and an adalimumab VL except for amino acid substitutions R16G in VH. D30S in VI I. T52S in VH. L54R in VL. and V83E in VL (also referred to herein as "the quintuple mutant" or XWU198). The nucleic acid and amino acid sequences of XWU199 are presented as SEQ II) NOs: 20 (VI I) and 22 (VL) and SEQ ID NOs: 21 (VH) and 23 (VL), respectively, and the nucleic acid and amino acid sequences of XWU198 are presented as SEQ ID NOs: 16 (VH ) and 18(VL) and SEQ ID NOs: 17 (VH) and 19 (VL), respectively.

(B) STABILIZED ANTI-TWEAK scFv MOLECULE

The present invention is also directed to a binding molecule comprising a binding region, which specifically binds to human TWEAK, wherein the binding region comprises a VH domain and/or a VL domain derived from an anti-TWEAK antibody, e.g., hP2D10. In one embodiment, a binding molecule of the present invention comprises a binding region comprising the hP2D10 VH except one or more stabilizing mutations. In another embodiment, a binding molecule of the invention comprises a binding region comprising the hP2D 10 VL domain except one or more stabilizing mutations. In other embodiments, a binding molecule comprising the hP2D10 VH except one or more stabilizing mutations or the hP2D 10 VL domain except one or more stabilizing mutations further comprises a VL domain or a VH domain, respectively, wherein the binding molecule specifically or preferentially binds to TWEAK.

[0122] In some embodiment, the binding molecule of the present invention acts to antagonize TWEAK activity. In certain embodiments, for example, a binding molecule of the present invention is administered to a host has at least one of the following activities: inhibits binding of TWEAK to a TWEAK receptor (e.g. , Fnl4); inhibits TWEAK-induced apoptosis; inhibits TWEAK-induced angiogenesis and/or endothelial cell proliferation; and/or inhibits TWEAK-induced cytokine secretion (e.g. , IL-6. IL-8).

[0123J In one embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein said scFv polypeptide comprises a heavy chain variable domain (VH) at least 80%, 85%), 90%, 95%, 96%, 97%, 98%, and 99% identical to hP2D10 VH (SEQ ID NO: 13) and a light chain variable domain (VL) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to hP2D 10 VL (SEQ ID NO: 15), wherein the scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of: (1) Asparagine (N) at Kabat position 60 in the VH, (2) Lysine (K) at Kabat position 62 of VH, and (3) Glutamic Acid (E) at Kabat position 12 in the VL.

[0124] In one embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein said scFv polypeptide comprises a VH at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to hP2D10 VI I (SEQ I D NO: 13) and a VL at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to hP2D10 VL (SEQ ID NO: 15), wherein said scFv polypeptide comprises stabilizing mutations or substitutions selected from the group consisting of the amino acids Asparagine (N) at Kabat position 60 in the VH, Lysine (K) at Kabat position 62 of VFI, and Glutamic Acid (E) at Kabat position 12 in the VL. In another embodiment, a binding molecule of the present invention is a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein said scFv polypeptide comprises a VH at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to hP2D l () VH (SEQ ID NO: 13) and a VL at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to hP2D10 VL (SEQ ID NO: 15), wherein said scFv polypeptide comprises stabilizing amino acid mutations or substitutions of the amino acids Asparagine (N) at Kabat position 60 in the VH, Lysine (K) at Kabat position 62 of VH, and Glutamic Acid (E) at Kabat position 12 in the VL.

[0125] In certain embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the scFv polypeptide comprises a heavy chain variable domain (VH). which comprises at least one. two. or three complementarity domain regions (CDRs) of hP2D10 VI I (SEQ ID NO: 13), and/or a light chain variable domain (VL), which comprises at least one, two. or three CDRs o hP2D 10 VL (SEQ ID NO: 15), except for one r more stabilizing mutations in the CDRs selected from the group consisting of P60N in the VH, T62K in the VH, P12E in the VL, and any combinations thereof. In other embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TWEAK, where the scFv polypeptide comprises a VH having three CDRs from hP2D10 VH and/or the scFv polypeptide comprises a VL having three CDRs of hP2D10, except P60N and/or T62K stabilizing mutations in the CDRs of in the VH. In other embodiments, a binding molecule of the invention further comprises a P12E stabilizing amino acid substitution in a framework region of VL.

[0126] In certain embodiments, a binding molecule is a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the scFv polypeptide comprises a VH comprising CDR1 , CDR2. and CDR3 of hP2D10 VI I except two stabilizing mutations wherein amino acid Threonine (T) at Kabat position 62 is replaced with Lysine (K) and amino acid Proline (P) at position Kabat position 60 is replaced with Asparagine (N), and a VL comprising CDRl , CD2, and CDR3 of hP2D 10 VI, except a stabilizing mutation wherein amino acid Proline (P) at Kabat position 12 is replaced with Glutamic Acid (E).

(0127] In one embodiment, a binding molecule of the present invention comprises a stabilized anti-TWEAK scFv molecule having the hP2D10 VII with one or more stabilizing mutations and the hP2D 10 VL. In another embodiment, a binding molecule of the present invention comprises a stabilized anti-TWEAK scFv molecule having the hP2D10 VL with one or more stabilizing mutation and the hP2D10 VH. The binding molecule specifically or preferentially binds to TWEAK. In other embodiments, the invention provides a binding molecule comprising a hP2D 10 VH domain with one or more stabilizing mutations and the hP2D10 VL domain with one or more stabilizing mutations, wherein the binding molecule specifically or preferentially binds to TWEAK. In certain embodiments, the binding molecule further comprises a scFv linker.

[01281 In one embodiment, a binding molecule of the invention is a single chain TWEAK binding molecule (e.g. , a scFv). An examples of a stabilized TWEAK scFv molecule is presented as SEQ ID NO: 64 (nucleotide sequence) and SEQ I D NO: 65 (amino acid sequence).

[0129] A stabilized anti-TWEAK scFv molecule of the invention comprises a VH or VL domain with an optional scFv linker with one stabilizing amino acid mutation in the VL domain, or one or two stabilizing amino acid mutations in the VH domain, or stabilizing amino acid mutation in the VL domain and one or two stabilizing amino acid mutations in the VH domain. In one embodiment, the stabilizing mutations or substitutions in the VH domain of the scFv molecule of the present invention are at amino acid 60 and amino acid 62. according to the Kabat numbering system. In other embodiments, the stabilizing mutation or substitution in the VL domain of the scFv molecule of the present invention is at amino acid 12, according to the Kabat numbering system.

[0130] In one embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the anti- TWEAK scFv polypeptide comprises an hP2D10 VH and an hP2D10 VL except for one or more stabilizing substitutions or mutations at Kabat position 60 in the VH, Kabat position 62 of VI I, and at Kabat position 1 2 in the VL. or any combinations thereof. In another embodiment, a binding molecule of the present invention comprises a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein the anti-TWEAK sci ^'V polypeptide comprises an hP2D10 VI I and an hP2D10 VL except for one or more stabilizing substitutions or mutations selected from the group consisting of Asparagine (N) at Kabat position 60 in the VI I, Lysine (K) at Kabat position 62 of VH. Glutamic Acid (E) at Kabat position 12 in the VL, and any combinations thereof. (C) scFV LINKER

[0131] A stabilized anti-TNF-alpha or anti-TWEAK scFv molecule of the invention can comprise a scFv linker interposed between a VH domain and a VL domain, wherein the VH and VL domains are linked by a disulfide bond between an amino acid in the VH and an amino acid in the VL domain. In one embodiment, a stabilized anti-TNF-alpha or anti-TWEA scFv molecule of the invention comprises a scFv linker having an optimized length or composition. In another embodiment, a scFv linker is a peptide chain. In one aspect, an scFv linker is (Gly)_n (SEQ ID NO: 78), (GlyAla),, (SEQ ID NO: 79), (Gly_nSer)_m (SEQ ID NO: 80), Ser(Gly₄Ser)_n (SEQ ID NO: 30) and (Gly₂Ser)_n(Gly₄Ser)n (SEQ ID NO: 31 ), wherein n and m are any integers selected from the group consisting of any numbers between 1 and 50, between 1 and 100, between 1 and 150, between 1 and 200, or between 1 and 300. In a specific embodiment, a stabilized anti-TNF-alpha or anti-TWEAK scFv polypeptide of the invention comprises the scFv linker (Gly₄ Ser)₄ (SEQ ID NO: 32). Other exemplary linkers comprise, consist essentially of. or consist of (Gly₄Ser)₂ (SEQ ID NO: 33), (Gly₄Ser)₃ (SEQ ID NO: 34), (Gly₄Ser)₅ (SEQ ID NO: 35), and (Gly₄Ser)₆ (SEQ ID NO: 36) sequences. ScFv linkers of the invention can be of varying lengths. In one embodiment, a scFv linker of the invention is from about 5 to about 50 amino acids in length. In another embodiment, a scFv linker of the invention is from about 10 to about 40 amino acids in length. In another embodiment, a scFv linker of the invention is from about 15 to about 30 amino acids in length. In another embodiment, a scFv linker of the invention is from about 17 to about 28 amino acids in length. In another embodiment, a scFv linker of the invention is from about 19 to about 26 amino acids in length. In another embodiment, a scFv linker of the invention is from about 21 to about 24 amino acids in length. ScFv linkers can be introduced into polypeptide sequences using techniques known in the art. For example, in one embodiment, PCR mutagenesis is used. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.

[0132] In some embodiments, a stabilized anti-TNF-alpha or anti-TWEAK scFv polypeptide is connected to a cleavable signal peptide sequence, which facilitates secretion of the scFv polypeptide out of the host cell into the cell culture medium. In one embodiment, a native signal peptide of the VH or VL is used. In another embodiment, a heterologous signal peptide is used, for example, a signal peptide of GPIII, OmpA, PhoA, STII, mouse IgK light chain or MGC class I Kb. Any known signal peptides can also be used for the purpose of this invention.

[0133J A stabilized anti-TNF-alpha or anti-TWEAK scFv molecule or a binding molecule comprising a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule of the invention has an improved stability. The stability of anti-TNF-alpha or anti-TWEAK scFv molecules of the invention or fusion proteins comprising them can be evaluated in reference to the biophysical properties (e.g. , thermal stability) of a conventional (non- stabilized) scFv molecule or a binding molecule comprising a conventional scFv molecule. In one embodiment, a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule of the present invention has an improved thermal stability compared to a conventional scFv molecule or a scFv molecule without the stabilizing mutations. Thermal stability can be measured, e.g. , by Differential Scanning Calorimetry, Differential Scanning Fluorimetry, Dichroism spectroscopy, or Fluorescence Emission Spectroscopy. In further embodiments, a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule of the invention exhibits an improved biophysical property, for example, an improved pH unfolding profile, expression level (% yield), or aggregation level; more stable removal of glycosylation; improved solubility, improved ligand binding affinity, or any combination of improved properties.

101341 In certain embodiments, a binding molecule of the invention comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, certain stabilized T F-alpha specific or TWEAK specific scFv polypeptides of the invention comprise a hinge connecting linker sequence, and/or are modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0135] In certain embodiments, a binding molecule comprising a stabilized anti-TNF- alpha or anti-TWEAK scFv polypeptide of the invention comprises a fusion protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences of the fusion protein normally exist in separate proteins that are brought together in the fusion polypeptide or they can exist in the same protein but are placed in a new arrangement in the fusion polypeptide. In certain embodiments, fusion proteins are created by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

IV. BINDING MOLECULES CONTAINING STABILIZED TNF-ALPHA SPECIFIC SCFV MOLECULE OR TWEAK SPECIFIC SCFV MOLECULE

(A) STABILIZED TNF-ALPHA scFv MOLECULE

[0136] The present invention is further directed to a binding molecule comprising at least one stabilized scFv molecule which specifically binds to TNF-alpha disclosed herein and a second binding region, which specifically binds to a heterologous epitope relative to the epitope bound by the at least one stabilized TNF-alpha specific scFv molecule.

[0137] In certain embodiments, a binding molecule of the invention is monovalent or multivalent or monospecific or multispecilic. e.g., it has at least one binding site comprising one of the stabilized anti-TNF-alpha scFv molecule disclosed herein and at least one heterologous (second) binding site that binds to a second, different target molecule or to a second, different epitope of TNF-alpha.

[0138] In another embodiment, a binding molecule of the invention is a mul timer comprising at least one stabilized TNF-alpha specific scFv polypeptide disclosed herein. For example, in one embodiment, a binding molecule of the invention is a dimer, wherein at least one monomer of the dimer is a stabilized TNF-alpha specific scFv polypeptide disclosed herein. In certain embodiments, dimers of the invention are homodimers, comprising two identical stabilized TNF-alpha specific scFv polypeptides disclosed herein. In other embodiments, dimcrs of the invention are heterodimers. comprising two non-identical monomeric subunits, wherein one monomer is a stabilized TNF-alpha specific scFv polypeptide disclosed herein. In certain embodiments, subunits of a dimer comprise one or more polypeptide chains. For example, in one embodiment, a dimer comprises at least two polypeptide chains, for example, two polypeptide chains or four polypeptide chains (e.g. , as in the case of native antibody molecules).

[0139] In various embodiments, a binding molecule of the invention is monovalent for each specificity or multivalent for each specificity, e.g., bivalent, trivalent, or tetravalent (quadrivalent). In one embodiment, a binding molecule of the invention comprises at least a stabilized anti-TNF-alpha scFv molecule and, in addition, a binding site that reacts with a second target molecule (e.g. a bispecific antibody molecule). In another embodiment, a binding molecule of the invention comprises two binding sites that react with TNF-alpha and two binding sites that react with a second target molecule, wherein at least one of the two TNF-alpha binding sites comprises one of the stabilized TNF-alpha specific scFv molecule disclosed herein.

[0140] The invention also provides more generally multivalent and/or multi specific binding molecules including an anti-TNF moiety. For example, the invention also provides TNF-TWEAK bispecific or multispecific molecules.

[01411 In one embodiment, a second binding region specifically binds to TNF-alpha

(alternate epitope), TWEAK, or IL6. In another embodiment, a binding molecule comprising a stabilized TNF-alpha specific scFv molecule is multivalent for TNF-alpha binding and/or binding to a second binding region, e.g. , TWEAK. For instance, a binding molecule comprising a stabilized TN F-alpha specific scFv molecule binds to two different epitopes on TNF -alpha and two different epitopes on TWEAK.

[0142] In certain aspects, multispecific binding molecules of the invention comprise a stabilized anti- TNF-alpha scFv molecule and a binding region that specifically binds to a T WEAK polypeptide or a fragment thereof. The amino acid sequence of human TWEAK is available in GenBank as Accession No. BAE16557 and presented as SEQ ID NO: 7.

[01431 In one embodiment, the second binding region specifically bi ding to TWEAK is an anti-TWEAK antibody or antigen binding fragment thereof including, but not limited to, P2D10, hP2D10, P5G9, humanized P5G9, or a stabilized anti-TWEAK scFv described herein. The amino acid and nucleotide sequences of hP2D10 are disclosed in U.S. Patent Publication No. 2008/0241 163, which is incorporated herein in its entirely. The nucleic acid and amino acid sequences of the heavy chain variable domain of hP2D10 are presented as SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The nucleic acid and amino acid sequences of the light chain variable domain of hP2D 10 are presented as SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0144] In one embodiment, the second binding region of the binding molecule which specifically binds to TWEAK comprises at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, or six CDRs of the hP2D10 antibody or P5G9 antibody. In another embodiment, the second binding region of the binding molecule which specifically binds to TWEAK comprises CDR3 of the VH domain. In other embodiments, the second binding region comprises the VH domain of hP2D10, P5G9 antibody, or fragments thereof or the VL domain of hP2D10, P5G9, or fragments thereof or both the VH and VL of hP2D10 or P5G9. In certain embodiments, the second binding region comprises the VH domain of P2D10, P5G9, or fragment thereof or the VL domain of P2D 10, P5G9, or fragment thereof or both the VH or VL of P2D10 or P5G9. In another embodiment, the second binding region comprises CDR1 , CDR2, and CDR3 of the VH domain of hP2D10 or P5G9 and a VL domain. Alternatively, the second binding region comprises CDR1 , CDR2, and CDR3 of the VL domain f hP2D10 or P5G9 and a VH domain. In certain embodiments, the second binding region of the binding molecule which specifically binds to TWEAK binds to the same T WEAK epitope as hP2D10 or P5G9 or competitively inhibits binding of hP2D10 or P5G9 to TWEAK. In other embodiments, the TWEAK binding region of the binding molecule binds to the same epitope as Fn 14 or competitively inhibits binding of Fnl4 to TWEAK. In a specific embodiment, the binding molecule of the present invention is a bispecifie antibody comprising the light chain amino acid sequence o SEQ ID NO: 19 and the heavy chain amino acid sequence of SEQ ID NO: 1 7 (XWU198) or 21 (XWU 1 99).

[01451 In certain embodiments, a stabilized TNF-alpha specific scFv molecule as described herein is connected to the second binding region via a hinge connecting linker. A hinge connecting linker can be any organic molecule or inorganic molecule. In one embodiment, the linker is polyethylene glycol (PEG). In another embodiment, the linker comprises amino acids. The linker can comprise, for example, 1-5 amino acids, 1 -10 amino acids, 1 -20 amino acids, 10-50 amino acids, 50-100 amino acids, or 100-200 amino acids. In one embodiment, the linker is the eight amino acid linker EFAGAAAV (SEQ ID NO: 37). Any of the linkers described herein can be used in the binding molecule of the invention, e.g. , in a ant i -TN F-T W ΕΛ bispecifie antibody.

[0146] In certain embodiments, a hinge connecting linker comprises the sequence (Gly)_n.

In other embodiments, a hinge connecting linker comprises the sequence (GlyAla)_n. In one embodiment, a hinge connecting linker comprises the sequence (GGS)_n or (GGS)n(GGGGS)n (SEQ ID NO: 31 ), wherein n is an integer from 1 - 10, 5-20. 10-30, 20- 50, 40-80, or 50-100. Other examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 38), GGSGGSGGSGGSGGG (SEQ ID NO: 39), GGSGGSGGGGSGGGGS (SEQ ID NO: 40), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 41), (GGGGSb (SEQ ID NO: 34), or (GGGGS)₄ (SEQ 11) NO: 32). In one embodiment, the hinge connecting linker is identical to the scFv linker. In another embodiment, the hinge connecting linker is different from the scFv linker. The hinge connecting linker need not eliminate or diminish the biological activity of the binding molecule. Optionally, the hinge connecting linker enhances the biological activity of the binding molecule, e.g. , stabilized anti-TNF-alpha scFv molecule, by further diminishing the effects of steric hindrance and making the stabilized anti-TNF-alpha scFv molecule more accessible to its target binding site. For example, a linker (e.g., GGGGS (SEQ ID NO: 46) or ( GGGGS )5 (SEQ ID NO: 35)) is used in a binding molecule (XWU242 or XWU243, respectively), which comprises the XWU198 VH in the heavy chian and hP2D10 V_L in the light chain.

[0147] In certain embodiments, a multispecific binding molecule of the present invention comprising a stabilized anti-TNF-alpha scFv molecule as described herein and a second binding region which specifically binds to a heterologous molecule simultaneously inhibits activities of TNF-alpha and the heterologous molecule. The inhibition of activities can be measured by any known techniques, which include but are not limited to the IL-8 release assay, the WiDr MTT combo assay, or the NFKB inhibition assay in NFKB Incite rase cellular assay. For example, a tetravalent anti- TNF-alpha and anti- TWEAK bispecific antibody as disclosed herein simultaneously inhibits TWEAK and TNF-alpha activity in the WiDr MTT combo assay. In another example, a tetravalent anti-TNF-alpha and anti-TWEAK bispecific antibody inhibits rabbit or human TNF-alpha activity. In other examples, a tetravalent anti-TNF-alpha and anti-TWEAK bispecific antibody inhibits TWEAK or TNF-alpha activity in the IL-8 release assay. In certain examples, a tetravalent anti-TNF-alpha and anti-TWEAK bispecific antibody inhibits WEHI cell death by inhibiting or reducing TWEAK or TNF-alpha activity in WEHI cytostatic assay. In a further example, an anti-TNF-alpha/TWEAK antibody can inhibit TNF-alpha and TWEAK induced NFKB activation in NFicB-luciferase transgenic mice and NFKB-luciferase expressing 293 cells.

[0148] Also provided in the present invention are tetravalent bispecific antibodies, which comprise two binding regions, where at least one binding region is a stabilized TNF-alpha specific scFv molecule as described herein, and at least one binding region is an antibody that specifically binds to a heterologous polypeptide, wherein the two binding regions are linked to the antibody. For example, the invention includes, but is not limited to two binding regions specifically binding to TNF-alpha and two binding regions that specifically bind to a heterologous polypeptide, e.g., TWEAK. For example, bispecific tetravalent antibodies comprise two stabilized TNF-alpha specific scFv molecules linked to the CH3 domain of the anti-TWEAK antibody (hP2D10) as shown in Figure 4. The stabilized anti-TNF-alpha scFv molecules are linked to the anti-TWEAK antibody (hP2D10) optionally via a hinge connecting linker disclosed herein.

[01491 In certain embodiments, a binding molecule of the present invention comprising a stabilized anti-TNF-alpha scFv molecule, which comprises the adalimumab VH and the adalimumab VL except for one or more stabilizing mutations described herein binds to a TNF-alpha t rimer at a ratio of 1 : 1 at equilibrium.

