TNF Antagonists

RELATED APPLICATIONS

[001] This patent application claims the benefit of U.S. Provisional Patent

Application Serial No. 61/178,594 filed May 15, 2009, the contents of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[002] This invention relates generally to compositions that contain multiple pharmaceutical regents, such as modulating agents, e.g., multiple neutralizing monoclonal antibodies or Fc-fusion receptors, that target CD3 on T cells and neutralize one or more biological activities of tumor necrosis factor alpha (TNF), such as CD3 modulators and anti- TNF antagonists, and methods of using these compositions in the treatment, amelioration and/or prevention of relapse of an autoimmune disease.

BACKGROUND OF THE INVENTION

[003] Tumor necrosis factor (also known as TNF, cachexin or cachectin and formally known as tumor necrosis factor-alpha) is a cytokine involved in systemic inflammation. Dysregulation and overproduction of TNF has been implicated in a variety of human diseases, as well as cancer. Accordingly, there exists a need for therapies that neutralize one or more biological activities of TNF.

SUMMARY OF THE INVENTION

[004] Both innate and adaptive immune components are involved in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and Crohn's Disease (CD). RA is characterized by leukocyte infiltration and chronic inflammation of joints that induces synovitis and erosion of cartilage and bone. Similarly, CD is associated with leukocyte induced chronic inflammation of the gut mucosa. A widely used model of RA is collagen induced arthritis (CIA) in the DBA mouse strain. In RA and CD patients as well as in CIA, T cell infiltration and high levels of TNF are observed in affected tissues. T cells are thought to exert their effect during the induction phase of the disease while TNF, produced by activated macrophages, plays a critical role during the effector phase. (Luross J.A & Williams N.A. The genetic and immunopathological process underlying collagen- induced arthritis. (2001) Immunology. 103: 407-416).

[005] TNF blocking strategies produce a rapid clinical improvement in many patients with RA and to a lesser extent with CD. However, in both RA and CD, some patients do not respond to TNF antagonists at all and with time, the use of anti-TNF reagents becomes less effective in a substantial percentage of patients who primarily responded to this therapy. These observations underscore the complexity of the mechanisms contributing to chronic pathogenesis. Therefore, modifying both TNF and T cell responses in combination could lead to a synergy that provides long term relief by resetting of the immune system's regulation. A clinically proven way to alter T cell function is by the administration of anti-CD3 antibodies. The mechanism involves modulation of the CD3/TCR complex from the T cell surface and a transient disappearance of lymphocytes from the circulation. Anti-CD3 treatment in NOD mice, a model of Type 1 diabetes, has been shown to not only eliminate pathogenic effector T cells but also induce concomitantly TGF-β dependent T regulatory cells. (Chatenoud L, Thervet E, Primo J, Bach JF. Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. (1994) PNAS. 91 : 123-127; You. S et al. Adaptive TGF-β- dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment. (2007) PNAS. 104: 6335-6340; and Perruche S, Bluestone J.A, Wanjun C et al. CD3-specific antibody-induced immune tolerance involves transforming growth factor-β from phagocytes digesting apoptotic T cells. (2008) Nature. Med. 14: 528-535). However, the efficacy of anti-CD3 treatment in arthritis remains controversial (Maeda T et al. Exacerbation of established collagen-induced arthritis in mice treated with an anti-T cell receptor antibody. (1994) Arthritis. Rheum. 37: 406-413; Hughes et al. Induction of T helper cell hyporesponsiveness in an experimental model of autoimmunity by using nonmitogenic anti-CD3 monoclonal antibody. (1994) J. Immunol. 153: 3319-3325; Pietersz GA et al. Inhibition of destructive autoimmune arthritis in FcgammaRIIa transgenic mice by small chemical entities. (2009) Immunol Cell Biol. 87:3- 12; and Malfait AM et al. Chronic relapsing homologous collagen-induced arthritis in DBA/1 mice as a model for testing disease-modifying and remission-inducing therapies. (2001) Arthritis. Rheum. 44: 1215-1224), once again underlining the multi-factorial nature of autoimmune diseases.