[0150] In some embodiments, a binding molecule of the present invention demonstrates cross-reactivity to TNF-alpha from different species. For example, the stabilized anti- TNF-alpha scFv molecule binds surface rabbit TNF-alpha or mouse TNF-alpha with a binding affinity greater than that of adalimumab.

[0151] In one embodiment, the present invention provides a multispecific binding molecule comprising a stabilized anti-TNF-alpha scFv molecule comprising SEQ ID NO: 17 (VH domain of XWU 198) and SEQ ID NO: 19 (VL domain of XWU198) and an anti- TWEAK antibody comprising three CDRs of SEQ ID NO: 13 and three CDRs of SEQ ID NO: 15. In another embodiment, a binding molecule of the present invention comprises a stabilized anti-TNF-alpha scFv polypeptide comprising SEQ I NO: 21 (VI I domain of XWU 199) and SEQ ID NO: 23 (VL domain of XWU 199) and an anti-TWEAK antibody comprising three CDRs of SEQ ID NO: 13 and three CDRs of SEQ ID NO: 15.

(B) STABILIZED ANTI-TWEAK scFV MOLECULE

[0152] The present invention is further directed to a binding molecule comprising at least one stabilized scFv molecule which specifically binds to TWEAK disclosed herein and a second binding region, which specifically binds to a heterologous epitope relative to the epitope bound by the at least one stabilized I WEAK specific scFV molecule.

[01531 In certain embodiments, a binding molecule of the invention is monovalent or multivalent or monospecific or multispecific, e.g. , it has at least one binding site comprising one of the stabilized anti-TWEAK scFv molecules disclosed herein and at least one heterologous (second) binding site that binds to a second, different target molecule, e.g. , T F-alpha or a second, different epitope of TWEAK.

[0154] In another embodiment, a binding molecule of the invention is a multimer comprising at least one stabilized TWEAK specific scFv polypeptide disclosed herein. For example, in one embodiment, a binding molecule is a dimer, wherein at least one monomer of the dimer is a stabilized TWEAK specific scFv polypeptide disclosed herein . In certain embodiments, dimers of the invention are homodimers, comprising two identical stabilized TWEAK specific scFv polypeptides disclosed herein. In certain embodiments, dimers of the invention are heterodimers, comprising two non-identical monomeric subunits, wherein one monomer is a stabilized TWEAK specific scFv polypeptide disclosed herein. In certain embodiments, subunits of a dimer comprise one or more polypeptide chains. For example, in one embodiment, a dimer comprises at least two polypeptide chains, for example, two polypeptide chains or four polypeptide chains (e.g. , as in the case of native antibody molecules).

[01551 In various embodiments, a binding molecule of the invention is monovalent for each specificity or multivalent for each specificity, e.g. , bivalent, trivalent, or tetravalent (quadrivalent). In one embodiment, a binding molecule of the invention comprises at least a stabilized anti-TWEAK scFv molecule and, in addition, a binding site that reacts with a second target molecule (e.g. , a bispecific antibody molecule). In another embodiment, a binding molecule of the invention comprises two binding sites that react with TWEAK and two binding sites that react with a second target molecule, wherein at least one of the two TWEAK binding sites comprises one of the stabilized TWEAK specific scFv molecules disclosed herein.

[0156] The invention also provides more generally multivalent and/or multispecific binding molecules including an anti-TWEAK moiety. For example, the invention also provides bispecific or multispecific molecules comprising a stabilized anti-TWEAK moiety and a stabilized or non-stabilized anti-TNF binding moiety. In one embodiment, a second binding region specifically binds to TWEAK (alternate epitope), TNF-alpha, or IL-6. In another embodiment, a binding molecule comprising a stabilized TWEAK specific scFv molecule is multivalent for TWEAK binding and/or binding to a second binding region, e.g. , TNF-alpha. For instance, a binding molecule comprising a stabilized TWEAK specific scFv molecule binds to two different epitopes on TWEAK and two different epitopes on TNF-alpha.

[0157] In certain aspects, multispecific binding molecules of the invention comprise a stabilized anti-TWEAK scFv and a binding region that specifically binds to TNA-alpha or a fragment thereof. In one embodiment, the second binding region specifically binding to TNF-alpha is an ami- l^'NF-alpha antibody or antigen binding fragment thereof including, but not limited to adalimumab.

[0158] In one embodiment, the second binding region of the binding molecule which specifically binds to TNF-alpha comprises at least one CDR, at least two CDRs. at least three CDRs, at least four CDRs, at least five CDRs, or six CDRs of adalimumab. In other embodiments, the second binding region comprises the VH domain of adalimumab or fragment thereof or the VL domain of adalimumab or fragment thereof, or both the VH and VL domain of adalimumab. In another embodiment, the second binding region comprises CDR1, CDR2, and CDR3 of the VH domain of adalimumab and a VL domain. Alternatively, the second binding region comprises CDR1, CDR2, and CDR3 of the VL domain of adalimumab and a VH domain. In certain embodiments, the second binding region of the binding molecule which specifically binds to TNF-alpha binds to the same epitope as adalimumab or competitively inhibits binding of TNF-alpha to a TNF-alpha receptor. In some embodiments, the second binding region of the molecule which specifically binds to TNF-alpha inhibits binding of TNF-alpha to TNF-alpha type 1 receptors, to TNF-alpha type 2 receptors, or to both types f receptors.

[0159] In one embodiment, the second binding region o the molecule which specifically binds to TNF-alpha comprises an adalimumab VH and an adalimumab VL except for one or more stabilizing substitutions or mutations at Kabat position 16 of VI I. Kabat position 30 of VH, Kabat position of VI I, Kabat position 49 of VH, Kabat position 52 of VII. Kabat position 57 of VH, Kabat position 61 of VH, Kabat position 64 of VH, Kabat position 70 of VH, Kabat position 77 of VH, Kabat position 77 of VH, Kabat position 77 of VH, Kabat position 82b of VH. Kabat position 84 of VH. or any combinations thereof.

[0160] In one embodiment, the second binding region of the molecule which specifically binds to TNF-alpha comprises an adalimumab VH and an adalimumab VL except for one or more stabilizing substitutions or mutations selected from the group consisting of (i) amino acid substitution at Kabat position 16 from Arginine(R) to Glycine (G) of VH (R16G), (ii) amino acid substitution at Kabat position 30 from Aspartic acid (D) to Serine (S) of VH (D30S), (iii) amino acid substitution at Kabat position 49 from Serine (S) to Glycine (G) of VH (S49G), (iv) amino acid substitution at Kabat position 49 from Serine (S) to Alanine (A) of VH (S49A), (v) amino acid substitution at Kabat position 52 from Threonine (T) to Serine (S) of VH (T52S), (vi) amino acid substitution at Kabat position 57 from Isoleucine (I) to Threonine (T) of VH (I57T), (vii) amino acid substitution at Kabat position 61 from Aspartic acid (D) to Proline (P) of VH (D61 P), (viii) amino acid substitution at Kabat position 64 from Glutamic acid (E) to Lysine (K) of VH (E64K), (ix) amino acid substitution at Kabat position 70 from Serine (S) to Glycine (G) of VI I (S70G), (x) amino acid substitution at Kabat position 77 from Serine (S) to Glutaminc (Q) of VH (S77Q), (xi) amino acid substitution at Kabat position 77 from Serine (S) to Threonine (T) of VH (S77T), (xii) amino acid substitution at Kabat position 77 from Serine (S) to Lysine (K) of VH (S77K), (xiii) amino acid substitution at Kabat position 82b from Serine (S) to Asparagine (N) of VH (S82bN), (xiv) amino acid substitution at Kabat position 84 from Alanine (A) to Proline (P) of VH (A84P) and any combinations thereof.

[01611 In one embodiment, the second binding region of the molecule which specifically binds to TNF-alpha comprises an adalimumab VH except for one or more stabilizing substitutions or mutations at Kabat positions 16, 30, and 52 of VH. In certain embodiments, the second binding region of the molecule which specifically binds to TNF-alpha comprises an adalimumab VH except for one or more stabilizing substitutions or mutations selected from the group consisting of (i) amino acid substitution at Kabat position 16 from Arginine(R) to Glycine (G) of VH (R16G), (ii) amino acid substitution at Kabat position 30 from Aspartic acid (D ) to Serine (S) of VH (D30S), (iii) amino acid substitution at Kabat position 52 from Threonine (T) to Serine (S) of VH (T52S), and any combinations thereof.

[0162] In a specific embodiment, the second binding region of the molecule which specifically binds to TNF-alpha comprises an adalimumab VI I with an amino acid substitution at Kabat position 16 of VI I from Arginine( R) to Glycine (G) (R16G). an amino acid substitution at Kabat position 30 of VH from Aspartic acid (D) to Serine (S) (D30S), and amino acid substitution at Kabat position 52 of VH from Threonine (T) to Serine (S) (T52S). [01631 In a specific embodiment, the binding molecule of the present invention is a bispecific antibody comprising the light chain sequence of adalimumab and the heavy chain amino acid sequence of SEQ ID NO: 67 (MJF149).

[0164] In certain embodiments, a stabilized TWEAK specific scFv molecule as described herein is connected to the second binding region via a hinge connecting linker. A hinge connecting linker can be any organic molecule or inorganic molecule. In one embodiment, the linker is PEG. In another embodiment, the linker comprises amino acids. The linker can comprise, for example, 1-5 amino acids, 1 -10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, or 100-200 amino acids. In another embodiment, the linker is the eight amino acid linked EFAGAAAV (SEQ ID NO: 37). Any of the linkers described herein can be used in the binding molecule of the invention, e.g., an anti-TWEAK- TNF bispecific antibody.

[01651 In certain embodiments, a hinge connecting linker comprises the sequence (Gly)_n.

In one embodiment, a hinge connecting linker comprises the sequence (GGS)_n or (GGS)_n(GGGGS)_n (SEQ ID NO: 31), wherein n is an integer from 1 -10. 5-20, 10-30. 20- 50, 40-80, or 50-100. Other examples of linkers include, but are not limited to, GGG. SGGSGGS (SEQ ID NO: 38). GGSGGSGGSGGSGGG (SEQ ID NO: 39), GGSGGSGGGGSGGGGS (SEQ ID NO: 40), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 41 ). (GGGGSb (SEQ ID NO: 34). or (GGGGS)₄ (SEQ ID NO: 32). In one embodiment, the hinge connecting linker is identical to the scFv linker. In another embodiment, the hinge connecting linker is different from the scFv linker. The hinge connecting linker need not eliminate or diminish the biological activity of the binding molecule. Optionally, the hinge connecting linker enhances the biological activity of the binding molecule, e.g. , stabilized anti-TWEAK scFv molecule, by further diminishing the effects of steric hindrance and making the stabilized anti-TWEAK scFv molecule more accessible to its target binding site. For example, a linker (e.g. , (GGGGS) (SEQ ID NO: 35 ) is used in a binding molecule (XWU245 ), which comprises the stabilized scFv of MJF149 and the Fc fragment of adalimumab.

[0166] In certain embodiments, a multispecific binding molecule of the present invention comprising a stabilized anti-TWEAK scFv molecule as described herein and a second binding region which specifically binds to a heterologous molecule simultaneously inhibits activities of TWEAK and the heterologous molecule. The inhibition of activities can be measured by any techniques known in the art. Also provided in the present invention are tetravalent bispecific antibodies, which comprise two binding regions, where at least one binding region is a stabilized TWEAK specific scFv molecule as described herein, and at least one binding region is an antibody that specifically binds to a heterologous polypeptide, wherein the two binding regions are linked to the antibody. For example, the invention includes, but is not limited to two binding regions specifically binding to TWEAK and two binding regions that specifically bind to a heterologous polypeptide, e.g., TNF-alpha. For example, bispecific tetravalent antibodies comprise two stabilized TWEAK specific scFv molecules linked to the CH3 domain of the anti- TNF antibody (e.g. , adalimumab). The stabilized anti-TWEA scFv molecules are linked to the anti-TNF-alpha antibody (adalimumab) optionally via a hinge connecting linker disclosed herein.

V. CHARACTERIZATION OF BINDING MOLECULES COMPRISING A

STABILIZED TNF-ALPHA SPECIFIC scFv MOLECULE OF THE

PRESENT INVENTION

A) STABILITY PROPERTIES

[0167] The stability properties of the stabilized anti-TNF-alpha or anti-TWEAK scFv polypeptide of the invention can be analyzed using methods known in the art. Stability parameters acceptable to those in the art can be employed. Non-limiting exemplary parameters are described in more detail below.

1) THERMAL STABILITY

[0168] The stability properties of the stabilized anti-TNF-alpha or anti-TWEAK scFv polypeptide of the invention can be analyzed using methods known in the art. Stability parameters acceptable to those in the art can be employed. Non-limiting exemplary parameters are described in more detail below.

[0169] The thermal stability of the binding molecule of the invention can be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by thermoanalytical techniques. An exemplary thermoanalytical method is Differential Scanning Calorimetry (DSC). DSC employs a calorimeter which is sensitive to the heat absorbances that accompany the unfolding of most proteins or protein domains (see, e.g. Sanchez-Ruiz, et al, Biochemistry, 27: 1648-52, (1988)). To determine the thermal stability of a protein, a sample of the protein is loaded into the calorimeter cell and the temperature is increased in a controlled fashion. Pronounced changes in the sample heat capacity, delta Cp, at defined temperatures, are monitored as indications of domain unfolding, such as the unfolding of scFv VH and VL domains, or cooperative unfolding of Fab fragments. The temperatures at which the different domains of a protein unfold are indicative of overall protein stability.

[0170] In certain embodiments, thermal stability is evaluated by analytical spectroscopy.

One exemplary analytical spectroscopy method is Circular Dichroism (CD) spectroscopy. CD spectrometry measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. A disordered or unfolded structure results in a CD spectrum very different from that of an ordered or folded structure. The CD spectrum reflects the sensitivity of the proteins to the denaturing effects of increasing temperature and is therefore indicative of a protein's thermal stability (see van Mierlo and Steemsma, J. Biotechnol, 79(3):281 -98, (2000)).

10171 ] Another exemplary analytical spectroscopy method for measuring thermal stability is Fluorescence Emission Spectroscopy (see van Mierlo and Steemsma. supra). Yet another exemplary analytical spectroscopy method for measuring thermal stability is Nuclear Magnetic Resonance (NMR) spectroscopy (see, e.g. van Mierlo and Steemsma. supra).

[0172] In other embodiments, the thermal stability of a composition of the invention is measured biochemically. An exemplary biochemical method for assessing thermal stability is a thermal challenge assay. In a "thermal challenge assay", a composition of the invention is subjected to a range of elevated temperatures for a set period of time. For example, in one embodiment, scFv polypeptides or molecules comprising scFv polypeptides are subject to a range of increasing temperatures, e.g. , for 1 -1.5 hours. The activity of the protein is then assayed b a relevant biochemical assay. For example, if the protein is a binding protein (e.g. , an scFv or scFv-containing polypeptide of the invention) the binding activity of the binding protein can be determined by a functional or quantitative ELISA. [0173] In certain embodiments, thermal stability is evaluated by measuring the melting temperature (Tm) of a binding molecule of the invention using any of the above techniques (e.g. , analytical spectroscopy techniques). See Miller B.R., et al.. Methods Mol. Biol. 525: 279-89 (2009). The melting temperature is the temperature at the midpoint of a thermal transition curve wherein 50% of molecules of a composition are in a folded state. In other embodiments, thermal stability is evaluated by measuring T50 of a binding molecule. The T50 value represents the temperature at which 50% of the scFv molecules retain their binding activity. See Michaelson J.S., et al , mAbs 1 : 128-141 (2009).

[0174 J In certain embodiments, stabilized TNF-alpha specific scFv molecules of the invention have a T50 of greater than 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66. 67. 68, 69, 70, 71 , 72. 73. 74. or 75 °C. In other embodiment, stabilized TNF- alpha specific scFv molecules of the invention have a T₅₀ of greater than 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, or 70 °C. In yet another embodiment, a binding molecule comprising a stabilized TNF-alpha specific scFv molecule of the invention demonstrates a thermal stability characterized by a VH melting temperature (Tm) of at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73. 74, or 75 °C. In certain embodiments, a binding molecule comprising a stabilized TNF-alpha specific scFv molecule demonstrates a thermal stabilit characterized by a VL Tm of at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, or 75 °C.

[0175] In certain embodiments, the thermal stabilit of TNF-alpha specific scFv polypeptide of the invention is characterized by a Tm of at least 60 "C as measured by differential scanning calorimetry. In other embodiments, the thermal stability of a TNF- alpha specific scFv polypeptide of the invention is characterized by a Tm of at least 65 °C as measured by differential scanning calorimetry. In some embodiments, the thermal stability of a TN F-alpha specific scFv polypeptide of the invention is characterized by a Tm of at least 67 °C as measured by differential scanning calorimetry.

[0176] In one embodiment, a binding molecule comprising a stabilized TNF-alpha specific scFv polypeptide of the invention is characterized by a T50 of at least 55°C as measured by differential scanning calorimetry. In another embodiment, a binding molecule comprising a stabilized TNF-alpha specific scFv polypeptide of the invention is characterized by a T50 of at least 60°C as measured by differential scanning calorimetry. In some embodiments, a binding molecule comprising a stabilized TNF-alpha specific scFv polypeptide of the invention is characterized by a T50 of at least 62°C as measured by differential scanning calorimetry.

[0177] In another embodiment, a binding molecule comprising a stabilized TWEAK specific scFv polypeptide of the invention has an improved thermal stability compared to a binding molecule comprising a TWEAK specific scFv polypeptide without the stabilizing mutations. For example, a binding molecule comprising a stabilized TWEAK specific scFv polypeptide can have a T₅₀ higher than a binding molecule comprising a conventional TWEAK specific scFv polypeptide (e.g., TWEAK specific scFv polypeptide without the stabilizing mutations). In addition, a binding molecule comprising a stabilized TWEAK specific scFv polypeptide can have a I^'m higher than a binding molecule comprising a conventional TWEAK specific scFv polypeptide (e.g., a TWEAK specific scFv polypeptide without the stabilizing mutations).

[0178] In some embodiments, the binding molecules of the invention (a binding moleculeTNF-alpha specific or TWEAK specific scFv molecule) have a thermal stability that is greater than about 0.1 , about 0.25, about 0.5, about 0.75, about 1 , about 1.25, about 1.5, about 1.75, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 degrees Celsius (°C) than a control binding molecule (e.g. , a conventional scFv molecule comprising the variable domains of adalimumab or P2D10 without any stabilizing mutations).

[01791 In other embodiments, thermal stability is evaluated by measuring the specific heat or heat capacity (Cp) of a composition of the invention using an analytical calorimetric technique (e.g., DSC). The specific heat of a composition is the energy (e.g. , in kcal/mol) required to raise by 1 C. the temperature of 1 mo I of water. As large Cp is a hallmark of a denatured or inactive protein composition. In certain embodiments, the change in heat capacity (ACp) of a composition is measured by determining the specific heat of a composition before and after its thermal transition, in other embodiments, thermal stability can be evaluated by measuring or determining other parameters of thermodynamic stability including Gibbs free energy of unfolding (AG), enthalpy of unfolding (AH), or entropy of unfolding (AS). [0180] In other embodiments, one or more of the above biochemical assays (e.g. , a thermal challenge assay) is used to determine the temperature (i.e., the Tc value) at which 50% of the composition retains its activity (e.g., binding activity).

2) BINDING AFFINITY

[0181] A wide variety of methods for determining binding affinity are known in the art.

An exemplary method for determining binding affinity employs surface pi asm on resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time bispecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BlAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson, U., et al., ( 1993 ) Ann. Biol. Chn. 51 : 19-26; Jonsson, U., et al., ( 1991 ) Biotechniques 1 1 :620-627; Johnsson, B., et al., ( 1995) J. Mol. Recognit. 8: 125- 13 1 : and Johnnson, B., et al. ( 1991 ) Anal. Biochern. 198:268-277.

[0182] In some embodiments, the stability of a stabilized TNF-alpha specific or TWEAK specific scFv polypeptide of the invention or binding molecule comprising a stabilized TNF-alpha specific scFv molecule is assessed by determining its target binding affinity. In certain embodiments, a binding molecule of the present invention comprising a stabilized TNF-alpha or TWEAK scFv specifically binds to at least one epitope of TNF- alpha, TWEAK, or fragment or variant, respectively, with an affinit characterized by a

2 2 3 dissociation constant KD of less than about 5 x 10^" M, about 10^" M, about 5 x 10^" M, about 10^"- M. about 5 x 10^"4 M, about 10^"4 M, about 5 x 10^"5 M, about 10^"5 M, about 5 x 10^" ⁶ M, about 10^"6 M. about 5 x 10^"7 M, about 10^"7 M, about 5 x 10^"8 M, about 10^"8 M, about 5 x 10^"9 M, about 10^"9 M, about 5 x 10^"10 M, about 10^"10 M, about 5 x 10^"" M. about lO^{"1 1} M, about 5 x 10^"12 M, about 10^"12 M, about 5 x 10^"13 M, about 10^"13 M, about 5 x 10^"14 M, about 10^"14 M, about 5 x 10^"15 M, or about 10^"15 M. In one embodiment, the binding molecule of the present invention comprising a stabilized TNF-alpha or TWEAK scFv specifically binds to at least one epitope of TNF-alpha, TWEAK, or fragment or variant, respectively, with an affinity characterized by a dissociation constant KD of less than about 250pM, 200pM, 190pM, 180pM, 17()pM. 160pM. 150 pM, 140pM, 130pM, 120pM, HOpM, 100 pM, 90pM, 8()pM. 7()pM. 60pM or 50pM. In another embodiment, the binding molecule of the present invention comprising a stabilized TNF-alpha or TWEAK scFv specifically binds to at least one epitope of TNF-alpha, TWEAK, or fragment or variant, respectively, with an affinity characterized by a dissociation constant KD of less than about 150pM.

[0183] Also included in the present invention is a binding molecule which binds to at least one epitope of TNF-alpha, TWEAK, or fragments or variant thereof with an off rate (k(off)) of less than or equal to 10^"1 sec^{" 1}, 5 X 10^"2 sec^"1, 10^"2 sec^"1, 5 X 10^"3 sec^"1, or 10^"3 sec^"1. Alternatively, a TNF-alpha or TWEAK binding molecule binds TNF-alpha or TWEAK polypeptides or fragments or variants thereof, respectively, with an off rate (k(off)) of less than or equal to 5 X 10^"4 sec^"1, 10^"4 sec^"1, 5 X 10^"5 sec^{" 1}, or 10^"5 sec^"1 5 X 10^" sec^" , 10 sec , 5 X 10^" sec^" or 10^" sec^" . In other embodiments, a TNF-alpha or TWEAK binding molecule binds TNF-alpha or TWEAK polypeptides or fragments or variants thereof, respectively, with an on rate (k(on)) of greater than or equal to 10^' M^" sec^"1, 5 X 10³ M^{" 1} sec^"1. 10⁴ M^"1 sec^"1 or 5 X 10⁴ M^"1 sec^"1. Alternatively, an TNF-alpha or TWEAK binding molecule binds TNF-alpha or TWEAK polypeptides or fragments or variants thereof, respectively, with an on rate (k(on)) greater than or equal to 10⁵ M^"1 sec^" '. 5 X 10^s M^{" 1} sec^"1. 10⁶ M^"1 sec^{" 1}, or 5 X 106 M^{" 1} sec^"1 or 10⁷ M^"1 sec^"1.

3) % A GGREGA TION

[0184] In certain embodiments, the stability of a binding molecule of the invention is determined by measuring its propensity to aggregate. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, the aggregation of a binding molecule of the invention can be evaluated using chromatography, e.g., Size-Exclusion Chromatograpy (SEC). SEC separates molecules on the basis of size. A column is filled with semi-solid beads of a polymeric gel that will admit ions and small molecules into their interior but not large ones. When a protein composition is applied to the top of the column, the compact folded proteins {i.e., non- aggregated proteins) are distributed through a larger volume of solvent than is available to the large protein aggregates. Consequently, the large aggregates move more rapidly through the column, and in this way the mixture can be separated or fractionated into its components. Each fraction can be separately quantified {e.g. , by light scattering) as it elutes from the gel. Accordingly, the % aggregation of a binding molecule of the invention can be determined by comparing the concentration of a fraction with the total concentration of protein applied to the gel. Stable compositions elute from the column as essentially a single fraction and appear as essentially a single peak in the elution profile or chromatogram.

[0185] In other embodiments, Size-Exclusion Chromatograpy (SEC) is used in conjunction with in-line light scattering (e.g. classical or dynamic light scattering) to determine the % aggregation of a composition. In one embodiment, static light scattering is employed to measure the mass of each fraction or peak, independent of the molecular shape or elution position. In another embodiment, dynamic light scattering is employed to measure the hydrodynamic size of a composition. Other exemplary methods for evaluating protein stability include High-Speed SEC (see e.g. , Corbett et aL Biochemistry. 23(8): 1888-94. 1984).

[018 j In one embodiment, the % aggregation is determined by measuring the fraction of protein aggregates within the protein sample. In another embodiment, the % aggregation of a binding molecule is measured by determining the fraction of folded protein within the protein sample.

[0187] In one embodiment, the % aggregation of a binding molecule comprising the stabilized anti-TNF-alpha or TWEAK scFv molecule of the invention is comparable or superior to a conventional scFv polypeptide or a binding molecule comprising the conventional scFv polypeptide. In one embodiment, populations of stabilized anti-TNF- alpha or TWEAK scFv molecules of the invention or molecules comprising the same are expressed as a monomcric (e.g., a stabilized anti-TNF alpha or anti-TWEAK scFv disclosed herein) or tetrameric (e.g. , anti-TNF and anti-TWEAK bi specific antibody disclosed herein), soluble protein of which is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, or 15% in dimeric, tetrameric, or otherwise aggregated form. In another embodiment, a binding molecule comprising the stabilized anti-TNF-alpha or anti-TWEAK scFv molecule is characterized by the % aggregation of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% less than a binding molecule comprising a conventional scFv molecule.

4 % YIELD

[0188] In other embodiments, the stability of a bindin molecule of the invention is evaluated by measuring the amount of protein that is recovered (herein the "% yield") following expression (e.g. , recombinant expression) of the protein. For example, the % yield can be measured by determining milligrams of protein recovered for every ml of host culture media (i.e. , mg/ml of protein). In another embodiment, the % yield is evaluated following expression in a mammalian host cell (e.g. , a CHO cell).

5) % LOSS

[0189] In certain embodiments, the stability of a binding molecule of the invention is evaluated by monitoring the loss of protein at a range of temperatures (e.g., from -80 to 25 °C) following storage for a defined time period. The amount or concentration of recovered protein can be determined using any protein quantification method known in the art, and compared with the initial concentration of protein. Exemplary protein quantification methods include SDS-PAGE analysis or the Bradford assay for (Bradford, et al., Anal. Biochem. 72, 248, (1976)). A method for evaluating % loss can employ any of the analytical SEC methods described supra. The percent loss measurements can be determined under any desired storage condition or storage formulation, including, for example, lyophilized protein preparations.

6) % PROTEOLYSIS

[0190] In other embodiments, the stability of a binding molecule of the invention is evaluated by determining the amount of protein that is proteolyzed following storage under standard conditions. In an exemplary embodiment, proteolysis is determined by SDS-PAGE analysis of a sample of the protein wherein the amount of intact protein is compared with the amount of low-molecular weight fragments which appear on the SDS- PAGE gel. In another exemplary embodiment, proteolysis is determined by Mass Spectrometry (MS), wherein the amount of protein of the expected molecular weight is compared with the amount of low-molecular weight protein fragments within the sample.

B) BIOCHEMICAL/BIOPHYSICAL PROPERTIES

1) CROSS-REACTIVITY TO TNF-ALPHA FROM

DIFFERENT SPECIES

[0191] Also provided in this invention is a binding molecule comprising a stabilized

TNF-alpha specific or TWEAK scFv molecule, e.g., an anti-TNF-TWEAK bispecific antibody (e.g. , XWU198 or XWU199) having TNF-alpha binding affinity different from that of a control antibody, e.g. , adalimumab, P2D1 ().. or scFvs directly derived therefrom. In one embodiment, a TNF-alpha specific or TWEAK specific binding molecule of the instant invention binds to murine, rat. rabbit, or monkey TNF-alpha with higher affinity than adalimumab or TWEAK with higher affinity than P2D10 . For example, an anti- TNF-TWEAK bispecific antibody (e.g., XWU198 and XWU199) binds to rabbit TNF- alpha or mouse TNF-alpha with binding affinity greater than that of adalimumab.

2) BINDING STOICHIOMETR Y

[0192] In certain embodiments, a binding molecule comprising a stabilized anti- TNF- alpha scFv molecule, e.g., an anti-TNF-TWEAK bispecific antibody (e.g. , XWU198 or XWU199) has different stoichiometry from its parent antibody, e.g., adalimumab.

[0193] Adalimumab is known to form large complexes with TNF. See Santora, L.C., et al, Analytical Biochemistry, 299: 1 19-129 (2001) and Tadahiko. K., et al, J. Investigative Dermatology Symposium Proceedings, 12: 5-8 (2007). Our observations and published data are consistent with a stoichiometry of two TNF-alpha trimers per three adalimumab antibodies, or with the stoichiometry of TNF-alpha and adalimumab at equilibrium of 1 : 1.5. When excess adalimumab antibodies are present, three adalimumab antibodies bind to one TNF-alpha trimer. When excess TNF-alpha is present compared to adalimumab, one adalimumab antibody binds to two TNF-alpha trimers. However, an anti-TNF-TWEAK bispecific antibody at equilibrium binds to one TNF trimer as shown in Figures 8-10. Therefore, the data suggests that the binding stoichiometry of an anti- TNF-TWEAK bispecific antibody is different from the adalimumab antibody.

3) OTHER BINDING STUDIES

[01 4] In some embodiments, the stability of a binding molecule of the invention is assessed by quantifying the binding of a labeled compound to denatured or unfolded portions of a binding molecule. In certain embodiments, the labeled composition is hydrophobic and binds or interacts with large hydrophobic patches of amino acids that are normally buried in the interior of a native protein, but which are exposed in a denatured or unfolded binding molecule. An exemplary labeled compound is the hydrophobic fluorescent dye, l-anilino-8-naphthalcne sulfonate (ANS). I. POLYNUCLEOTIDES ENCODING STABILIZED ANTI-TNF- ALPHA scFv MOLECULES, STABILIZED ANTI-TWEAK scFv MOLECULES, AND MULTISPECIFIC BINDING MOLECULES [0195] The present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the binding molecule of the invention disclosed herein. In one embodiment, the present invention is directed to a nucleic acid molecule comprising a polynucleotide encoding a stabilized anti-TNF-alpha scFv molecule comprising the VH domain of adalimumab and/or a VL domain of adalimumab except one or more stabilizing mutations, wherein the scFv molecule acts to antagonize TNF-alpha activity. In another embodiment, the present invention is directed to a nucleic acid molecule comprising a polynucleotide encoding a binding molecule comprising at least one stabilized anti-TNF-alpha scFv molecule comprising the VH domain of adalimumab and/or a VL domain of adalimumab except one or more stabilizing mutations, wherein the scFv molecule acts to antagonize TNF-alpha activity.

[0196] The present invention also provides a nucleic acid molecule comprising a polynucleotide encoding a stabilized anti-TWEAK scFv molecule comprising the VI I domain of P2D10 and/or a VL domain of P2D10 except one or more stabilizing mutations, when the scFv molecule acts to antagonize TWEAK activity.

[0197] In other embodiments, the present invention is drawn to a nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule which comprises a binding region comprising the adalimumab or P2D10 VH with one or more stabilizing mutations and the adalimumab or P2D10 VI. with one or more stabilizing mutations. The binding molecule specifically or preferentially binds to TNF-alpha or TWEAK, respectively.

[0198] In one embodiment, the present invention is drawn to a nucleic acid molecule comprising a polynucleotide encoding a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule of the invention and further comprises a nucleotide sequence encoding an scFv linker situated between a VI I encoding and VL encoding sequences.

[0199] In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding a stabilized TNF-alpha specific or TWEAK specific scFv molecule of the invention comprises a nucleotide sequence encoding a VH or VL domain with an optional scFv linker, wherein the stabilized scFv molecule comprises at least one. at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 1 1, or at least 12 stabilizing amino acid mutation(s) or substitution(s). In one embodiment, a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule comprises a VH or VL domain with a single mutation (mutant) or double, triple, quadruple, quintuple (or pentuple), sextuple (or hextuple), septuple, octuple, nonuple, decuple, hendecuple (or undecuple), or duodecuple mutations (or mutants). The stabilizing mutations or substitutions in the VH domain of the stabilized anti-TNF-alpha scFv molecule of the present invention can be at a position selected from the group consisting of amino acid 16, amino acid 30, amino acid 49, amino acid 52, amino acid 64, amino acid 77, amino acid 82b, amino acid 84, and any combinations thereof, according to the Kabat numbering system. The stabilizing mutations or substitutions in the VI. domain of the stabilized anti-TNF-alpha scFv molecule of the present invention can be at a position selected from the group consisting of amino acid 3, amino acid 54, amino acid 83, and any combinations thereof, according to the Kabat numbering system.

[0200] In another embodiment, a nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule of the present invention comprises a nucleotide sequence encoding a stabilized anti-TNF-alpha scFv polypeptide which specifically binds to human TNF-alpha, wherein the stabilized anti-TNF-alpha scFv polypeptide comprises an adalimumab VI I and an adalimumab VL except for one or more stabilizing substitutions or mutations selected from the group consisting of (i) amino acid substitution at Kabat position 3 from Q to P of VL; (ii) amino acid substitution at Kabat position 16 from R to G of VL (R16G); (iii) amino acid substitution at Kabat position 54 from L to R of VL (L54R); (iv) amino acid substitution at Kabat position 83 from V to E of VL ( V83E); and any combinations thereof.

102011 In certain embodiments, a nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule of the present invention comprises a nucleotide sequence encoding a stabilized scFv polypeptide which specifically binds to human TNF- alpha. wherein the scFv polypeptide comprises an adalimumab VI I and an adalimumab VL except for one or more stabilizing substitutions or mutations selected from the group consisting of (i) S77Q in VH and I57T in VH; (ii) R 16G in VH and S70G in VH; (iii) R16G in VH and S70G in VH; (iv) T52S in VII and V83E in VL: (v) S77T in VH and V83E in VL; (vi) S77K in VH and V83E in VL; (vii) R16G in VH, S70G in VII. and V83 in VL; (viii) S49A in VH and V83E in VL; (ix) T52S in VI I. L54R in VL, and V83E in VL; (x) R16G in VI I. D30S in VH, L54R in VL, V83E in VL; (xi) S49A in VI I, L54R in VL, and V83E in VL; (xii) R16G in VH, 03 OS in VH, S77T in VI I, L54R in VL, and V83E in VL: (xiii) R16G in VH. D30S in VH. E64K in VI I, L54R in VL, and V83E in VL, (xiv) R16G in VI I, D30S in VH, A84P in VH, L54R in VL, and V83E in VL, and (xv) R16G in VI I. D30S in VII, T52S in VI I. L54R in VL. and V83E in VL

(0202] In certain embodiments, a nucleic acid molecule comprising a polynucleotide encoding a binding molecule of the present invention comprises a nucleotide sequence encoding a stabilized scFv polypeptide which specifically binds to human TNF-alpha. wherein the scFv polypeptide comprises an adalimumab VH and an adalimumab VL except for amino acid substitutions Rl 6G in VH, D30S in VH, L54R in VL, and V83E in VL (also used herein as the quadruple mutant or XWU 199). In another embodiment, a polynucleotide encoding a binding molecule of the present invention comprises a nucleotide sequence encoding a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein the scFv polypeptide comprises an adalimumab VH and an adalimumab VI, except for amino acid substitutions R 16G in VI I, D30S in VH, T52S in VH, L54R in VL, and V83E in VL (also used herein as "the anti-TNF/TWEAK BsAb." the quintuple mutant or XWU198).

[0203] The stabilizing mutations or substitutions in the VI I domain of the stabilized anti-

TWEAK scFv molecule of the present invention can be at a position selected from the group consisting of amino acids 60, 62, and both, according to the Kabat numbering system. The stabilizing mutations or substitutions in the VL domain of the stabilized anti-TWEAK scFv molecule of the present invention can be at amino acid 12. according to the Kabat numbering system.

102041 In another embodiment, a nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule of the present invention comprises a nucleotide sequence encoding a stabilized anti-TWEAK scFv polypeptide which specifically binds to human T WEAK, wherein the stabilized anti-TWEAK scFv polypeptide comprises a P2D10 VH and a P2D10 VL except for one or more stabilizing substitutions or mutations selected from the group consisting of (i) amino acid substitution at Kabat position 12 from P to E of VL (P12E); (ii) amino acid substitution at Kabat position 60 from P to N of VL (P60N); (iii) amino acid substitution at Kabat position 62 from T to K of VL, (T62K); and any combinations thereof.

[02051 In other embodiments, a nucleic acid molecule comprising a polynucleotide sequence encoding the stabilized TNF-alpha or TWEAK binding scFv molecule is altered without altering the amino acid sequence encoded thereby. For instance, the sequence can be altered for improved codon usage in a given species, to remove splice sites, or the remove restriction enzyme sites. Sequence optimizations such as these are described in the examples and are well known and routinely carried out by those of ordinary skill in the art. Codon usage tables are readily available, for example, at http://phenotype.biosci.umbc.edu/codon/sgd/index.php or at http://www.kazusa.or.jp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y.. et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).

[0206] Also included in the present invention is a nucleic acid molecule comprising a polynucleotide sequence encoding a multispecific binding molecule disclosed herein. For example, in one embodiment, the present invention includes a nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule that is monovalent, multivalent, monospecific, or multispecific. In another embodiment, a polynucleotide of the invention comprises a nucleotide sequence encoding a multispecific binding molecule comprising a first binding region comprising a stabilized anti-TNF-alpha or anti-TWEAK scFv molecule disclosed herein which specifically binds to TNF-alpha or TWEAK and a second binding region which specifically binds to a different epitope or a heterologous (different) molecule. For example, the present invention includes a polynucleotide sequence comprising a nucleotide sequence encoding a bispecific anti-TNF-alpha and TWEAK antibody which comprises a stabilized TNF-alpha binding scFv molecule disclosed herein and an anti-TWEAK antibody (e.g., hP2D10 or P5G9) or a stabilized TWEAK binding scFv molecule disclosed herein and an anti-TNF-alpha antibody (adalimumab).

1 2071 The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 77:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PGR. [0208] Once the nucleotide sequence and corresponding amino acid sequence of the binding molecule, fragment, variant, or derivative thereof is determined, its nucleotide sequence can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PGR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons. NY ( 1998), which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

[0209] A nucleic acid molecule comprising a polynucleotide sequence encoding a binding molecule of the present invention can be composed of a polyribonucleotide or polydeoxribonucleotide. which can be unmodified RNA or DNA or modified RNA or DNA. For example, a nucleic acid molecule comprising a polynucleotide sequence encoding a stabilized TNF-alpha or TWEAK binding scFv molecule can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA. and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, a polynucleotide encoding a stabilized TNF-alpha or TWEAK binding scFv molecule can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding a stabilized TNF- alpha or TWEAK binding scFv molecule can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0210] A nucleic acid molecule comprising a polynucleotide sequence encoding a non- natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PC R -mediated mutagenesis. Conservative amino acid substitutions are made at one or more non-essential amino acid residues.

V II. VECTORS/HOST CELLS/METHODS OF MAKING BINDING MOLECULES

[021 1 ] Following manipulation of the isolated genetic material to provide binding molecules of the invention, polynucleotides encoding binding molecules of the invention are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired quantity of TNF-alpha or TWEAK binding molecule.

[0212] Recombinant expression of a binding molecule comprising one or more stabilized anti-TNF-alpha or anti-TWHA scFv molecules that specifically bind to TNF-alpha or TWEAK requires construction of an expression vector containing a polynucleotide that encodes the binding molecule. Once a polynucleotide encoding a binding molecule (or a chain or portion thereof) of the invention has been obtained, the vector for the production of the binding molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a binding molecule encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing binding molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, but are not limited to. in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention thus provides replicable vectors comprising a nucleotide sequence encoding a binding molecule o the invention, or a chain or domain thereof, operably linked to a promoter. Such vectors include, but are not limited to, the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036: and U.S. Pat. No. 5, 122.464, which are incorporated by reference in their entireties) and the nucleotide encoding the binding molecule (or chain or domain thereof) can be cloned into such a vector for expression of the entire binding molecule.

[021 1 Where a binding molecule of the invention is a dimer, the host cell can be co- transfected with two expression vectors of the invention, the first vector encoding a first polypeptide monomer and the second vector encoding a second polypeptide monomer. The two vectors can contain identical selectable markers which enable equal expression of the monomers. Alternatively, a single vector may be used which encodes both monomers. In embodiments the monomers are antibody light and heavy chains, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler. Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the monomers of a binding molecule may comprise cDNA or genomic ON A. The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such vectors include, but are not limited to, plasmids. phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

[0214] For the purposes of this invention, numerous expression vector systems can be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baeulovirus. retroviruses (RSV, MM TV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of trans fected host cells. Certain markers provide for prototrophy to an auxotrophic host, biocide resistance (e.g. , antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can cither be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements for optimal synthesis of mRNA can also be included. These elements include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0215] In other embodiments the cloned variable region genes are inserted into an expression vector in fusion with heavy and light chain constant region genes (e.g. , human) as discussed above. In one embodiment, this is effected using a proprietary expression vector of Biogen ID FX', Inc.. referred to as NEOSPLA (disclosed in U.S. patent 6,159,730, which is incorporated by reference in its entirety). This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G41 8 containing medium and methotrexate amplification. Of course, any expression vector which is capable of eliciting expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to plasmids pcDNAS, pHCMV/Zeo, pCR3.1 , pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each o which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., > 30 pg/cell/day. Other exemplary vector systems are disclosed e.g. , in U.S. Patent Nos. 6.41 3.777 and 7.569.362. incorporated herein by reference in their entireties.

[0216] In other embodiments the binding molecules of the invention are expressed using polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety. In these novel expression systems, multiple gene products of interest such as heavy and light chains of antibodies are produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of TNF-alpha or TWEAK binding molecules thereof in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6.193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of TNF-alpha or I WEAK binding molecules disclosed in the instant application.