[006] Combination therapy with two monoclonal antibodies (mAbs), one that targets CD3 on T cells and one that neutralizes TNF, produces a potent synergy that reduces disease severity and prevents disease relapse, as shown in Figures 1-4B. These finding provide the basis to support using such a combination strategy to obtain an effective long- term treatment for RA, CD and other autoimmune diseases where TNF therapies are ultimately found limiting.

[007] The combination therapies provided herein are useful for treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease by administering a combination of reagents such as modulating agents to a subject in need thereof in an amount sufficient to treat, delay the progression of, prevent a relapse of, or alleviate the symptom of the autoimmune disease in the subject, wherein the combination of modulating agents comprises a modulating agent that binds to or otherwise interacts with CD3 and an antagonist that binds to or otherwise interacts with TNF. As used herein, the term "modulating agent" refers to a reagent that binds to or otherwise interacts with a target, e.g., CD3, and alters at least one biological property and/or biological activity of that target. The terms "modulating agent" and "modulator" are used interchangeably herein. The CD3 modulators, also referred to herein as CD3 modulating agents and modulators of CD3, bind CD3 and alter or otherwise modify at least one biological property and/or biological activity of that target. In some embodiments, the CD3 modulators used in the combination therapies provided herein have an inhibitory or otherwise neutralizing effect on at least one biological property and/or biological activity of CD3 and also have a stimulatory effect on at least a second biological property and/or biological activity of CD3. For example, in some embodiments, the CD3 modulator binds or otherwise interacts with CD3 and alters (e.g., decreases) the cell surface expression level or activity of CD3 or the T cell receptor (TcR). In some embodiments, exposure to the CD3 modulating agent removes or masks CD3 and/or TcR without affecting cell surface expression of CD2, CD4 or CD8. The masking of CD3 and/or TcR results in the loss or reduction of T-cell activation, which is desirable in autoimmune diseases where uncontrolled T-cell activation occurs. Antigenic modulation refers to the redistribution and elimination of the CD3-T cell receptor complex on the surface of a cell, e.g., a lymphocyte. Decrease in the level of cell surface expression or activity of the TcR on the cell is meant that the amount or function of the TcR is reduced. Modulation of the level of cell surface expression or activity of CD3 is meant that the amount of CD3 on the cell surface or function of CD3 is altered, e.g., reduced. The amount of CD3 or the TcR expressed at the plasma membrane of the cell is reduced, for example, by internalization of CD3 or the TcR upon contact of the cell with the CD3 modulator. Alternatively, upon contact of a cell with the CD3 modulating agent, CD3 or the TcR is masked.

[008] In some embodiments, the modulator of CD3 is an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is a monoclonal antibody (mAb). In some embodiments, the anti-CD3 antibody is a mouse, chimeric, humanized and/or fully human mAb.

[009] In some embodiments, the anti-CD3 antibody is the fully human anti-CD3 mAb referred to herein as "28Fl 1," which includes a heavy chain CDRl having the amino acid sequence GYGMH (SEQ ID NO:5), a heavy chain CDR2 having the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:6), a heavy chain CDR3 having the amino acid sequence QMGYWHFDL (SEQ ID NO: 7), a light chain CDRl having the amino acid sequence RASQSVSSYLA (SEQ ID NO:8), a light chain CDR2 having the amino acid sequence DASNRAT (SEQ ID NO: 9), and a light chain CDR3 having the amino acid sequence QQRSNWPPLT (SEQ ID NO: 10). In some embodiments, the 28Fl 1 antibody comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 2 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 4.

>28F11 VH nucleotide sequence: (SEQ ID NO: 1)

CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCT CCTGTGCAGCGTCTGGATTCAAGTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTC C AGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAAGAAATACTATGT A GACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTG C AAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAAATGGGCT A CTGGCACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

>28F11 VH amino acid sequence: (SEQ ID NO: 2)

QVQLVESGGGVVQPGRSLRLSCAASGFKFSpYGMHlWVRQAPGKGLEWVAlVIWYDG SKKYYV

DSVKGIRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR^GYWHFDLIWGRGTLVTVS S

>28F11 VL nucleotide sequence: (SEQ ID NO: 3)

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCC ACCC TCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTG G CCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAG G TTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAA G ATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGCTCACTTTCGGCGGAG G GACCAAGGTGGAGATCAAA