[0217] More generally, once the vector or DNA sequence encoding a monomeric subunit of the TNF-alpha or TWEAK binding molecule has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped ON A. microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors. Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction into the host is via electroporation. The host cells harboring the expression construct are grown under conditions appropriate to the production of the binding molecule, and assayed for binding molecule synthesis. Exemplary assay techniques include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence- activated cell sorter analysis (FACS), immunohistochemistry and the like.

[0218] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a binding molecule for use in the methods described herein. Thus, the invention includes host cells containing a polynucleotide encoding a binding molecule of the invention, or a monomer or chain thereof, operably linked to a heterologous promoter. In other embodiments for the expression of double-chained or dimeric binding molecules, vectors which separately encode binding molecule chains are co-expressed in the host cell for expression of the entire binding molecule, as detailed below.

[0219] As used herein, "host cells^'" refers to cells which harbor vectors constructed using recombinant ON A techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of binding molecules from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of binding molecule unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" means either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.

102201 A variety of host-expression vector systems can be utilized to express binding molecules for use in the methods described herein. Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can. when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g. , E. colx, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing binding molecule coding sequences; yeast (e.g. , Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing binding molecule coding sequences: insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing binding molecule coding sequences: plant cell systems infected with recombinant virus expression vectors (e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing binding molecule coding sequences; or mammalian cell systems (e.g.. COS, CHO, BLK, 293, 3 13 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. , metal lothionein promoter) or from mammalian viruses (e.g. , the adenov irus late promoter; the vaccinia virus 7.5 promoter). For example, mammalian cells such as Chinese hamster ovary cells (CHO) in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus are an effective expression system for antibodies and other binding molecules (Foecking et ai, Gene 45: 101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).

[02211 The host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB l 1 (Chinese Hamster Ovary lines. DHFR minus). 1 1 HI . A (human cervical carcinoma). CVI (monkey kidney line). COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney). MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma). P3x63-Ag3.653 (mouse myeloma). BFA- l c l BPT (bovine endothelial cells), RAJ I (human lymphocyte) and 293 (human kidney). In certain embodiments, the host cell used for the invention is CHO cells. I lost cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.

[02221 In certain embodiments, a host cell strain is chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. , glycosylation) and processing (e.g. , cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

[0223] For long-term, high-yield production of recombinant proteins, stable expression can be used. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements {e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are allowed, e.g., to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method is advantageously used, for example, to engineer cell lines which stably express the binding molecule.

[0224] In various embodiments, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro. which confers resistance to hygromycin. Methods commonly known in the art o recombinant DNA technology which can be used are described in Ausubcl et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopo!i et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons. NY ( 1 94): Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981), which are incorporated by reference herein in their entireties.

[0225] The expression levels of a binding molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system expressing the binding molecule is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the binding molecule, production of the binding molecule will also increase (Grouse et al., Mol. Cell. Biol. 3:251 (1983)).

102261 In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.

[0227] Genes encoding TNF-alpha or TWEAK binding molecules of the invention can also be expressed non-mammalian cells such as bacteria or insect: or yeast or plant cells. Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae. such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of binding molecules are desired, the subunits will then self-assemble into tetravalent binding molecules (e.g. tetravalent antibodies (WO02/096948A2, which is incorporated herein by reference in its entirety)).

[0228] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the binding molecule being expressed. For example, when a large quantity of such a protein is to be produced for the generation of pharmaceutical compositions of a binding molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the binding molecule coding sequence is ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 73:3101-3109 (1985); Van Heeke & Schuster, J. Biol Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0229] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g., Pichia pastor is.

[§230] For expression in Saccharomyces, the plasmid YRp7, for example. (Stinchcomb et al., Nature 252:39 (1979); ingsman et al., Gene 7: 141 (1979); Tschemper et al., Gene 7 : 1 57 ( 1980)) is commonly used. This plasmid already contains the TRP 1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics #5:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0231] In an insect system, Autographa calif arnica nuclear polyhidrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0232] Once a binding molecule of the invention has been recombinantly expressed, it can be purified by any method known in the art for puri fication of a binding molecule, for example, by chromatography (e.g. , ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Alternatively, a method for increasing the affinity of binding molecules (e.g. antibodies) of the invention is disclosed in US 2002 0123057 Al .

VIII. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION METHODS OF

THE INVENTION

[0233] Methods of formulating and administering a binding molecule of the present invention, e.g., a stabilized TNF-alpha binding molecule, a stabilized TWEAK-binding molecule, or anti- TNF-alpha and TWEAK bispecific antibody or immunospecific fragment thereof to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the binding molecule, e.g., binding polypeptide, e.g., anti- TNF-alpha and TWEAK bispecific antibody or immunospecific fragment thereof can be, for example, oral, parenteral, by inhalation or topical. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration including, without limitation, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrathecal, rectal, vaginal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intracranial, intranasal, intraspinal, epidural and intrasternal injection and infusion. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. In some methods, antibodies are administered as a sustained release composition or dev ice, such as a Medipad™ device.

1 2 4] Usually, a suitable pharmaceutical composition for injection comprises a buffer

(e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, binding molecules, e.g. , binding polypeptides, e.g. , anti- TNF-alpha and TWEAK bispecific antibodies or immunospecific fragments thereof can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.

102 51 Preparations for parenteral administration include without limitation sterile aqueous or non-aqueous solutions, microemulsion, dispersion, liposome, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01 -O. l M and 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers. electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives are typically also present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0236] More particularly, pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation o sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action o microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol. polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy), Lippincott Williams & Wilkins. 21 st ed. (2005).

[0237] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. 10238] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g. , a binding molecule of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods f preparation include vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations are typically packaged and sold in the form of a kit such as those described in co-pending U.S. S.N. 09/259.337 (US-2002-0102208 Al), which is incorporated herein by reference in its entirety. Such articles of manufacture ordinarily have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or inflammatory disorders.

[0239] Effective doses of the compositions of the present invention, for treatment of inflammatory or autoimmune disorders as described herein, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non- human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0240] For treatment of inflammatory or autoimmune disorders with an antibody or fragment thereof, the dosage can range, e.g. , from about 0.0001 to 100 mg/kg, of the host body weight.

[0241 ] TNF-alpha or TWEAK specific binding molecules disclosed herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of target polypeptides or target molecules in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 μg/ml. Alternatively, binding molecules can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. The half-life of a binding molecule can also be prolonged via fusion to a stable polypeptide or moiety, e.g., albumin or PEG. In one embodiment, the binding molecules of the invention can be administered in unconjugated form, In another embodiment, the binding molecules for use in the methods disclosed herein can be administered multiple times in conjugated form. In still another embodiment, the binding molecules of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.

102421 The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising antibodies or a cocktail thereof are administered to a patient not already in the disease state or in a pre-disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose.^" In this use, the precise amounts again depend upon the patient's state of health and general immunity. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

10243] In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of binding molecule, e.g., antibody per dose, at relatively short intervals is sometimes required until progression o the disease is reduced or terminated, or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

102441 In one embodiment, a subject can be treated with a nucleic acid molecule encoding a TNI ^'-alpha or TWEAK specific antibody or immunospecific fragment thereof (e.g. , in a vector, e.g., a viral vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μ« DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.

102451 In certain embodiments, a binding molecule of the present invention is used to replace any existing agents that a subject has been taking or is known to be resistant or to be non-responsive to. [0246] In one embodiment, a binding molecule of the present invention is administered to non-responders to existing TNF-alpha inhibitors, for example, etanercept, infliximab, certolizumab pegol, golimumab, adalimumab, pentoxyfylline, or bupropion. In one aspect, subjects who have rheumatoid arthritis, or who are at risk for RA, or who have or at risk for another disorder described herein, can be evaluated for a parameter predictive of their ability to respond to a particular agent (e.g., a biologic DMARD), e.g., their ability to respond to a TNF-alpha inhibitor such as etanercept, infliximab, certolizumab pegol, golimumab, adalimumab, pentoxyfylline, or bupropion. For example, the parameter can be the presence or absence of a nucleotide in a gene encoding TNF-alpha. Subjects who are indicated to be less or non- responsive to a particular agent can be administered an alternative agent. In another aspect, certain subjects have failed to respond to any one of the TNF-alpha inhibitors or have developed resistance to any one of other TNF-alpha inhibitors. For example, subjects who are indicated as non- responsive to etanercept or have developed resistance to etanercept would be administered a bispecific TNF-alpha/TWEAK antibody of the present invention either in combination with, or instead of, etanercept.

[0247] In addition to the TNF-alpha inhibitors, a variety o treatments for inflammatory or autoimmune diseases, e.g., inflammatory bowel disease (IBD), are available. For example, traditional therapeutics known to treat IBD include, but are not limited to. aminosalicylates, corticosteroids, immunomodulators, antibiotics, or TNF antagonists.

[0248] A binding molecule comprising stabilized TNF-alpha or TWEAK specific scFv polypeptide can also be administered in lieu of or in combination with any treatments used for IBD such as aminosalicylates, e.g., 5- aminosalicylic acid (5--ASA), sulfasalazine (AZULFIDINE^®), olsalazine (DIPENTUM^®), mesalamine (ASACOL^®, PENTASA^®, APRISO™, LIALDA^®), and balsalazide (COLAZAL™).

[0249] Another example of the treatment used for IBD that can be used in lieu of or in combination with a binding molecule of the present invention is corticosteriods. Examples of corticosteroids are prednisone (DELTASONE®), methylprednisolone (MEDROL^®), budesonide (ENTOCORT^® EC), or hydrocortisone.

[0250] Additional examples of the treatment used for IBD that can be used instead of or in combination with a binding molecule of the present invention include, but are not limited to, immunomodulators. e.g., zathioprine (IMURAN^®, AZASAN^®), 6- mercaptopurine (6-MP, PURINETHOL ), cyclosporine A (SANDIMMUNE , NEORAL^®) tacrolimus (PROGRAF^®), or methotrexate (MTX^®, RHEUMATREX^®, or MEXATE^®) or antibiotics, e.g., metronidazole (FLAGYL^®) or ciprofloxacin (CIPRO^®).

[02511 In certain embodiments, a binding molecule of the present invention can be administered instead of or in combination with any treatments used for RA, e.g. a TWEAK antagonist, e.g., an anti-TWEAK antibody, e.g. , a binding molecule comprising a stabilized anti-TWEAK scFv molecule derived from hP2D10.

[0252] An example of the therapeutics used for RA is disease modifying anti-rheumatic drugs ( DMARDs). which include, but arc not limited to. PLAQUENIL® (hydroxychloroquine), AZULFIDINE® (sulfasalazine), ARAVA® (leflunomide), RHEUMATREX® (methotrexate), IMURAN® (azathioprine), CYTOXAN® ( cyclophosphamide ). D-penicillamine. minocycline, or cyclosporine. For example, it has been observed that the withdrawal rate from DMARD treatment in rheumatoid arthritis increases with the length of time the patient has been receiving the drug and that a number of these withdrawals relate to loss of efficacy (see, e.g., Annals of the Rheumatic Diseases (2003) 62:95-96). Accordingly, a binding molecule of the present invention can be administered to a subject either instead of the traditional therapeutics for IBD or the DMARD, or in combination with it.

[0253] A binding molecule comprising stabilized TNF-alpha or TWEAK specific scFv polypeptide can also be administered in lieu of or in combination with nonsteroidal antiinflammatory drugs including salicylates (NSAIDs), such as aspirin.

[02541 In some patients, gold may produce clinical remission and decrease the formation of new bony erosions. Parenteral preparations include gold sodium thiomalate or gold thioglucose. Gold should be discontinued when signs of toxicity appear. Minor toxic manifestations (e.g. , mild pruritus, minor rash) may be eliminated by temporarily withholding gold therapy, then resuming it cautiously about 2 weeks after symptoms have subsided. A binding molecule of the invention can be administered when gold is being discontinued or when a gold chelating drug (such as dimercaprol) is being administered to counteract gold toxicity.

[0255] Hydroxychloroquine is known to control symptoms of mild or moderately active inflammatory or autoimmune disease, e.g., RA. Toxic effects of hydroxychloroquine usually are mild and include dermatitis, myopathy, and generally reversible corneal opacity. However, irreversible retinal degeneration has been reported. Accordingly, hydroxychloroquine can be withdrawn or reduced and replaced or administered in combination with an anti-TNF-alpha and TWEAK bispecific antibody, e.g., upon detection of one or more of these side effects.

[0256] Oral penicillamine may have a benefit similar to gold. Side effects requiring discontinuation are more common than with gold and include marrow suppression, proteinuria, nephrosis, other serious toxic effects (e.g., myasthenia gravis, pemphigus. Goodpasture's syndrome, polymyositis, a lupus-like syndrome), rash, and a foul taste. Oral penicillamine can be withdrawn or reduced and replaced or administered in combination with an anti-TNF-alpha and TWEAK bispecific antibody, e.g., upon detection f one or more of these side effects.

[0257] Steroids are highly effective short-term anti-inflammatory drugs. However, their clinical benefit for inflammatory diseases or autoimmune diseases often diminishes with time. Steroids do not predictably prevent the progression of joint destruction. Furthermore, severe rebound often follows the withdrawal of corticosteroids in active disease. Accordingly, a binding molecule of the invention can be administered, prior to withdrawal, during withdrawal, or subsequent to complete withdrawal. Other side effect which can trigger withdrawal and use of a binding molecule of the present invention include peptic ulcer, hypertension, untreated infections, diabetes mellitus, and glaucoma.

[0258] Immunosuppressive drugs can be used in management of severe, active inflammatory diseases or autoimmune disease. However, major side effects can occur, including liver disease, pneumonitis, bone marrow suppression, and, after long-term use of azathioprine. malignancy. Withdrawal from immunosuppressive drags can include administering a binding molecule of the present invention, e.g. , upon detection, of a side effect. Alternatively or in combination with a binding molecule of the present invention can be administered to a subject who is receiving another treatment for RA, e.g., one of the above treatments. The combination of the treatment and the a binding molecule of the present invention can be used to achieve additional therapeutic benefit and, optionally, to reduce the dosage of the other treatment. As result, side effects and other issues can be mitigated.

[0259] In certain embodiments, a pharmaceutical composition comprising a binding molecule of the present invention disclosed herein further comprises an additional TNF- alpha inhibitor disclosed above, DMARDs disclosed above or any other agents (e.g., NSAIDs, Gold, Hydroxychloroquine, Oral penicillamine, steroids, or any other immunodepressive drugs).

[0260] The pharmaceutical composition described herein, e.g. , an anti-TNF-alpha and

TWEAK bispecific antibody monotherapy or a combination therapy, can be used to treat a subject who has one or more severe complications of inflammatory disorder, e.g. , RA.

[0261] In this regard, a combination of a binding molecule comprising a stabilized TNF- alpha specific scFv molecule or a stabilized TWEAK specific scFv molecule disclosed above and another pharmaceutical active ingredient is administered in any order and within any time frame that provides a therapeutic benefit to the patient. In one embodiment, the TNF-alpha inhibitor or DMARDs and a TNF-alpha specific binding molecule or a TWEAK specific binding molecule of the present invention are administered in any order or concurrently. In some embodiments, TNF-alpha or TWEAK specific binding molecules of the present invention will be administered to patients that have previously undergone treatments with DMARDs or TNF-alpha inhibitors. In other embodiments, binding molecules of the present invention are administered substantially, simultaneously, concomitantly, sequentially, extensively, contemporaneously, or concurrently with DMARDs or TNF-alpha inhibitors. In other embodiments, binding molecules of the present invention are administered within 10, 8, 6, 4, or 2 months of any DMARDs or TNF-alpha inhibitors administration. In still other embodiments binding molecules are administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment. In certain embodiments a binding molecule is administered within 5, 4, 3, 2 or 1 days f the selected DMARDs or TNF-alpha inhibitors. The two agents or treatments can be administered to the patient within a matter of hours or minutes (i.e. substantially simultaneously).

[0262] The amount o the traditional therapeutics for IBD, DMARDs or other TNF-alpha blocking agents to be used in combination with a TNF-alpha or TWEAK specific binding molecule of the present invention varies by subject and is administered according to what is known in the art.

[0263] In keeping with the scope of the present disclosure, a binding molecule of the present invention can be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The binding molecules of the present invention can be administered to such human or other animal in a conventional dosage form prepared by combining the binding molecule of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of binding molecules according to the present invention may prove to be particularly effective.

IX. METHODS OF TREATMENT USING COMPOSITIONS COMPRISING TNF- ALPHA OR TWEAK BINDING MOLECULES OF THE INVENTION

102641 One embodiment of the present invention provides methods for reducing or decreasing the number of active TNF-alpha or TWEAK in circulation in an animal in need thereof by administering an effective amount of a binding molecule of the invention.

In another embodiment, the present invention includes methods of reducing or inhibiting

TNF-alpha or TWEAK activity or binding to its receptors in an animal in need thereof.

Also included are methods of treating an inflammatory or autoimmune disease or disorder, e.g., . rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, Sjogren's syndrome, inflammatory myositis, I .angerhans-cell histiocytosis, adult respiratory distress syndrome/bronchiolitis obliterans. Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, noninfectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplasia syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, and a combination of two or more of said diseases or disorders,, in an animal suffering from such disease or predisposed to contract such disease, the method comprising administering to the animal an effective amount of a binding molecule or composition of the invention described herein. The conditions, diseases, or disorders that can be prevented, treated, or ameliorated by a binding molecule of the present invention is also described in U.S. Application Publication No.

2008/0187544, which is incorporated by reference in its entirety . [0265] A binding molecule comprising a stabilized TNF-alpha or TWEAK specific scFv molecule of the invention can be prepared and used as a therapeutic agent that reduces or decreases the TNF-alpha level or the TWEAK level or prevents or inhibits TNF-alpha or TWEAK activity involved in an inflammatory disease or response, respectively. Binding molecules according to the invention can be used in combination with other therapeutic agents.

[0266] The present invention provides methods for inhibiting TNF-alpha receptor activation or preventing binding of TNF-alpha to TNF-alpha receptor I or II in a mammal, comprising administering to the mammal an effective amount of a binding molecule which comprises a stabilized TNF-alpha specific scFv polypeptide of the invention, e.g. , a bi speci ic anti-TNF-alpha/TWEAK tetravalent antibody comprising two stabilized anti- TNF-alpha scFv polypeptides specifically or preferentially binding to TNF-alpha. The present invention also provides methods for inhibiting TWEAK receptor activation or preventing binding of TWEAK to its receptor in a mammal, comprising administering to the mammal an effective amount of a binding molecule which comprises a stabilized TWEAK specific scFv polypeptpidc of the invention, e.g.. a bi specific anti- TWEAK/TNF-alpha tetravalent antibody comprising two stabilized TWEAK scFv polypeptides specifically or preferentially binding to TWEAK.

[0267] The present invention is more specifically directed to a method of TNF-alpha inactivation comprising administering to the mammal an effective amount of a binding molecule which comprises a stabilized TNF-alpha specific scFv polypeptide of the invention, e.g. , a bispecifie a n t i - T N F -a 1 p h a/T W E A K tetravalent antibody comprising two stabilized anti-TNF-alpha scFv polypeptides specifically or preferentially binding to TNF-alpha. The present invention is also directed to a method of TWEAK inactivation comprising administering to the mammal an effective amount of a binding molecule which comprises a stabilized TWEAK specific scFv polypeptide of the invention, e.g. , a bispecifie anti-TWEAK/TNF-alpha tetravalent antibody comprising two stabilized anti- TWEAK scFv polypeptides specifically or preferentially binding to TWEAK.

[0268) In one embodiment, the present invention is more specifically directed to a method of reducing the toxicity of TNF-alpha inactivation in a mammal comprising administering to the mammal an effective amount of a binding molecule which comprises a stabilized TNF-alpha specific scFv polypeptide of the invention, e.g. , a bispecifie anti- TNF-alpha/TWEAK tetravalent antibody comprising two stabilized anti-TNF-alpha scFv polypeptides specifically or preferentially binding to TNF-alpha, wherein the toxicity is reduced compared to administration of a conventional TNF-alpha blocking agents, e.g. , adalimumab. Examples of toxicities mediated by TNF-alpha blocking agents include, but are not limited to serious infection caused by viruses, fungi, or bacteria, e.g. , tuberculosis, occurrence of solid tissue cancers, lymphoma, or cardiac failure. In another embodiment, the present invention is more specifically directed to a method of reducing the toxicity of TWEAK inactivation in a mammal comprising administering to the mammal an effective amount o a binding molecule which comprises a stabilized TWEAK specific scFv polypeptide of the invention, e.g. , a bispeci fie anti-T WE AK/TN F-alpha tetravalent antibody comprising two stabilized anti-TWEAK scFv polypeptides specifically or preferentially binding to TWEAK, wherein the toxicity is reduced compared to administration of a conventional TWEAK blocking agents, e.g. , P2D 10.

X. DIAGNOSTIC OR PROGNOSTIC METHODS USING TNF-ALPHA-SPECIFIC scFv BINDING MOLECULES AND NUCLEIC ACID AMPLIFICATION ASSAYS

[0269] Stabilized scFv molecules that specifically bind to TNF-alpha or TWEAK, binding molecules comprising the scFv molecules, or compositions of the invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of TNF-alpha or TWEAK. TNF-alpha expression or TWEAK expression is increased in inflammatory disease and other autoimmune conditions.

[0270] The binding molecules are useful for diagnosis, treatment, prevention and/or prognosis o inflammatory disorders in mammals, e.g. , humans. Such disorders include, but are not limited to, rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis, Sjogren's syndrome, inflammatory myositis, Langerhans-cell histiocytosis, adult respiratory distress syndrome/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, noninfectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplasia syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, a combination of two or more of said diseases or disorders, and/or as described under elsewhere herein.

[0271] In certain embodiments, a binding molecule comprising a stabilized TWEAK specific scFv prevent or treat a neoplastic disorder, e.g., a cancer in a subject. In one embodiment, the cancer is a solid cancer, e.g., a carcinoma, e.g., an adenocarcinoma. In another embodiment, the cancer can be selected from the group consisting of pancreatic cancer, breast cancer, lung cancer, prostate cancer, colon cancer, colorectal cancer, skin cancer, ovarian cancer, cervical cancer, renal cancer, or adenocarcinoma. In some embodiments, the cancer is metastatic, advanced and/or late-stage (e.g., stage III or later). In other embodiments, the cancer is in stage II or earlier.

[02721 Binding molecules of the invention can be used to detect particular tissues expressing increased levels of TNF-alpha or TWEAK. These diagnostic assays can be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.

[0273] Thus, the invention provides a diagnostic method useful during diagnosis of an inflammatory disorder or other autoimmune disorders, which involves measuring the expression level of TNF-alpha or TWEAK protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with standard TNF-alpha or TWEAK expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.

[0274] TNF-alpha specific or TWEAK specific binding molecules of the present invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g.. see Jalkanen. et al., J. Cell. Biol. 707:976-985 (1985); Jalkanen, et al, J. Cell Biol. 705:3087-3096 (1987)). Other antibody-based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^mIn), and technetium (⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Suitable assays are described in more detail elsewhere herein. [0275] In an additional embodiment, a binding molecule of the invention is used to quantitatively or qualitatively detect the presence of TNF-alpha or TWEAK gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoresence techniques employing a fluorescently labeled binding molecule coupled with light microscopic, flow cytometric, or fluorimetric detection.

XI. IMMUNOASSAYS

[0276] A stabilized anti-TNF-alpha or TWEAK scFv molecule disclosed herein or a binding molecule comprising the stabilized anti-TNF-alpha or TWEAK scFv molecule can be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and noncompetitive assay systems using techniques such as western blots, radioimmunoassays, El . ISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g. , Ausubel et al, eds. Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated by reference herein in its entirety).

[0277] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al , ed., Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al , ed., Cold Springs Harbor Laboratory. New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed.. ( 1984); Mullis et al. U.S. Pat. No: 4.683.195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.. ( 1 987): Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y., (1986); and in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

[02781 General principles of antibody engineering are set forth in Antibody Engineering,

2nd edition. C.A.K. Borrebaeck. Ed.. Oxford Univ. Press ( 1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al.. Eds., IRL Press at Oxford Univ. Press, Oxford. Eng. ( 1995 ). General principles of antibodies and antibody-hapten binding are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed.. Sinauer Associates, Sunderland, MA ( 1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall. New York. NY (1984). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (eds) , Basic and Clinical -Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).

[0279] Standard reference works setting forth general principles of immunology include

Current Protocols in Immunology, John Wiley & Sons. New Y ork; Klein. J., Immunology.' The Science of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R., et al, eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., "Monoclonal Antibody Technology" in Burden, R., et al , eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology 4^th ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, 1 1. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D., Immunology 6^th ed. London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier Health Sciences Division (2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003).

All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

EXAMPLES

Example 1. Construction of a Conventional anti-TNF-alpIia scFv molecule

[0281] The conventional anti-TNF scFv molecule derived from adalimumab was synthesized at Codon Devices, based on the sequence of MAb D2E7 (adalimumab, HUMIRA^®) found in U.S. Patent No. 6,258,562 (Bl), which is incorporated herein by reference in its entirely. The resulting plasmid OBP01 H4L was cut with Ndel and Sail and ligated into pIEH095 to generate plasmid pIEH254. Plasmid pIEH254 encodes a molecule containing an amino terminal 18 residues derived from gplll to direct the scFv polypeptides to the cytosol, the VI I domain of D2E7, a linker consisting of four repeats of GGGGS (SEQ I NO: 46). the D2E7 VL domain, and a C-terminal VDHl lHUIII I sequence (SEQ ID NO: 47) used as a detection tag. The nucleic acid sequence of the insert in pIEH254 is presented as SEQ ID NO: 24. and the amino acid sequence encoded by the insert is presented as SEQ ID NO: 25.

Example 2. Construction of a Stabilized anti-TNF scFv molecule with

Improved Thermal Stability

A) Thermostability Screening - Identification of Stabilizing Mutations in VH

[0282] As shown in Figure I B. initial characterization of the wild-type D2E7 scFv, pIEH254, by thermal challenge assay, revealed a T₅₀ of 52 °C, well below the target T₅₀ of ~ 65 °C. Analysis of puri fied D2E7 scFv pIEH254 by differential scanning calorimetry (DSC) revealed a large deficit in thermostability relative to the parental Fab fragment of adalimumab. DSC thermal melt profiles of the D2E7 scFv and adalimumab Fab are shown in Figure 1 Λ. While the Adalimumab Fab unfolds cooperatively, with a melting temperature, Tm. o 74 °C, the D2 E7/adal i mumab scFv unfolds as two species (VH and VL), with Tms of 57 and 62 °C, also below the target Tm of ~ 65 °C. Therefore. D2E7 scFv required stabilization mutagenesis before incorporation into a bispeeific antibody.

[0283J In order to obtain an scFv molecule with the desired themodynamic property (e.g.,

Tm >65 °C), a thermostability screen was performed by the thermal challenge assay, essentially as described in U.S. Patent Publication No. US 2008-0050370 A 1. which is incorporated herein by reference in its entirety. A number of stabilized mutants were identified from the screen (pBRM034, pBRM035, pIEH263, pi Fl 1264. pIEH265, pIEH266, pIEH270, pIEH271. pIEH273, pIEH274, pIEH275, pIEH277, pIEH278, pIEH279, pIEH280, pIEH281). See Table 3. All but three of the mutations were in the VH domain, and the increases in T₅₀ were between 1 and 6 °C. In the wild-type D2E7 scFv, VH is the domain of lower thermostability and thus the domain required to be stabilized to see an increase in assay Τ₅₀ (Table 5). Stabilization of VH alone would not be sufficient to fully stabilize the scFv, as VL has a DSC Tm of 62 °C. Any VL- stabilizing mutations, unless they improved VH stability, would be invisible in the thermal challenge assay. For this reason, full stabilization of the molecule required a separate approach to identify VL mutations.

Table 3. Mutation Sequences of scFv Polypeptides containing Adalimumab VH

B) Thermostability Screening - Identification of Stabilizing Mutations in VL

[0284] To identify stabilizing mutations in VL, a small set of mutants in the list of VL designs from sequence-based methods (covariation analysis, consensus analysis) was targeted for analysis. Selection was based on the number of covariation links gained by each mutation, and/or the relative frequency of the wild-type residue at a given position. The assumption was made that mutations with a larger number of gained covariation links would have a higher probability of stabilizing the VL domain. Similarly, mutations at positions where the wild-type amino acid relative frequency is lower than at other positions were assumed to have a greater probability of stabilization. Six mutations were selected, at four di ferent positions in VL (Table 4). A 2^nd tier of VI, mutants, as well as four VH mutants, were also chosen, but were not tested owing to the success of the initial set of VL mutants. D2E7 VL mutants were cloned by site-direct mutagenesis of pIEH254, expression in E. coli strain W31 10 (ATCC, Manassas, Va. Cat. # 27325), and purification by conventional chromatography.

Table 4. Prioritization and Selection of Potentially Stabilizing Mutations in

Adalimumab scFv VL and VH domains for biophysical analysis screen

[0286] Purified D2E7 scFv VL mutants L54R, L54Y, V83E, V83T, V85D and R90Q

(Kabat numbering) were subjected to differential scanning calorimetry, A summary is shown in Table 5.

[0287] Tabic 5. Thermostability and TNF-alpha binding of Adalimumab VI 1 and VI . mutants

Plasmid Mutation T50 T_m (V_H) T_m AT_m

(°C) (°C) (V_H) (Vu) (V_L) (TNFct

(°C) (°C) (°C) ) (P )

NA Adalimumab Fab 74.1 74.1

plEH254 Wild-type scFv 52 57 0 62 0 138 ±

24

(n=4)

pDAAOOI VL L54R ND 56.7 -0.3 65 3 134

pDAA002 VL L54Y ND 53.6 -3.4 53.6 -8.4 ND

pDAA003 VL V83E 53 56.4 -0.6 65.8 3.8 146

pDAA004 VL V83T ND 56.6 -0.4 59 -3 ND

pDAA005 VL V85D ND 53.2 -3.8 53.2 -8.8 ND

pDAA006 VL R90Q ND 44.3 -12.7 68.9 6.9 753

plEH290 VH R16G, D30S ND 63.3 6.3 63.3 1.3 76

VL L54R

plEH285 VH R16G, D30S ND 64.5 7.5 64.5 2.5 149

VL V83E

plEH282 VH T52S VL V83E 57.4 61.8 4.8 61.8 -0.2 254

plEH286 VH S49A VL V83E 57.3 61.5 4.5 61.5 -0.5 184

plEH291 VH S49A VL L54R 56.5 60.9 3.9 60.9 -1.1 155

PIEH293 VH R16G, D30S 60.2 63.0 6,0 70.0 8.0 104

VL V83E L54R

PIEH294 VH S49A VL V83E 58.2 60.6 3.6 69.9 7.9 ND

L54R

PIEH298 VH T52S, VH 62 66.8 9.8 69.2 7.2 131

R16G, VH D30S,

VL V83E, VL L54R

0288] Three mutants, L54R, V83E and R90Q, were found to have VL domains stabilized by 3.0, 3.8 and 6.9 °C, respectively. While the VH domains of L54R and V83E were relatively unperturbed in their thermostabilities, the VH Tm of R90Q was decreased by 12.7 °C. Solution affinity biacore analysis of the binding of 0.8 nM TNF to scFv showed that neither L54R nor V83E is perturbed in TNFa binding affinity. The assay is described in further detail in section C. Wild-type scFv binds with a dissociation constant, KD, of 138 ± 24 pM (n = 4) (Table 5 and Figure 3). L54R and V83E scFv affinities are 134 and 146 pM, respectively (Table 5). R90Q, however, has a weaker affinity of 753 pM. Therefore, L54R and V83E were selected for combination with the VH mutations already identified in the thermal challenge screen. These were tested in further rounds of the thermal challenge assay.

102891 The resulting scFv molecule-encoding constructs, and the accompanying T50 values in the thermal challenge assay, are listed in Table 3 including plasmid pIEH298, which contains five mutations relative to pIEH254. The DSC profiles of a number of the combination VH and VI. mutants, as well as the single VL, mutants, are shown in Figure 2. These mutations are: VH T52S, R16G, and D30S, VL V83E and L54R, according to the Kabat numbering system of each individual variable domain. This quintuple mutant has the greatest thermostability of the tested 1)2 E7 mutants. The ON A sequence of the scFv-encoding insert in pi E l 1298 and the amino acid sequence encoded by the insert are presented as SEQ ID NO: 26 and SEQ ID NO: 27, respectively. The scFv polypeptide expressed by pi EI 1298 is referred to hereinafter as "1EH298".

C) Binding Affinity of adalimumab scFv mutants

102901 E. coli strain W3110 (ATCC, Manassas, Va. Cat. # 27325) was transformed with plasmids encoding conventional and engineered TNF-alpha scFv molecules pi EI 1254 (listed in Table 5) under the control of an inducible ara C promoter. Transformants were grown overnight in expression media consisting of SB (Teknova, Half Moon Bay, CA. Cat. # SO 140) supplemented with 0.02% arabinose and 50 ng m carbenieillin at 30°C. The next morning, the cells were isolated by centrifugation, lysed by microfluidization, and purified by affinity chromatography on a Ni-ΝΤΛ column.

[0291] The binding affinities of the conventional and stabilized anti-TNF scFvs were analyzed by solution phase Biacore for their affinity towards human TNF-alpha as follows. In an equilibrium solution binding assay, test molecules were incubated at varying concentrations with or without recombinant TNF-alpha. The amount of ligand- free test molecule was measured by passing the samples over a S treptav id i n-coated biacore chip bearing biotiny latcd TNF-alpha. The initial rate of binding was used as a measure of the concentration of ligand-free protein. Samples containing only the test molecule at various concentrations were used to build standard curves. [0292] As described above, the single mutations that were incorporated into IEH298 did not perturb binding. Figure 2 shows solution affinity data for binding of 0.8 nM human TNF-alpha to the adalimumab conventional scFv and quintuple mutant stabilized scFv expressed by pIEH298. The binding profiles are identical; conventional scFv binds with KD = 138+/-24 pM (n=4) and the stabilized scFv binds with D = 131 pM. Therefore, the mutations that increase the thermostability of both the VH and VI, domains of the scFv do not perturb TNF-alpha binding. From these data it was concluded that the stabilizing mutations in the quintuple mutant scFv had no appreciable effect on the TNF- alpha binding affinity.

Example 3. Construction of Anti-TNF-alpha and TWEAK Bispecific

Antibodies

[0293] TNF-TWEAK is a bispecific antibody comprising a glycosylated human IgGl heavy chain and a human kappa light chain. The structure of the BsAb is such that the stability engineered anti-TNF-alpha scFv (derived from adalimumab) is appended onto the C-terminus of the hP2D10 anti-TWEAK heavy chain via a (G₄S)3 linker sequence (SEQ ID NO: 34). A schematic of the TNF-TWEAK bispecific antibody (BsAb) is shown in Figure 4.

A) Construction of the TNF-TWEAK BsAb

[0294] The TNF-TWEAK BsAb was constructed by the following method. Construction of the heavy chain-linker-stabilized scFv vector involved several intermediate steps. First, the heavy chain plus some 3' flanking sequence was excised from pCN313 by digestion with Not I. and Bam I II. and this fragment was ligated to pBS SK+ (Stratagene, See Michaelson J.S., et al, niAbs 1 : 128- 141 (2009)) cut with same enzymes. The resulting plasmid pIEH257 was then digested with BsrGl and B ami 11 to remove a portion of the CH3 domain, the stop codon, and the 3' noncoding sequence derived from pCN313. A 0.3 kbp BsrG l -BamH l fragment derived from Hercules BsAb heavy chain plasmid pXWU033 (See U.S. Patent Publication No. 2008-0050370, which is incorporated herein by reference in its entirety) was ligated to the BsrGl -BamHl backbone of pi EI 1257 to create pIEH258. In essence, this recreates the IgGl CH3 domain, removes the stop codon, and adds half of the (G₄S)₃ (SEQ ID NO: 34) linker between the end of CH3 and the scl^'v. [0295] The scFv insert from pIEH298 was amplified with the primers 5' Gly/Ser linker and 3' Add Notl and Hindlll listed in Table 6, and the resulting amplification product was digested with BamHI and Hind III (relevant restriction sites are underlined). This fragment was gel purified and ligated into pIEH258 in order to generate pIEH300. After confirmation of the coding sequence of the hP2D10 HC-(G₄S)₃ linker-scFv encoded by pIIT LlOO, the primers IEH303-F and IEH303-R (Table 3) were used to amplify this coding sequence from pIEFBOO. After digestion of the resulting PGR product with Notl and Dral, the fragment was ligated to pV90rnod (pV90 modified to include a unique 3' Dral site) to generate a pXWU198. The entire sequence of pXWU198 was verified prior to transfer to CCE.

Table 6: Oli onucleotide names and sequences

10296] The nucleotide and amino acid sequences of the light chain and the heavy chain of the anti-TNF-TWEAK BsAb are presented as SEQ ID NO: 18 and SEQ ID NO: 19 (light chain) and SEQ ID NO: 16 and SEQ ID NO: 17 (heavy chain), respectively. Amino acids 1 -238 of SEQ ID NO: 19 contain the light chain sequence. Amino acids 1 -19 of SEQ ID NO: 19 contain the native light chain signal peptide. The mature N-terminus begins with amino acid 20 (Asp). Amino acids 16-474 of SEQ ID NO: 1 7 is the mature heavy chain sequence of hP2D10. Amino acids 1 - 19 (nucleotides in lower case) is the heavy chain signal peptide. The mature N-terminus begins with amino acid 20 (Gly). Amino acids 475-490 is the hinge connecting linker, and amino acids 491 -738 is the stabilized anti- T F-alpha scFv derived from pIEI 1298. All of the references cited above, as well as all references cited herein, arc incorporated herein by reference in their entireties.

B) Construction of the TNF-TWEAK BsAb

[0297] Vectors for expression of the TNF-TWEAK BsAb were constructed as follows.

The heavy and light chain expression cassettes for expression of the TNF-TWEAK BsAb were constructed to be expressed from separate plasmids in a single host cell. [0298] The plasmid expressing the light chain, pCN307, contains an expression cassette for the neomycin phosphotransferase gene (neo) containing the SV40 early promoter and the SV40 polyadenylation site (poly A). The light chain expression cassette contains the human CMV immediate early promoter and first intron as well as the variant human growth hormone polyadenylation sequence. The remaining sequences are for propagation in E. coli.

[02991 The plasmid expressing the heavy chain. pXWU 198- 1 , contains an expression cassette for the dhfr gene which was used as a selectable and amplifiable marker. The dhfr expression cassette contains the SV40 early promoter and SV40 polyadenylation sequence. The heavy chain expression cassette contains the CMV major immediate early promoter and first intron as well as the variant human growth hormone polyadenylation sequence.

[0300] Plasmids pCN307 and pXWU 198 were sequenced in their entirety by the Biogen

Idee DNA Sequencing Group and found to be consistent with the electronically assembled hypothetical sequences.

C). Construction of the TNF-TWEAK BsAb-expressing Cell Line

[03011 The cell line used for expression o the TNF-TWEAK BsAb was a Chinese hamster ovary (CHO) dihydrofolate reductase (dhfr) deficient host cell line.

[0302] Plasmids pCN307 and pXWU 198-1 expressing the light chain of hP2D 10 and heavy chains of the TNF-TWEAK BsAb, respectively, were trans fee ted into the host cell line by electroporation. Transfcctants expressing dhfr were selected using a chemically- defined media deficient in a nucleosides, and the highest producing cell lines were selected for amplification with methotrexate (M I X).

Example 4: Culturing and Purification of the TNF-TWEAK BsAb

[0303] The TNF-TWEAK BsAb was purified from TNF-TWEAK-expressing CHO cells by the following method. The culture supernatant prepared as noted above was applied to a MAbSelect Protein A column attached to an Akta FPLC system (GE Healthcare), and bound antibodies were eluted according to manufacturer specifications. The elution fractions were analyzed by analytical SEC chromatography on an Agilent 1 100 series HPLC equipped with a TOSOH TSK3000SW column and was shown to be pure with 7.7% soluble aggregate. The yield of protein in the MAbSelect pool was 442 mg at 3.7 mg/ml as determined by Abs (280 nm). The pool was dialyzed against PBS. The dialyzed pool was concentrated using an Amicon pressure cell concentrator with a Diaflo 10K MWCO membrane. After 0.22 μ ι filtration, the material was loaded onto a 300 ml Superdex 200 column, pre-equilibrated in PBS, at a flow rate of 3 ml/min. Fractions were collected and analyzed by A280, and fractions containing 100% (H-scFv)2L2 monomer, were pooled. The pure TNF-TWEAK fractions were pooled and concentrated as above. The final material was characterized, for example, by reducing and non-reducing SDS- PAGE, analytical size exclusion chromatography, and N-terminal sequencing. The purified material was filtered, aliquoted and frozen at -70 ^°C.

Example 5. Binding Characteristics of the TNF-TWEAK BsAb

A) Solution Affinity Binding ofthe TNF-TWEAK BsAb to TNF-alpha and TWEAK

103041 The same solution affinity Biacore assay that was used in Example 2 above to test binding affinity o the stabilized scFv to TNF-alpha was also used to measure TNF-alpha and TWEAK solution binding to the TNF-TWEAK BsAb. The results are shown in Figures 5A - 5D. Using 0.8 nM human TNF-alpha and TWEAK, binding curves were measured with 0 to 0.8 nM of the purified TNF-TWEAK BsAb, Adalimumab, and anti- TWEAK hP2D10. The binding curves, fit by quadratic binding functions, indicate Ko < 10 pM for both TNF-alpha and TWEAK binding. This assay, compared to the kinetic surface binding assays described below, has the advantage of measuring binding in solution, thus avoiding contributions of surface avidity effects to the determined affinity that can result in aberrantly tight affinity figures.