>28F11 VL amino acid sequence: (SEQ ID NO: 4)

E IVLTQS PATLSLS PGERATLSCIRASQSVS SYLAJWYQQKPGQAPRLLIYIDASNRATIGI PAR

FSGSGSGTDFTLT I S SLEPEDFAVYYCQQRSNWPPLT FGGGTKVE IK

[0010] In some embodiments, the 28Fl 1 antibody further includes a mutation in the heavy chain at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof, and reduces the release of cytokines from a T-cell. In some embodiments, the mutation results in an alanine or glutamic acid residue at the position. In some embodiments, the 28Fl 1 antibody is an IgGl isotype and contains at least a first mutation at position 234 and a second mutation at position 235, wherein the first mutation results in an alanine residue at position 234 and the second mutation results in a glutamic acid residue at position 235.

[0011] In some embodiments, the anti-CD3 modulating agent is a fully human anti-

CD3 mAb. Suitable antibodies for use in the combination therapies and methods provided herein include, by way of non-limiting example, those antibodies described in PCT Publication No. WO 05/118635, the contents of which are hereby incorporated by reference in their entirety, or an anti-CD3 antibody that binds to the same epitope as those antibodies described in PCT Publication No. WO 05/118635. Other suitable anti-CD3 mAbs for use in the combination therapies and methods provided herein include, but are not limited to, Orthoclone OKT3, human OKT3γl (HOKT3γl), ChAglyCD3 and Nuvion® (Visilizumab), or antibodies that bind to the same epitope as Orthoclone OKT3, human OKT3γl (HOKT3γl), ChAglyCD3 or Nuvion® (Visilizumab).

[0012] In some embodiments, the anti-CD3 antibody contains an amino acid mutation. For example, the mutation is in the constant region. Preferably, the mutation results in an antibody that has an altered effector function. An effector function of an antibody is altered by altering, i.e., enhancing or reducing, the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. By altering an effector function of an antibody, it is possible to control various aspects of the immune response, e.g., enhancing or suppressing various reactions of the immune system. For example, the mutation results in an antibody that is capable of reducing cytokine release from a T-cell. For example, the mutation is in the heavy chain at amino acid residue 234, 235, 265, or 297 or combinations thereof. Preferably, the mutation results in an alanine residue at either position 234, 235, 265 or 297, or a glutamate residue at position 235, or a combination thereof. The term "cytokine" refers to all human cytokines known within the art that bind extracellular receptors expressed on the cell surface and thereby modulate cell function, including but not limited to IL-2, IFN-gamma, TNF-a, IL-4, IL-5, IL-6, IL-9, IL- 10, and IL-13.

[0013] The release of cytokines can lead to a toxic condition known as cytokine release syndrome (CRS), a common clinical complication that occurs, e.g., with the use of an anti-T cell antibody such as ATG (anti-thymocyte globulin) and OKT3 (a murine anti- human CD3 antibody). This syndrome is characterized by the excessive release of cytokines such as TNF, IFN-gamma and IL-2 into the circulation. The CRS occurs as a result of the simultaneous binding of the antibodies to CD3 (via the variable region of the antibody) and the Fc Receptors and/or complement receptors (via the constant region of the antibody) on other cells, thereby activating the T cells to release cytokines that produce a systemic inflammatory response characterized by hypotension, pyrexia and rigors. Symptoms of the CRS include fever, chills, nausea, vomiting, hypotension, and dyspnea. Thus, the anti-CD3 antibody used in some embodiments of the combination therapy contains one or more mutations that prevent heavy chain constant region-mediated release of one or more cytokine(s) in vivo.

[0014] The anti-CD3 antibodies of the invention include, for example, a L ²³⁴ L ²³⁵ ->

A ²³⁴ E ²³⁵ mutation in the Fc region, such that cytokine release upon exposure to the anti- CD3 antibody is significantly reduced or eliminated, as described in PCT Publication No. WO 05/118635. Other mutations in the Fc region include, for example, L ²³⁴ L ²³⁵ -» A ²³⁴ A ²³⁵ , L ²³⁵ ^ E ²³⁵ , N ²⁹⁷ ^ A ²⁹⁷ , and D ²⁶⁵ ^ A ²⁶⁵ .