B) Kinetic Biacore-based Binding of TNF and TWEAK to the TNF-TWEAK BsAb

[0305] Binding assays measuring association and dissociation of TNF-alpha and

TWEAK analytes with Biacore chip-associated TNF-TWEAK BsAb, adalimumab and hP2D10 (anti-TWEAK monoclonal antibody) were used as an orthogonal method to the solution methods described above. Streptavidin SA chips (GE Healthcare) were coated with biotinylated Goat anti-human IgG Fc, and then used to capture the TNF-TWEAK BsAb, hP2D10, and adalimumab. In the first experiment (Figure 6), studying serial binding of TNF-alpha and TWEAK to the bispecific, 33 nM recombinant His-tagged TNF-alpha and 33 nM recombinant, His-tagged TWEAK were bound to chip-captured TNF-TWEAK BsAb, hP2D 10 and adalimumab. As expected, adalimumab bound only TNF-alpha, while hP2D10 bound only TWEAK. The TNF-TWEAK BsAb was the only molecule to which both cytokines bound. Serial binding demonstrated that the bispecific could bind simultaneously to both TNF and TWEAK. The magnitude of the binding signal was identical when TNF-alpha was bound first (Figure 6A.) or second (Figure 6B.). Accounting for the masses of the different binding components, the magnitudes of the binding signals can be used to deduce binding stoichiometry. The resonance unit Biacore signal, which is directly proportional to the mass of analyte associated with the chip, was identical for TWEAK binding to the bispecific and hP2D10, showing that TWEAK binds to both molecules with the same stoichiometry.

[0306] Interestingly, TNF-TWEAK bound less TNF-alpha per molecule than adalimumab ( Figure 6 A and 6B). The stoichiometry difference is consistent with a single monomer binding to the two scFvs of one bispecific antibody, with two monomers associating separately with the two Fab arms of a single adalimumab molecule.

[0307] In order to evaluate the binding kinetics of the two cytokines to the TNF-TWEAK

BsAb and its two parental antibodies, a second set of kinetic Biacore experiments was then performed, as follows. Various concentrations of TNF-alpha and TWEAK were bound to chip-captured TNF-TWEAK BsAb, hP2D10 and adalimumab. The results are shown in Figure 7A-B (TNF binding to TNF-TWEAK and adalimumab) and 7C-D (TWEAK binding to TNF-TWEAK and hP2D10). TNF and TWEAK showed extremely slow dissociation. However, the association rates of TNF binding were identical between TNF- (^"WEAK (ko„ = 1.6 x 10⁶ M-l s-1) and adalimumab (k_on = 0.95 x 10⁶ M-l s-1) as they were for TWEAK binding to TNF-TWEAK (k_on = 2 x 10" M-l s- 1 ) and hP2D10 (k_on = 2 x 10⁶ M-l s-1). The slow dissociation rates are consistent with an off-rate of at least 1 x 10^"4 s- 1. or slower. This works out to a minimum apparent TNF affinity of - 60 pM for TNF-TWEAK and -100 pM for adalimumab. and minimum TWEAK affinities of ~ 50 pM for both TNF-TWEAK and hP2D10. The stoichiometries (from RU signals and Molecular Weights) are consistent with 0.87 TNF-alpha per the TNF-TWEAK BsAb and 1.5 TNF-alpha per adalimumab, or a roughly two-fold difference. The TWEAK binding stoichiometries were measured to be 1.7 TWEAK molecule per TNF-TWEAK BsAb and hP2D10 molecule. C) Stoichiometry of TNF-alpha binding by the TNF-TWEAK Bispecific Antibody

[0308] To further characterize TNF-TWEAK complexes with TNF-alpha and the stoichiometry of binding, TNF-TWEAK and adalimumab (5,0 μΜ) were dialyzed into 10 niM citrate. 160 inM argininc. pH 6.0, and mixed with 10 μ.Μ TNF-alpha in the same buffer. Samples were incubated for 3 days at 4 C and analyzed by simultaneous analytical size exclusion chromatography (SEC) and static light scattering (LS). The results are shown in Figure 8A and 8B. The SEC profile of TNF-TWEAK + TNF in Figure 8 A shows 5.0 μΜ TNF-TWEAK forms predominantly a species consistent with the stoichiometry of 1 TNF-TWEAK molecule: 1 TNF-alpha trimer. A small amount of larger complexes in the MW range 400,000 - 600,000 are also present. In contrast, adalimumab complexes with TNF-alpha are predominantly of the larger 400,000-600,000 MW class, and no 1 : 1 complex is observed (Figure 8B).

[0309] The stoichiometry of TNF-alpha binding was studied further using the biacore resonance unit analysis method described above. Using the streptavidin SA biacore chip coated with polyclonal goat anti-huIgG Fc, varying amounts of TNF-TWEAK BsAb and adalimumab were captured on the surface. Different volumes of 6.5 nM TNF-TWEAK BsAb or adalimumab were flowed over the biacore chip surface to capture varying resonance units (RUs) of BsAb or antibody, and the signal of saturating TNF-alpha binding was measured. Figure 9A shows that the TNF-TWEAK BsAb (XWU198) captures 1 mole of TNF-alpha trimer per mole of TNF-TWEAK BsAb over the range of captured BsAb amounts. This is consistent with one BsAb being able to bind simultaneously through its two scFv fragments to two monomers of a single TNF-alpha trimer (see Figure 10), which is what is also observed above by SEC/LS analysis. In contrast, adalimumab captured >1.5 mol TNF-alpha irimer per antibody molecule. In the model for TNF-alpha binding to adalimumab, shown in Figure 10, the antibody is unable to bind through its two Fab fragments to two monomers of a single TNF-alpha trimer. The TNF: adalimumab binding ratios in Figure 9A show that, as one extrapolates to infinitely low adalimumab surface density, the stoichiometry ratio is two TNF-alpha trimers per one adalimumab molecule. As the adalimumab density is increased, single TNF-alpha trimers are increasingly crosslinked by multiple adalimumab molecules, thus lowering the TN F-al ph a: adal imumab ratio. Theoretically, the ratio for fully crosslinked TNF-alpha trimer would be 0.75. Figure 9B illustrates the proposed binding model on the biacore chip for both molecules. While we do not wish to be bound by theory, these data strongly support the alternative model of binding illustrated in Figure 10 of TNF-alpha to the TNF-TWEAK BsAb from that of adalimumab.

D) Differential Scanning Calorimetry (DSC)

[0310] The TNF-TWEAK BsAb (XWU198) was analyzed by Differential Scanning

Calorimetry on a VP-DSC capillary cell microcalorimeter (Microcal). The bispecific was dialyzed against a buffer consisting of 10 mM Citrate, 160 niM Arginine, pH 6.0, assayed for protein concentration by UV absorbance scan, and then diluted to 1.0 mg/ml in the dialysis buffer. 3 x 450 μΐ samples of dialyzed TNF-TWEAK BsAb and 1 x 450 μΐ sample of non-dialyzed TNF-TWEAK BsAb were subjected to DSC analysis (10 - 1 10 °C, 120 C/hr. 10 minute pre-scan per sample, 8 second filtering period, low feedback mode). Raw data were analyzed and fit to determine the thermostabilities of the different immunoglobulin domains within the bispecific antibody samples. The data are summarized in Table 7. The scFv and CH2 domains melt in one peak with TM = 66 ^°C, while the Fab and CI 13 domains melt at 78.9 ^°C and 86.7 ^°C, respectively.

Table 7: Summary of TNF-TWEAK BsAb Tms

Example 6. Cross-Species Binding by the TNF-TWEAK Bispecific Antibody

1031 11 This example demonstrates that the TNF-TWEAK bispecific antibody exhibits different cross-species cross-reactivity than adalimumab. The TNF species specificity o the TNF-TWEAK BsAb was determined by multiple methods. A multiple sequence alignment of mouse, rat, rabbit (two polymorphs), cynomolgus monkey and human TNF- alpha is shown in Figure 1 1. Mouse, rat and rabbit TNF-alpha are all roughly 80% identical to human TNF-alpha, while cynomolgus monkey TNF-alpha is highly homologous to human TNF-alpha, with 96% shared identity. Rabbit TNF-alpha was tested for TNF-TWEAK BsAb and adalimumab binding by solution affinity biacore under the same conditions as those used with human TNF-alpha in Example 2C. The results are shown in Figure 12. Adalimumab demonstrated undetectable binding to 0.8 nM rabbit TNF-alpha in solution. Under these conditions, adalimumab binding affinity is therefore weaker than KD = 10 nM, compared to KD < 10 pM for human TNF-alpha (Figure 12, right). In contrast, the TNF-TWEAK BsAb showed clearly detectable binding under the same conditions, with a KD of 175 pM (Figure 12, left). Adalimumab has been described to bind only very weakly to rodent TNF-alpha. Our experiments confirm that adalimumab binds rodent TNF-alpha in in vitro only very weakly, and that adalimumab is poorly active in neutralizing rodent TNF-alpha in cell-based assays. By similar analysis, TNF-TWEAK shows equal binding to human and cyno TNF-alpha, with tight dissociation constants of at least 10 pM. Mouse TNF-alpha binds more weakly (KD = 14 nM), while rat TNF shows barely detectable binding (KD > 7 μΜ).

The difference between TNF-TWEAK BsAb and adalimumab in species specificity was also tested in cellular cytotoxicity assays. MTT assays in the WEHI mouse fibrosarcoma cell line and the WiDr human carcinoma cell line were used to evaluate the abilities f TNF-TWEAK BsAb and adalimumab to neutralize TN F-alpha of different species. The MTT assay is described in further detail in Example 7. Recombinant mouse or human TNF-alpha (5pM) triggered a cytotoxic or cytostatic response in WiDr and WEHI cells, respectively (Figures 13A and 13C). The response was measured after a 4-day incubation with cytokine and the relevant BsAb/Antibody/Fc- Receptor fusion. In the WiDr cytotoxicity assay, the cells were also incubated with 80 units/ml human I nterfe ro n- gam ma , to potentiate the response. A murine TNFR-humanFc fusion protein was used as a positive control in the murine WEHI assay; it fully neutralized mouse TNF-alpha with an IC50 of - 10 pM. At the highest concentration tested, 1.0 μΜ, adalimumab only partially neutralized murine TNF-alpha. In contrast, TNF-TWEAK BsAb fully neutralized mouse TNF-alpha with IC50 = 1.5 nM. Similarly, in a rabbit TNF-alpha-mediated WiDr cell killing assay (Figure 13B), adalimumab was completely unable to neutralize rabbit TNF-alpha (5.0 pM) at up to 1.0 μΜ, while TNF- TWEAK BsAb fully inhibited rabbit TNF-alpha with IC50 - 600 pM. In the same assay, human TNF-alpha (5 pM) cell killing activity (Figure 13C) was completely inhibited by TNF-TWEAK BsAb with IC50 = 2 pM. The adalimumab IC50 is approximately 10-fold greater in the same assay.

Example 7. Functional inhibition/stimulation assays

[0313] Several in vitro assays were employed to demonstrate functional inhibition of both

TNF and TWEAK by the TNF-TWEAK BsAb. These include (1) IL-8 release assay in A375 cell line (2) ME T assay in WiDr cell line (3) Luciferase assay in NFkB-luc expressing 293 cells.

A) IL-8 release assay in A375 cells

[0314] The IL-8 cytokine release assay was previously used to demonstrate activity of anti-TWEAK (hP2D10). In this assay, IL-8 is released into the supernatant from A375 cells following exposure to TNF or TWEAK. Thus, functional neutralization of TNF or TWEAK can be measured by assaying IL-8 release in response to TNF (or TWEAK) in the presence of a TNF (or TWEAK) inhibitor. In this case, the ability of the TNF- TWEAK b i spec i lie antibody to inhibit IL-8 release in response to TNF or TWEAK was measured. The potency of the bispecific antibody in neutralizing TNF was compared to inhibition with the parental anti-TNF antibody (adalimumab), and the potency in neutralizing TWEAK was compared to the parental anti-TWEAK antibody (hP2D10).

[0315] The protocol is as follows: Serial 1 :3 dilutions of anti-TNF mAb (adalimumab). anti-TWEAK mAb (hP2D10) or The TNF-TWEAK BsAb were created in A 75 cell medium at 4X the final desired antibody concentration in 96-well plates, 100 μΐ per well. A TNF or TWEAK solution was prepared in A375 cell medium at 4 nM each (again, 4X the final desired concentration). 100 μΐ of the 4X cytokine solutions were added to the mAb titrations, resulting in 2X final concentrations of mAb and cytokine. This mixture was incubated at 37 °C for approximately 30 minutes. During the incubation. A375 cells were prepared by harvesting the cells and adjusting their concentration to 4 X 10 cells/ml. Then, 50 μΐ of the mAb-cytokine combinations were transferred to new 96 well plates followed by addition of 50 μΐ of cell suspension. This resulted in a mAb titration starting at 10 nM and serially diluted 1 :3, a cytokine concentration of 1 nM, and 20,000 cells per well. The cells were incubated at 37 ^°C for 40 hours followed by harvesting of the cell supernatant. Finally, the IL-8 concentration in the supernatants was assayed by ELISA (R&D Systems). [0316] The results, shown in Figure 14, show that the TNF-TWEAK BsAb is equivalent to hP2D 10 in inhibiting TWEAK-stimulated IL-8 release, and is slightly superior to adalimumab in neutralizing TNF-induced IL-8 release.

B) MTT assay in WiDr cell line - (dual inhibition assay)

[0317] The MTT assay has classically been employed in the literature to measure TNF activity. See, e.g., Browing J. and Ribolini A., J. immunol. 143(6): 1859-67 (1989). In this assay, treatment of WiDr cells with TNF (or TWEAK) results in a loss of cell viability. In this case, we employed a dual inhibition MTT assay in WiDr to demonstrate simultaneous inhibition of both TNF and TWEAK activities with the bi speci ic antibody. This assay was used to show that the bi specific antibody was required to neutralize the combined activities of TNF and TWEAK, whereas each individual mAb could only neutralize activity of its cognate ligand.

[0318] The protocol was as follows. Serial 1 :3 dilutions of anti-TNF mAb (adalimumab). anti-TWEAK mAb (hP2D10), anti-TNF mAb + anti-TWEAK mAb (adalimumab + P2D10). or the TNF-TWEAK BsAb were created in WiDr cell media at a 4X final concentration desired for the antibodies in a volume of 100 μΐ per well in 96- we 11 plates. A solution of TNF, TWEAK or TNF + TWEAK was prepared in WiDr cell media, also at a 4X final concentration, with I X TNF = 4 pM, and I X TWEAK = 15 pM. The cytokine solutions (100 μΐ) were added to the mAb titrations and allowed to incubate at 37 C for 30 minutes. Then, 50 μΐ of the mAb/cytokine mixtures were transferred to a new 96 well plate and 50 μΐ of a 100,000/mL WiDr cell suspension was added to the wells to give IX mAb/cytokine combination and 5000 cells/well. The media contains 80U/ml hlFN- gamma final cone. Cell viability was measured at day 4 of the assay using the Promega CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent.

[0319] The results are shown in Figure 15. As expected, hP2D 10 neutralized activity induced by TWEAK but not TNF, and adalimumab neutralized activity of TNF but not TWEAK. On the other hand, the TNF-TWEAK BsAb simultaneously neutralized both TNF and TWEAK. Moreover, the activity of the bispecific was superior to that of the combination of antibodies, and this was specifically seen with respect to ability of the bispecific to inhibit TNF rather than TWEAK. C) Luciferase assay in 293/NFkB-luc cells - (dual inhibition assay)

[0320] An NFkB-luciferase reporter cell line was employed to provide a more biologically relevant assay. Employing 293 cells transfected with an NFkB-luc reporter allows for direct readout of NFkB pathway activity. NFkB activity, as measured by luciferase activity, is induced in 293 NFkB-luc cells when the cells are exposed to TNF and/or TWEAK. The ability of inhibitory mAbs to neutralize activity is measured as a reduction in luciferase activity. In this case, a dual inhibition assay was employed to demonstrate simultaneous inhibition of both TNF and TWEAK activities with the bi specific antibody. This assay was used to show that the bi specific antibody was required to neutralize the combined activity of TNF and TWEAK, whereas each individual mAb could only neutralize activity of its cognate ligand.

103211 The protocol is as follows. Serial 1 :3 dilutions of ant i -TNF (adalimumab), anti-

TWEAK (hP2D10), anti-TNF + anti-T WEAK (adalimumab + P2D10), or the TNF- TWEAK BsAb were created in cell specific media at a 4X final concentration in a volume of 100 ul per well in 96-wcll plates. Solutions o TNF, TWEAK or TNF + TWEAK were prepared in cell specific media at a 4X final concentration ( I X TNF = 5 pM, IX TWEAK = 139 pM), and 100 μΐ was added to the mAb titrations and allowed to incubate at 37 °C for 30 minutes. 50 uL of this mAb/eytokine mixture is transferred to a 96 well iso-plaic white-walled TC plate, and 50 ul of a 10⁶/ml 293/NFkB-Luc cell suspension was added to each well, resulting in IX mAb/cytokine combination and 50.000 cells/well. Luciferase expression was measured at 24 hours using Promega Steady-Glo Luciferase Assay Reagent. Briefly, the plates were brought to room temperature and 100 ul of reconstituted reagent was added to each well of the 100 ul cell supernatant, and cells. Luminescence was measured approximately 1 hour later in the MicroBeta Jet plate reader. The results are presented in Figure 16.

[0322] As expected, the results show that hP2D10 neutralized luciferase activity induced by TWEAK but not TNF, and adalimumab neutralized luciferase activity induced by TNF but not TWEAK. On the other hand, the TNF-TWEAK BsAb simultaneously neutralized both TNF and TWEAK. Moreover, the activity of the bispecific was superior to that of the combination of antibodies, and this was specifically a function of the superior ability of the bispecific to inhibit TNF.

Example 8: Effector Function A) Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay

[0323] ADCC activity of the TNF-TWEAK BsAb was measured in vitro using 3T3 cells overexpressing membrane-bound TNF-alpha (3T3/TNF-alpha) as targets and primary NK cells as effectors. To generate the 3T3/TNF-alpha cell line, 3T3 cells were stably transfected with a construct designed for expression of a non-cleavable form of human membrane TNF-alpha. The TNF-TWEAK BsAb was tested and compared to the TNF- alpha inhibitors adalimumab and etanercept (ENBREL^®). Previous reports have shown ADCC activity with both of these inhibitors. See. e.g., Nesbitt A., et al, Inflamm. Bowel Dis. 13(1 1 ): 1323-32 (2007); Mitoma H., et al, Arthritis Rheum. 58(5): 1248-57 (2008); and Arora T.. et al.. Cytokine 45(2): 124-31 (2009). Negative controls included hlgGl and the human anti-Tweak antibody hP2D10 and Adalimumab Fab.

[0324] 3T3/TNF-alpha cells were plated the day before at 20,000 cells per well in a 96 well plate. NK cells were isolated from whole blood using RosetteSep (Stem Cell Technologies). Briefly, whole blood was incubated with human NK cell enrichment cocktail for 20 minutes at room temperature. The blood was then diluted and layered over ficoll-paque and NK cells were obtained by density gradient centrifugation. Target cells were incubated with various concentrations of test agents before being combined with NK cells at an effector to target ratio of 5: 1. The cells were incubated for 4 hours at 37 °C. After the incubation, supernatant was taken from each sample, and cell lysis was measured by G6PD detection using a Vybrant kit (Invitrogen). Fluorescence was read on a plate reader. Control samples included spontaneous release (3 T3 /TNF-alpha cells only), maximum lysis (Triton-X). and no antibody wells (3T3/TNF-alpha and NK cells only ). The percentage of cell lysis was quantified as follows:

Experimental samples - Spontaneous release X 100

Maximum Release - Spontaneous Release

[0325] As shown In Figure 17A-B, the TNF-TWEAK BsAb can mediate ADCC. The activity of the TNF-TWEAK BsAb in this assay was comparable to etanercept, while adalimumab had elevated activity compared to both the TNF-TWEAK BsAb and etanercept. As expected hlgGl , hP2D 10. and adalimumab Fab negative controls did not display any ADCC activity. The absolute levels of ADCC varied by donor, presumably - I l l - due to differences in FcR expression on NK cells. However, the trends observed remain similar. Shown below are examples from two different donors.

B) Complement-Dependent Cytotoxicity (CDC)

103261 CDC activity of the TNF-TWEAK BsAb was measured using the 3T3/TNF-alpha cell line described above. As with ADCC, adalimumab and etanercept have been reported to have CDC activity, and were tested alongside the TNF-TWEAK BsAb. Human IgGl and hP2D10 were used as negative controls.

[0327] 3T3/TNF-alpha cells were plated the day before at 50,000 cells per well in a 96 well plate. The following day, the cells were washed and placed in phenol red free medium and incubated with varying concentrations of test agent, diluted rabbit serum complement (Cedarlane), and diluted propidium iodide (lmg/ml, Biogen Idee) at 37 °C for 1 hour. The propidium iodide signal was read on a fluorescent plate reader. As a maximum lysis positive control, Triton-x was added to wells. Negative controls include no antibody (to determine background lysis) and no complement wells. Percentage of cell lysis was calculated as follows:

Experimental samples - Background lysis X 100

Maximum lysis- Background lysis

[0328] The results are presented in Figure 18. The results show that the TNF-TWEAK

BsAb can mediate significant levels of CDC activity. Adalimumab demonstrated the highest levels of CDC activ ity, while the TNF-TWEAK BsAb trended towards slightly elevated levels relative to etanercept. The negative controls hlgGl and hP2D10 did not give a signal in this assay.

Example 9: In- Vivo Efficacy Study

A) Rationale for selection of in vivo models

[0329] NFkB-luciferase transgenic mice express a luc if erase reporter gene under the control of NFkB (three tandem DNA binding sites) to enable real time imaging of NFkB activity in mice. The NFkB-luciferase mouse model was selected for demonstration of in vivo activity at both ends of the bispecific antibody. Since both TNF and TWEAK are known to induce the NFkB pathway, these mice provide an in vivo readout for combinatorial activity of TNF and TWEAK and for blockade of these pathways with antibody inhibitors. Since the mice can be administered human TNF-alpha. this provides a unique opportunity for testing the activity of the TNF-TWEAK BsAb in rodents since the bispecific has limited cross-reactivity to mouse TNF.

B) Primary in vivo efficacy model results, NFkB-lucif erase mouse model:

[0330] The goal of these studies was to demonstrate that the TNF-TWEAK BsAb could simultaneously inhibit NFkB activ ity induced by both TN F and TWEAK. Preliminary experiments were performed to determine an optimal dose for TNF and for TWEAK to induce NFkB activation with the luciferase readout and specifically to see combinatorial activity. Additional preliminary experiments were performed to demonstrate that induction of NFkB activity by TWEAK could be blocked by dosing with hP2D10, and activity induced by TNF could be blocked by administration of adalimumab. The key experiment, which is described below, was to demonstrate that the TNF-TWEAK BsAb could completely inhibit NFkB activity induced by TNF plus TWEAK, whereas the individual antibodies could only partially inhibit the NFkB response induced by the combinat ion of cytokines.

[0331 ) Female NFkB-luciferase transgenic mice (n = 3 or 4 per group) were pre-treated with the TNF-TWEAK BsAb (13.3 mg/kg), hPD210 (10 mg/kg) or adalimumab (10 mg/kg) injected IP at day -1. A background measurement of luciferase activity was also taken on day - 1 . On day 0, the mice were injected with liT F-alpha (20 ug/kg) and niFc- TWEAK (1 mg/kg), and then imaged at 2 and 6 hours. Additional images were also captured at 24. 48, 96 and 168 hours. The results are graphed in Figure 19, imaging results are not shown.

[0332 J The results showed that the TNF-TWEAK BsAb completely inhibited luciferase activity induced by TNF plus TWHAK at all timepoints. In contrast, adal i mumab-treated mice showed inhibition of luciferase activity at early timepoints (which previous experiments demonstrated was due to TNF-alpha) but the luciferase signal at later timepoints remained, whereas hP2D10 treated mice retained the early luciferase signal (due to TNF-alpha) but were inhibited at later timepoints (where TWEAK is active). A control Ig was not effective in blocking the TNF plus TWEAK signal (data not shown). Thus, activity of the bispeci fic antibody on both the adalimumab scFv and hP2D10 ends was shown to be active in vivo. Example 10: Construction of Anti-TNF-alpha/IL6 Bispecific Antibodies

[0333] Anti-TNF-alpha and IL6 bispecific antibodies are constructed by a method similar to the anti-TNF-alpha and TWEAK bispecific antibodies as shown in Example 3. First, the heavy chain-linker-stabilized scFv vector is constructed by ligating an anti-IL6 antibody heavy chain into a plasm id to add a scFv linker. A scFv insert from pIEH298 described in Example 2 is amplified with primers, and the resulting product is digested and ligated into the plasmid containing the nucleotides encoding the IL6 antibody heavy chain and scFv linker. The resulting coding sequences are digested and ligated into pV90mod. See Example 3.

Equivalents

[0334] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments o the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Example 11 - TNF and TWEAK Binding Stoichiometrics of various anti-TNF and anti-TWEAK Constructs

A) Stoichiometry ofTNF-alpha and TWEAK binding to other anti-TNF and anti-

TWEAK constructs, as measured by Analytical Size Exclusion Chromatography

[0335] A set of TNF- and TWEAK-binding constructs (Adalimumab. XWU 198

(TNF-TWEAK BsAb), MJF 149 (TWEAK-TNF BsAb), M J IT 93, hP2D10 (anti-TWEAK hlgGl), XWU242, and XWU243. all at 5 μΜ in Phosphate Buffered Saline (PBS), were mixed with recombinant, His-tagged TN Fa or TWEAK at concentrations of 10 μΜ. Samples were incubated for 1 week at 4 °C, then subjected to tanderm analytical size exclusion chromatography (SEC) and static light scattering (LS). Results are shown in Figure 20.

[0336] Complex sizes of the various constructs with TNF showed the same trend as what had been observed between adalimumab and XWU198 in Example 5. Adalimumab again formed only large constructs, and no 1 : 1 complexes, with TNFa (Figure 20A). Tike adalimumab, the TWEAK-TNF bispecific, MJF149, which is composed o adalimumab light chain and the adalimumab heavy chain fused to a (G₄S)₃ linker plus a C-terminal. stabilized, anti-TWEAK hP2D10 scFv, formed only large complexes with TNF (Figure 20 B). In contrast, the XWU198 bispecific formed 1 : 1 complexes with TNFa (Figure 20C). Like the TNF-TWEAK bispecific. the MJF 193 construct, containing a hlgGl Fc fused at its C-terminus to a (G₄S)₃ linker plus the C-terminal, stabilized, adalimumab scFv. formed 1 : 1 complexes with TNFa ( Figure 20D). The scFv-Fc constructs. XWU242 and XWU243 show that linker length is key in determining complex size. XWU242, containing on a single G₄S repeat between the N-terminal stabilized adalimumab scFv and a hlgGl Fc, did not form 1 : 1 complexes with TNFa (Figure 20E). XWU243. containing 5 repeats of the Gly-Ser repeat in the scFv-Fc linker, like the XWU198 and Μ.Π 193 constructs, does form 1 :1 complexes with TNFa (Figure 20F).

103371 TWEAK binding was analyzed similarly. The data are shown in figure 21 . hP2D 10 anti-TWEAK hlgG 1 (Figure 21 A) and XWU 198 bispecific antibody ( Figure 2 I B), which bind to TWEAK through hP2D10 anti-TWEAK Fabs. form only large complexes with the trimeric TWEAK cytokine. In contrast, the MJF 149 bispecific. in which the anti-TWEAK scFv is connected to the C-terminus of the hlgGl Fc via a (G₄S)₃ linker, formed 1 : 1 complexes with TWEAK, in addition to larger complexes (Figure 21 C).

103381 These results show that chelation of the TNF-alpha and TWEAK cytokines and consequent formation of 1 : 1 complexes of construct with trimeric TNF and TWEAK constructs is enabled by longer linker lengths between the cytokine binding modules and the construct Fc region.

B) Stoichiometry of TNF-alpha and TWEAK binding to other anti-TNF and anti-TWEAK constructs, as measured by Surface Plasmon Resonance binding assay

[0339] The stoichiometry of TNF-alpha and TWEAK binding to the different anti-TNF- alpha and TWEAK constructs was studied further using the biacore resonance unit analysis method described in example 5. Using the strcptavidin SA biacore chip coated with biotinylated polyclonal goat anti-huIgG Fc, varying amounts o XWU198 TNF- TWEAK BsAb. MJF 149 TWEAK-TNF BsAb, anti-TNF-alpha MJF 193 Fc-(G₄S)₃-scFv, XWU242 short linker anti-TNF-alpha scFv-Fc construct, XWU243 long linker anti-TNF- alpha scFv-Fc construct, adalimumab, and hP2D10 anti-TWEAK antibody, were captured on the biacore chip surface over a set of binding experiments. In the first experiment, the constructs were captured on the anti-human IgG Fc chip by flowing 15 microliters over a defined biacore flow cell at 6.5 nM. 100 microliters of 33 nM his-tagged, recombinant human TNF-alpha and/or TWEAK were injected. The two cytokines were injected in series; in one set of experiments, first TNF-alpha, then TWEAK was injected, while in the second set the order was reversed. For each experiment, the binding responses of construct, TNF-alpha and TWEAK were all recorded. By accounting for the molecular weight of each molecule, the molar ratios of binding were calculated. The data are presented in Figure 22. The parental anti-TNF-alpha antibody, adalimumab, bound 1.4 - 1.5 TNF-alpha trimers per divalent hlgG. while the parental anti-TWEAK antibody. hP2D10, bound 1.4 - 1.5 TWEAK trimers per divalent hlgG. Similarly, in the cases where TNF-alpha and TWEAK binding occurs through Fab-hinge-based constructs (TNF-TWEAK / XWU198 and TWEAK-TNF / MJF149), cytokine bound with molar ratios between 1.4 and 1 .5. In contrast, where the TNF and TWEAK-binding scFvs were connected via long (G₄S) linkers (3 Gly-Ser repeats in XWU198, MJF149 and the "Fabless" MJF 193. and 5 Gly-Ser repeats in the anti-TNF scFv-Fc construct .XWU243). the molar ratio was between 1.0 and 1.1. Shortening the linker in the anti-TNF scFv-Fc construct in XWU242 resulted in molar ratios between 1.6 and 1.7, similar to that observed with Fab-hinge-based binding. These experiments show both that chelation of the trimeric TNF and TWEAK cytokines can only occur with constructs with longer or more flexible linkers, and that the chelation effect occurs not only with TNF-alpha, but also with TWEAK. For all of the experiments, serial binding of TNF-alpha and TWEAK gave the same binding stoichiometrics regardless of the order of cytokine binding, demonstrating that the cytokine binding to one end of the construct does not change binding stoichiomctry at the other end.

In a second set of surface plasmon resonance-based experiments, cytokine binding to constructs captured on the biacore chip at varying densities. Adalimumab, TNF- TWEAK / XWU 198, and TWEAK-TNF / MJF 149 were evaluated for TNF-alpha binding with varying capture density, while hP2D10 anti-TWEAK, TNF-TWEAK / XWU 198. and TWEAK-TNF / MJF149 were evaluated for TWEAK binding with varying capture density. Different volumes (5 - 125 microliters) of 7 nM construct were flowed over the anti-hlgG Fc biacore chip surface (described above) to capture varying resonance units (RUs) of BsAb or antibody, and the signal of saturating TNF-alpha and TWEAK binding was measured, in the same manner as the experiments shown in Figure 9 and described in example 5C. Figure 23 shows that the TNF-TWEAK BsAb (XWU198) captures 1 mole of TNF -alpha trimer per mole of TNF - TWEAK BsAb over the range of captured BsAb densities. This result is consistent with one BsAb being able to bind simultaneously through its two scFv fragments to two monomers of a single TNF-alpha trimer (see Figure 10), which is what is also observed above by SEC/LS analysis. In contrast, adalimumab and TWEAK-TNF / MJF149, both of which contain anti-TNF-alpha Fab domains, captured ~ 1.5 mo! T F-alpha trimer per antibody molecule. In the model for TNF-alpha binding to adalimumab. shown in Figure 10, the antibody is unable to bind through its two Fab fragments to two monomers of a single TNF-alpha trimer. We hypothesize that TWEAK-TNF / Ml F 149 binds in the same manner as adalimumab. Most important, adalimumab and MI F 149 show the same ligand binding ratio, while XWU198 shows its distinct, 1 :1, binding ratio.

[0341] Figure 24 shows the measured TWEAK binding ratios for hP2D 10 anti-TWEAK hlgGl, TNF-TWEAK / XWU198, and TWEAK-TNF / MJF149. In this case, hP2D10 and XWU198 bind TWEAK with similar molar ratios, at 1.5 - 2 TWEAK trimers per antibody or bispecific molecule. In contrast, M.I I T 49 binds ~ 1 TWEAK trimer per bispecific antibody molecule. Thus, as with TNF-alpha binding to XWU198, TWEAK binding to MJF149 through its linker-scFv region is distinct from its binding to the parental, cytokine-binding antibody.

C) Stoichiometry of TNF-alpha and TWEAK binding to alternative anti-TNF scFv

bispecific constructs

103421 Two alternative TN -binding IgG-like bispecific antibodies, both carrying the same stabilized adalimumab scFv as XWU198 on their C-tcrmini, were tested for their stoichiometry of TNF binding. The first, mP5G9-TNF (construct MJF260/EAG1944) contains chimeric mP5G9 anti-TWEAK Fabs, and the second, IL-6- TNF (construct MJF258/MJF259), contains anti-IL-6 Fabs, both in their IgG portion. The same biacore method described in example 5 for measuring the stoichiometries of TNF-alpha and TWEAK binding to XWU 198. adalimumab and hP2D10 was used to analyze mP5G9-TNF and IL-6-TNF. As is shown in Figure 25. TNF bound to TNF- TWEAK / XWU198, IL-6-TNF and mP5G9-TNF at a stoichiometry of 0.9 to 1.0 TNF- alpha trimers per bispecific. TWEAK bound to XWU198 and mP5G9-TNF both bound with molar ratios of ~ 1.4 TWEAK trimers per bispecific antibody. As expected, IL-6- TNF did not bind to TWEAK. This experiment shows that TNF-alpha binds to all three stabilized adalimumab scFv-containing bispecifics with the same unique 1 : 1 stoichiometry.

Example 12 - Functional Assays Using Various Anti-TNF and Anti-TWEAK Constructs

A) NFkB Luciferase Assay

[0343] The NFkB luciferase assay was conducted similar to Example 7(C) above.

Specifically. anti-TNF antibody (HUMIRA®), anti-TWEAK antibody (mP5G10), anti- TNF/TWEAK bispecific antibody (BIIB40). anti-TNF/ Γ WEAK bispecific antibody (TNF-mP5G9), and anti-TNF/IL-6 antibody (TNF/IL-6) were titrated and combined with 5pM TNF-alpha and/or 133pM FcTWEAK and incubated at 37°C for 30 minutes. Then, 293/NFkB-Luc cells were added in the wells, resulting in 50,000/well and incubated for 24 hours. Luminescence was measured using Promega Steady-GLo Luciferase assay systems. The results are presented in Figures 26-28.

[0344] As expected, the results in Figure 26 show that anti TNF/TWEAK bispecific antibody (BIIB40), anti- Γ Ε/Τ WEAK bispecific antibody (TNF/mP5G9), anti-TNF/IL-6 bispecific antibody (TNF/IL-6), and HUMIRA^® neutralized luciferase assay induced by TNF-alpha. The anti - TN F/" I W E A K bispecific antibody TNF/mP5G9 and anti-TNF/IL-6 bispecific antibody show TNF inhibition similar to BIIB40 (anti- I NF/TWEAK bispecific antibody ).

[0345] In addition. Figure 27 shows that the level of TWEAK inhibition by the TNF- mP5G9 bispecific antibody is similar to BIIB40. Figure 28 also shows that the level of TNF and TWEAK inhibition by the TNF/mP5G9 bispecific antibody is similar to the inhibition by BIIB040.

(B) WiDr Cell Cytotoxicity Assay

[0346] The cytotoxicity assay was conducted similar to Example 7(B) above.

Specifically, anti-TNF antibody (HUMIRA^®), anti-TWEAK antibody (mP5G10), anti- TNF/TWEAK bispecific antibody (BIIB40). anti -TN F/^" [^'WEA bispecific antibody (TNF-mP5G9). and anti-TNF/IL-6 antibody (TNF/IL-6) were titrated and combined with 20pM TNF-alpha and/or 200pM hisTWEAK and incubated at 37°C for 30 minutes. Then, cells were added and incubated for four days in media containing 80u/mL hlFNg. Cell viability was measured at OD490nm using MTT reagent.

The results are shown in Figures 29-31. Figure 29 shows that TNF-mP5G9,

BIIB040, Humira, and ^'F F- 11.6 neutralized TNF activity and then improved cell survival. Figure 30 shows that the cell survival by TNF-mP5G9 was similar to BIIB40 at hisTWEA inhibition. Figure 31 also shows that the cell survival by inhibiting TNF and hisTWEAK together using TNF-mP5G9was similar to the cell survival using B1 IB040.

Claims

WHAT IS CLAIMED IS:

1. A binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein said scFv polypeptide comprises a heavy chain variable domain (VII) at least 90% identical to SEQ I D NO: 9 and a light chain variable domain (VL) at least 90% identical to SEQ I D NO: 1 1 , wherein said scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of:

(i) Glycine (G) at Kabat position 16 in the VH,

(ii) Serine (S) at Kabat position 30 in the VH,

(iii) Glycine (G) at Kabat position 49 in the VH,

(iv) Alanine (A) at Kabat position 49 in the VH.

(v) Serine (S) at Kabat position 52 in the VI I.

(vi) Threonine (T) at Kabat position 57 in the VH,

(vii) Proline (P) at Kabat position 61 in the VH,

(viii) Lysine (K) at Kabat position 64 in the VH,

(ix) Gly cine (G) at Kabat position 70 in the VH.

(x) Glutamine (Q) at Kabat position 77 in the VH.

(xi) Threonine (T) at Kabat position 77 in the VI I.

(xii) Lysine (K) at Kabat position 77 in the VH,

(xiii) Asparagine (N) at Kabat position 82b in the VH.

(xiv) Proline (P) at Kabat position 84 in the VH,

(xv) Proline (P) at Kabat position 3 in the VL,

(xvi) Arginine (R) at Kabat position 54 in the VL. (xvii) Glutamic acid (E) at Kabat position 83 in the VL, and any combinations thereof.

2. The binding molecule of claim 1 , wherein said VH is 95% identical to SEQ ID NO: 9 and said VL is 95% identical to SEQ ID NO: 1 1.

3. The binding molecule of claim 1 or 2, wherein said VH is 99% identical to SEQ ID NO: 9 and said VL is 99% identical to SEQ ID NO: 1 1.

4. The binding molecule of any one of claims 1-3, wherein said stabilized scFv polypeptide further comprises a scFv linker connecting the VH and the VL.

5. The binding molecule of any one of claims 1 -4, wherein said stabilizing amino acid substitutions are Glycine (G) at Kabat position 16 in the VI I. Serine (S) at Kabat position 30 in the VI I, Arginine ( R) at Kabat position 54 in the VL. and Glutamic acid (E) at Kabat position 83 in the VL.

6. The binding molecule of claim 5, further comprising amino acid Serine (S) at Kabat position 52 in the VH.

7. A binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TNF-alpha, wherein said scFv polypeptide comprises a heavy chain variable domain (VH), which comprises three complementarity domain regions (CDRs) of SEQ ID NO: 9. and a light chain variable domain (VL), which comprises three CDRs of SEQ ID NO: 1 1 , except for one or more stabilizing mutations.

8. The binding molecule of claim 7, wherein the VH is the amino acid sequence of SEQ ID NO:

9 and the VL is the amino acid sequence of SEQ ID NO: 1 1.

9. The binding molecule of claim 7 or 8, wherein said stabilized scFv polypeptide further comprises a scFv linker connecting the VI I and the VL.

10. The binding molecule of any one of claims 7-9, wherein said one or more stabilizing mutations comprise amino acid substitutions in the VH at any combination of Kabat positions 16, 30, 49, 52, 57, 61, 64, 70, 77, 82b, and 84.

1 1. The binding molecule of claim 10, wherein said amino acid substitutions are selected from the group consisting of R 160. D30S, S49G, S49A, T52S, I57T, D61 P, E64K, S70G. S77Q, S77T, S77K, S82bN, A84P. and any combination thereof.

12. The binding molecule of any one of claims 7-1 1, wherein said one or more stabilizing mutations comprise amino acid substitutions in the VL at any combination of Kabat positions 3, 54, and 83.

13. The binding molecule o claim 12. wherein said amino acid substitutions are selected from the group consisting of Q3P, L54R and V83E.

14. The binding molecule o any one o claims 7-1 3. comprising amino acid substitutions R16G and 1)3 OS in VI I and 1.54 and V83E in VL.

15. The binding molecule of claim 14. further comprising amino acid substitution T52S in VH.

16. The binding molecule of any one of claims 4-6 and 9-15, wherein said scFv linker comprises a disulfide bond between an amino acid in the VH and an amino acid in the VL.

17. The binding molecule of any one of claims 4-6 and 9-16, wherein said scFv linker comprises one or more amino acids.

18. The binding molecule of claim 17, wherein said scFv linker is a peptide chain at least 5 amino acids in length.

19. The binding molecule of claim 1 8. wherein said peptide chain is at least 15 amino acids in length.

20. The binding molecule o claim 19. wherein said peptide chain is at least 25 amino acids in length.

21. The binding molecule of any one of claims 18-20, wherein said peptide chain comprises an amino acid sequence selected from the group consisting of (Gly)„ (SEQ ID NO: 78), (GlyAla)„ (SEQ ID NO: 79), (Gly_nSer)_m (SEQ ID NO: 80), Ser(Gly₄Ser)_n (SEQ ID NO: 30) and (Gly₂Ser)_n(Gly₄Ser)_n (SEQ ID NO: 31), wherein said n and m are any integers between 1 and 100.

22. The binding molecule of claim 21 , wherein said peptide chain comprises (Glv^Ser).}.

23. The binding molecule of any one of claims 1 -22, wherein said stabilized scFv polypeptide is characterized by an improved biophysical property compared to an scFv polypeptide without said one or more stabilizing mutations.

24. The binding molecule of claim 23. wherein said biophysical property is selected from the group consisting of thermal stability, pi I unfolding profile, stable removal of glycosylation, solubility, ligand binding stoichiometry , ligand binding affinity, and a combination f two or more of said biophysical properties.

25. The binding molecule of any one o claims 1 -24. wherein thermal stability of said scFv polypeptide is characterized by a melting temperature (Tm) of at least 60 °C as measured by differential scanning calorimetry.

26. The binding molecule of claim 25. wherein thermal stability of said scFv polypeptide is characterized by a Tm of at least 65 °C as measured by differential scanning calorimetry.

27. The binding molecule of claim 26. wherein thermal stability of said scFv polypeptide is characterized by a Tm of at least 67 °C as measured by differential scanning calorimetry.

28. The binding molecule of any one of claims 1 -27, wherein said scFv poly peptide binds to human TNF-alpha with a dissociation constant of 150 pM or less.

29. The binding molecule of any one of claims 1 -28. wherein at equilibrium the binding stoichiometry of said binding molecule to a TNF-alpha trimer is 1 : 1 .

30. The binding molecule of any one of claims 1 -29, wherein said binding molecule binds surface rabbit TNF-alpha w ith a binding affinity greater than that of adalimumab.

3 1 . The binding molecule o any one of claims 1 -30. further comprising a CI 12 domain and a CI 13 domain of an immunoglobulin constant region.

32. The binding molecule of any one of claims 1 -3 1 . wherein the binding specificity for TNF- alpha is multivalent.

33. The binding molecule of any one of claims 1-32, comprising at least two copies of said TNF- alpha-specific scFv polypeptide.

34. The binding molecule of any one of claims 1 -33. further comprising a second binding region which specifically binds to a heterologous epitope relative to the epitope bound by said scFv polypeptide.

35. The binding molecule of claim 34, which is multispecific.

36. The binding molecule of claim 34 or claim 35, which is bispecific.

37. The binding molecule of any one of claims 34-36, wherein said second binding region comprises an Fab polypeptide.

38. The binding molecule of any one of claims 34-37, wherein said heterologous epitope is a TNF-alpha epitope.

39. The binding molecule of any one f claims 34-37. wherein said heterologous epitope is a non- TNF-alpha epitope.

40. The binding molecule of any one of claims 34-37. wherein said second binding region speci fically binds to a molecule selected from the group consisting of TWEAK or IL6.

41. The binding molecule of claim 40, wherein said second binding region is a TWEAK binding region which specifically binds TWEAK.

42. The binding molecule of claim 40 or claim 41 , which binds to the same TWEAK epitope as hP2D10, or competitively inhibits binding of hP2D10 to TWEAK.

43. The binding molecule of claim 42. wherein said second binding region is the binding molecule according to claim 41 or 42.

44. The binding molecule of claim 43, wherein said second binding region comprises a heavy chain variable domain (VH) at least 90% identical to SEQ ID NO: 13 and a light chain variable domain (VL) at least 90% identical to SEQ ID NO: 15, wherein said second binding region comprises one or more amino acid substitutions selected from the group consisting of:

(i) Asparagine (N) at Kabat position 60 in the VH, (ii) Lysine (K) at Kabat position 62 in the VI I. and

(iii) Glutamic Acid (E) at Kabat position 12 in the VL, and any combinations thereof.

45. The binding molecule of claim 40 or 41 , where said second binding region binds to the same TWEAK epitope as P5G9, or competitively inhibits binding of P5G9 to TWEAK.

46. The binding molecule of claim 45, wherein said second binding region comprises a heavy chain variable domain (VH) at least 90% identical to SEQ ID NO: 82 and a light chain variable domain (VL) at least 90% identical to SEQ ID NO: 60.

47. The binding molecule of claim 46. wherein said second binding region comprises at least one. at least two, or at least three of SEQ ID NO: 82 or at least one, at least two, at least three CDRs of P5G9 VL (SEQ ID NO: 60).

48. The binding molecule of claim 40 or claim 41 , which binds to the same epitope as Fnl4 or competitively inhibits binding of Fn 14 to TWEAK.

49. The binding molecule of any one of claims 40-48, comprising at least two TWEAK binding regions.

50. The binding molecule of claim 49. comprising at least two T F-alpha binding regions.

51. The binding molecule of claim 49 or claim 50. wherein said at least two TWEAK binding regions each comprise a heavy chain polypeptide and a light chain polypeptide.

52. The binding molecule of claim 51 , wherein the heavy chain polypeptides of said at least two TWEAK binding regions each comprises at least one CDR amino acid sequence of SEQ I D NO: 13.

53. The binding molecule of claim 5 1 or claim 52. wherein the light chain polypeptides of said at least two TWEAK binding regions each comprise at least one CDR amino acid sequence of SEQ ID NO: 15.

54. The binding molecule of claim 52 or 53, wherein said at least two TWEAK binding regions each comprise the CDR1 , CDR2, and CDR3 amino acid sequences of SEQ ID NO: 13 and the CDR1 , CDR2, and CDR3 amino acid sequences of SEQ ID NO: 1 5.

55. The binding molecule of claim 54. wherein said at least two TWEAK binding regions each comprise a heavy chain with the amino acid sequence of SEQ I D NO: 1 3 and a light chain with the amino acid sequence of SEQ ID NO: 15.

56. The binding molecule of any one of claims 49-55, wherein said at least two TWEAK binding regions each comprise a human heavy chain constant region, or fragment thereof and a human light chain constant region or fragment thereof.

57. The binding molecule of any one of claims 40-56. wherein one or more of said TWEAK binding regions is connected to the N-terminus of the heavy chain polypeptide of one or more of said scFv polypeptides.

58. The binding molecule of any one of claims 40-56. wherein one or more of said TWEAK binding regions is connected to the C-terminus of the heavy chain polypeptide of one or more of said scFv polypeptides.

59. The binding molecule of any one of claims 40-56, wherein one or more of said TWEAK binding regions is connected to the N-terminus of the light chain polypeptide of one or more o said scFv polypeptides.

60. The binding molecule of any one of claims 40-56. wherein one or more of said TWEAK binding regions is connected to the C-terminus of the heavy chain polypeptide of one or more of said scFv polypeptides.

61 . The binding molecule of any one of claims 40-60. wherein one or more of said TWEAK binding regions is connected to one or more of said scFv polypeptides by a hinge connecting linker.

62. The binding molecule of claim 61 . wherein said hinge connecting linker is a peptide comprising an amino acid sequence selected from the group consisting of (Gly)_n (SEQ ID NO: 78), (GlyAla)_n (SEQ ID NO: 79), (Gly_nSer)_m (SEQ ID NO: 80), Ser(Gly₄Ser)_n (SEQ ID NO: 30) and (Gly₂Ser)_n(Gly₄Ser)_n (SEQ 11) NO: 31), wherein said n and m are any integers between 1 and 100.

63. The binding molecule of claim 61 or claim 62, wherein said linker comprises the amino acid sequence (Gly₄Ser)₄ (SEQ ID NO: 32).

64. The binding molecule of any one of claims 40-63, which inhibits TWEAK and TNF-alpha activity in the II. -8 release assay.

65. The binding molecule of any one f claims 40-63. which simultaneously inhibits TWEAK and TNF-alpha activity in the WiDr MTT combo assay.

66. The binding molecule of any one of claims 40-63, which, when administered to a NF-kB- luciferase transgenic mice, simultaneously inhibits TWEAK and TNF-alpha activity in the NFkB-luciferasc combination inhibition assay.

67. A binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein said scFv polypeptide comprises a heavy chain variable domain (VH) at least 90% identical to SEQ ID NO: 13 and a light chain variable domain (VL) at least 90% identical to SEQ ID NO: 15, wherein said scFv polypeptide comprises one or more stabilizing amino acid substitutions selected from the group consisting of:

(i) Asparagine (N) at Kabat position 60 in the VH,

(ii) Lysine (K) at Kabat position 62 in the VH.

(iii) Glutamic Acid (E) at Kabat position 12 in the VL. and any combinations thereof.

68. The binding molecule of claim 106, wherein said VH is at least 95%, 96%. 97%>, 98%, or 99% identical to SEQ ID NO: 13. and said VL is at least 95%, 96%. 97%, 98%, or 99% identical to SEQ ID NO: 15.

69. The binding molecule of claim 67 or claim 68, wherein said stabilized scFv polypeptide further comprises a scFv linker connecting the VH and the VL.

70. The binding molecule of any one of claims 67-69, wherein said stabilizing amino acid substitutions are Asparagine (N) at Kabat position 60 in the VH, Lysine (K) at Kabat position 62 in the VH, and Glutamic Acid (E) at Kabat position 12 in the VL.

71. A binding molecule comprising a stabilized scFv polypeptide which specifically binds to human TWEAK, wherein said scFv polypeptide comprises a heavy chain variable domain (VH). which comprises three CDRs of SEQ ID NO: 13, and a light chain variable domain (VL), which comprises three CDRs of SEQ ID NO: 15, except for one or more stabilizing mutations.

72. The binding molecule of claim 71 , wherein said stabilized scFv polypeptide further comprises a scFv linker connecting the VH and the VL.

73. The binding molecule of claim 71 or claim 72, wherein said one or more stabilizing mutations comprise amino acid substitutions in the VI I at Kabat positions 60. 62. or both.

74. The binding molecule of claim 73, wherein said amino acid substitutions are P60N, T62K, or both.

75. The binding molecule of any one of claims 71-74, wherein said one or more stabilizing mutations comprise an amino acid substitution in the VL at Kabat position 12.

76. The binding molecule of claim 75. wherein said amino acid substitution is P12E.

77. The binding molecule of any one of claims 69, 70, and 72-76, wherein said scFv linker comprises a disulfide bond between an amino acid in the VH and an amino acid in the VL.

78. The binding molecule of any one of claims 69. 70, and 72-77, wherein said scFv linker comprises one or more amino acids.

79. The binding molecule of claim 78, wherein said scFv linker is a peptide chain at least 5, 15, or 25 amino acids in length.

80. The binding molecule of claim 79, wherein said peptide chain comprises an amino acid sequence selected from the group consisting o (Gly)_n (SEQ ID NO: 78). (GlyAla),, (SEQ ID NO: 79), (Gly_nSer)_m (SEQ ID NO: 80), Ser(Gly₄Ser)_n (SEQ ID NO: 30) and (Gly2Ser)_n(Gly₄Ser)_n (SEQ ID NO: 31), wherein said n and m are any integers between 1 and 100.

81. The binding molecule of claim 80, wherein said peptide chain comprises (Gly4Ser)4.

82. The binding molecule of any one of claims 67-81 , wherein said stabilized scFv polypeptide is characterized by an improved biophysical property compared to an scFv polypeptide without said one or more stabilizing mutations.

83. The binding molecule of claim 82. wherein said biophysical property is selected from the group consisting of thermal stability, pH unfolding profile, stable removal of glycosylalion. solubility, ligand binding stoichiometry. ligand binding affinity, and a combi nation of two or more of said biophysical properties.

84. The binding molecule of any one of claims 67-83, further comprising a CH2 domain and a CI 13 domain of an immunoglobulin constant region.

85. The binding molecule of any one of claims 67-84. wherein the binding specificity for TWEA is multivalent.

86. The binding molecule of any one of claims 67-85. comprising at least two copies of said TWEAK-specific scFv polypeptide.

87. The binding molecule of any one of claims 67-86, further comprising a second binding region which speci fically binds to a heterologous epitope relative to the epitope bound by said scFv polypeptide.

88. The bindin molecule of claim 87. which is multispecific.

89. The binding molecule of claim 87 or claim 88, which is bispeeific.

90. The binding molecule of any one o claims 87-89. wherein said second binding region comprises an Fab polypeptide.

91. The binding molecule of any one of claims 87-90, wherein said heterologous epitope is a TWEAK epitope.

92. The binding molecule of any one of claims 87-90, wherein said heterologous epitope is a non-TWEAK epitope.

93. The binding molecule of claim 93, wherein said second binding region is a TNF-alpha binding region which specifically binds to TNF-alpha.

94. The binding molecule of claim 93, wherein said second binding region binds to the same TNF-alpha as adalimumab or competitively inhibits binding of adalimumab to TNF-alpha.

95. The binding molecule of claim 93 or claim 94, wherein said TNF-alpha binding region is selected from the group consisting of the binding molecules according to claims 1 -39.

96. A nucleic acid molecule comprising a polynucleotide encoding the binding molecule of any one of claims 1 -95 or a binding site or v ariable region thereof.

97. The nucleic acid molecule of claim 96, further comprising a promoter operably associated with said polynucleotide.

98. A vector comprising the nucleic acid molecule of claim 96 or claim 97.

99. A host cell comprising the vector of claim 98.

100. The host cell of claim 99, wherein the cell is a Ci lO cel l or an NSO cell.

101. A pharmaceutical composition comprising (i) the binding molecule of any one of claims 1 -95, (ii) the nucleic acid o claim 6 or claim 97. (iii) the vector of claim 98, (iv) the host cell of claim 99 or claim 100, or a combination thereof.

102. The composition of claim 101 , further comprising a pharmaceutical ly acceptable carrier.

103. The composition of claim 101 or claim 102, which further comprises a heterologous binding molecule.

104. The composition o claim 103, wherein said heterologous binding molecule specifically binds to an immuriomodulating agent.

105. The composition of claim 104, wherein said immunomodulating agent is TNF-alpha, TWEAK, or IL6.

106. The composition of claim 104 or claim 105, wherein said heterologous binding molecule is an antibody specifically binds to TWEAK, TNF-alpha, or IL-6.

107. The composition of claim 106, wherein said heterologous binding molecule is selected from the group consisting of an anti-TWEAK antibody, an anti-TNF-alpha antibody, an anti- !L-6 antibody, and any combinations thereof.

108. The composition of an one of claims 101 -107, which is administered once a week, once in two weeks, once in three weeks, once in four weeks, once in five weeks, once in 10 days, once in 20 days or once in 30 days.

1 09. The composition of any one o claims 101 - 108 which is administered subeutaneously. intravenously, intraarterially. intraperitoneal!}', intramuscularly, rectal ly, or vaginally.

1 10. The composition of any one of claims 101 -109, wherein said composition is administered to a subject who does not respond to or has developed resistance to one or more therapeutics for inflammatory bowel disease (IBD).

1 1 1 . The composition o claim 1 10, wherein said therapeutics are selected from the group consisting of 5-aminosalicylic acid (5-ASA), sulfasalazine, olsalazine, mesalamine, balsalazide, prednisone, m e t hy 1 p re d n i s o 1 o n e . budesonide. hydrocortisone, zathioprine, 6- mercaptopurine, cyclosporine A. tacrolimus, methotrexate, metronidazole, and ciprofloxacin.

1 12. The composition of any one of claims 101 - 1 1 1. wherein said composition is administered to a subject who does not respond to or has developed resistance to at least one TNF-alpha or TWEAK antagonist.

1 13. The composition of any one of claims 1 01 - 1 12, wherein said binding molecule is administered with one or more therapeutics for IBD.

1 14. The composition of claim 1 13, wherein said therapeutics are selected from the group consisting of 5-aminosalicylic acid (5-ASA), sulfasalazine, olsalazine, mesalamine, balsalazide, prednisone, methyl predni so lone, budesonide, hydrocortisone, zathioprine. 6- mercaptopurine. cyclosporine A, tacrolimus, methotrexate, metronidazole, and ciprofloxacin,.

115. The composition of any one of claims 101-114, wherein said binding molecule is administered with one or more TNF-alpha antagonists.

1 16. The composition of claim 1 15, wherein said TNF-alpha antagonist is selected from the group consisting of infliximab, adalimumab, etanercept, certolizumab pegol, golimumab and any combinations thereof.

1 17. The composition of any one of claims 101- 1 16, which is administered to a subject in need thereof to prevent, ameliorate, or treat a disease or disorder associated with an inflammatory response or autoimmune response.

1 18. The composition of claim 1 17, wherein said disease or disorder associated with an inflammatory response or autoimmune response is selected from the group consisting of rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), psoriasis. Sjogren's syndrome, inflammatory myositis. Langcrhans-cell histiocytosis, adult respirator distress syndrome/bronchiolitis obliterans. Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dcrmatomyositis, polymyositis, noninfectious sclcritis. chronic sarcoidosis with pulmonary involvement, myelodysplastic syndromes/refractory anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, and a combination o two or more of said diseases or disorders.

119. The composition of claim 1 18, wherein said disease or disorder is rheumatoid arthritis, juvenile ideopathic arthritis, ankylosing spondylitis. Crohn's disease, psoriasis, psoriatic arthritis, ulcerative col itis, refractory asthma, or a combination of said diseases or disorders.

120. A method of producing the binding molecule of any one o claims 1 -95. comprising: (i) culturing the host: cell of claim 99 or claim 100 such that the binding molecule is secreted in host cell culture media and (ii) isolating the binding molecule from the media.

121. A method of reducing or inhibiting an activity of TNF-alpha or TWEAK, binding of TNF alpha to a TNF alpha receptor or binding of TWEAK to a TWEAK receptor in a subject in need thereof comprising administering to the subject (i) the binding molecule of any one of claims 1 -95, (ii) the nucleic acid of claim 96 or 97. (iii) the vector of claim 98. (iv) the host cell of claim 99 or 100, or (v) the composition of any one of claims 101 -120.

122. The method of claim 121 , which simultaneously reduces or inhibits an activity of TNF- alpha or TWEAK, binding of TWEAK to a TWEAK receptor, or binding o TNF-alpha to a TNF-alpha receptor in said subject.

123. T he method of claim 121 or claim 122, which is administered to a subject in need thereof to prevent, ameliorate, or treat a disease or disorder associated with an inflammatory response or autoimmune response.

124. A method of treating, preventing, or ameliorating an auto-immune or inflammatory disease or disorder in a subject, comprising administering to a subject in. need thereof (i) the binding molecule of claims 1 -95. (ii) the nucleic acid of claim 96 or claim 97. (iii) the vector of claim 98. (iv) the host cell of claim 99 or claim 100. or (v) the composition of claims 101 - 1 23 in an effective amount to treat, prevent, or ameliorate the auto-immune disease or disorder.

125. The method of claim 124, wherein said disease or disorder is selected from the group consisting o rheumatoid arthritis, juvenile ideopathic arthritis, pediatric psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease ( including ulcerative colitis and Crohn's disease), psoriasis. Sjogren's syndrome, inflammatory myositis. Langcrhans-cell histiocytosis, adult respiratory distress syndromc/bronchiolitis obliterans, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis. polymyositis, noninfectious scleritis. chronic sarcoidosis with pulmonary involvement, myelodysplasia sy n d ro m e s/re f rac to ry anemia with excess blasts, ulcerative colitis, moderate to severe chronic obstructive pulmonary disease, giant cell arteritis, and a combination of two or more of said diseases or disorders.

126. The method of claim 124 or claim 125, wherein said disease or disorder is rheumatoid arthritis, juvenile ideopathic arthritis, ankylosing spondylitis. Crohn's disease, psoriasis, psoriatic arthritis, ulcerative colitis, refractory asthma, or a combination thereof.

1 27. The method of any one of claims 121 - 126. wherein said method reduces one or more side effects compared to the side effects induced by administration of adalimumab.

128. The method of any one of claims 121-127, wherein said binding molecule, composition, nucleic acid, vector, or host cell is administered at a dosage of 0.01 mg/kg - 100 mg/kg.

129. The method of any one of claims 121 -128, wherein said binding molecule, composition, nucleic acid, vector, or host cell is administered parenterally.

130. The method of claim 1 29, wherein binding molecule, composition, nucleic acid, vector, or host cell is administered intravenously, intra-arterially. intraperitoneally, intramuscularly, subcutaneously, rectal ly or vaginally.

131 . The method of any one of claims 121 - 1 30, wherein said administration is once a week, once in two weeks, once in three weeks, once in four weeks, once in five weeks, once in 10 days, once in 20 days or once in 30 days.

132. The method of any one of claims 121 -131 , wherein said subject does not respond to or has developed resistance to one or more therapeutics for inflammatory bowel disease (IBD).

1 33. The method of claim 1 32. wherein said therapeutics are selected from the group consisting of 5 -aminosalicylic acid (5-ASA), sulfasalazine, olsalazine, mesalamine. balsalazide. prednisone, mcthylprednisolone. budesonide. hydrocortisone, zathioprine, 6- mercaptopurine. cyclosporine A, tacrolimus, methotrexate, metronidazole, and ciprofloxacin.

134. The method of any one of claims 121 - 133. wherein said subject does not respond to or has developed resistance to at least one TNF-alpha antagonist.

135. The method of claims 121 - 1 34, which further comprises administering one or more therapeutics or one or more TNF-alpha antagonists or combinations thereof to said subject.

1 36. The method of claim 1 35, wherein said therapeutics are selected from the group consisting of 5-aminosalicylic acid (5-ASA), sul fasalazine, olsalazine, mesalamine, balsalazide, prednisone, methylprednisolone. budesonide. hydrocortisone, zathioprine. 6- mercaptopurine. cyclosporine A, tacrolimus, methotrexate, metronidazole, and ciprofloxacin.

137. The method of claim 136 wherein said TNF-alpha antagonist is selected from the group consisting of infliximab, adalimumab, etanercept. certolizumab pegol, golimumab, and any combinations thereof.

138. The method of claim 136, wherein said therapeutics or TNF-alpha antagonist is administered concurrently or sequentially with said binding molecule, said nucleic acid, said vector, said host cell, or said composition.

139. A method of treating, preventing, or ameliorating a neoplastic disorder comprising adminstering to a subject in need thereof (i) the binding molecule of claims 1 -95, (ii) the nucleic acid of claim 96 or claim 97. (iii) the vector of claim 98, (iv) the host cell of claim 99 or claim 100, or (v) the composition of claims 101 -123 in an effective amount to treat, prevent, or ameliorate the neoplastic disorder.

140. The method of claim 139. wherein said neoplastic disorder is a cancer selected from the group consisting of pancreatic cancer, breast cancer, lung cancer, prostate cancer, colon cancer, colorectal cancer, skin cancer, ovarian cancer, cervical cancer, renal cancer, or adenocarcinoma.

141 . T he method of claim 140. wherein said neoplast ic disorder is pancreatic cancer.