[0015] In some embodiments, the modulator of CD3 is a modified antibody reagent or a non-antibody-based reagent. Such modulators include advanced antibody therapeutics, such as bispecific antibodies, immunotoxins, and radiolabeled therapeutics; peptide therapeutics; gene therapies, particularly intrabodies; oligonucleotide therapeutics such as aptamer therapeutics, antisense therapeutics, interfering RNA therapeutics; and small molecules.

[0016] In some embodiments, the antagonist of TNF is an anti-TNF antibody. In some embodiments, the anti-TNF antibody is a mAb. For example, the anti-TNF antibody is a chimeric, humanized and/or fully human mAb. [0017] In some embodiments, the anti-TNF antagonist is an anti-TNF mAb.

Suitable antibodies for use in the combination therapies and methods provided herein include, but are not limited to, Remicade® (Infliximab (Centocor)), and those antibodies described, for example, in U.S. Patent No. 6,835,823; 6,790,444; 6,284,471; 6,277,969; 5,919,452; 5,698,195; 5,656,272; and 5,223,395 and in EP Patent No. 0610201, the contents of each of which are hereby incorporated by reference in their entirety, or antibodies that bind to the same epitope as Remicade®. Others suitable anti-TNF antibodies for use in the combination therapies and methods provided herein are, by way of non- limiting example, Humira (Adalimumab (Abbott Laboratories, Esai)) as described in U.S. Patent No. 6,090,382; 6,258,562; or 6,509,015 and related patents and applications, the contents of which are hereby incorporated by reference in their entirety; Simponi™ (Golimimab, CNTO 148 (Centocor)) as described in PCT Publication No. WO 02/12502 and related patents and applications, the contents of which are hereby incorporated by reference in their entirety; ART621 (Arana Therapeutics), SSS 07 (Epitopmics and 3SBio) or antibodies that bind to the same epitope as Humira, Simponi, ART621 or SSS 07. [0018] In some embodiments, the TNF antagonist is a fusion protein. Suitable fusion proteins for use in the combination therapies and methods provided herein include, but are not limited to, Enbrel (Etanercept (Amgen)) and other fusion proteins or fragments thereof described in U.S. Patent No. 5,712,155, PCT Publication No. WO 91/03553, and related patents and applications, the contents of which are hereby incorporated by reference in their entirety.

[0019] In some embodiments, the antagonist of TNF is a modified antibody antagonist or a non-antibody-based antagonist. Such antagonists include advanced antibody therapeutics, such as antibody fragments including, but not limited to, Cimzia™ (Certolizumab pegol, CDP870 (Enzon)), bispecific antibodies, Nanobodies® such as ABX 0402 (Ablynx), immunotoxins, and radiolabeled therapeutics; peptide therapeutics; gene therapies, particularly intrabodies; oligonucleotide therapeutics such as aptamer therapeutics, antisense therapeutics, interfering RNA therapeutics; vaccines such as anti- TNF kinoid (Neovacs); and small molecules such as LMP-420 (LeukoMed) as described in EP Patent No. 0767793, and related patents and applications, the contents of which are hereby incorporated by reference in their entirety.

[0020] The combination therapies and methods of use thereof are preferably administered to human subjects. In some embodiments, the subject is non-responsive, less responsive over time, or has otherwise exhibited a decrease in responding to treatment with an anti-TNF antagonist, or is at risk for becoming non-responsive or less responsive to treatment with an anti-TNF antagonist. The anti-TNF antagonist to which the subject has become or is likely to become non-responsive or less responsive can be the same or a different anti-TNF antagonist that the anti-TNF antagonist to be administered in conjunction with a CD3 modulating agent.

[0021] In some embodiments, the autoimmune disease is rheumatoid arthritis or

Crohn's disease. In other embodiments, the autoimmune disease is selected from ankylosing spondylitis, asthma, Behcet's syndrome, glomerular nephritis, graft-versus-host disease, grave's disease, Hashimoto's thyroiditis, hidradenitis suppurativa, juvenile rheumatoid arthritis, luminal and fistulizing Crohn's disease, polyarticular juvenile arthritis, polymyositis/myositis/giant cell myocarditis and dermatomyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, undifferentiated polyarthritis, and uveitis.

[0022] In some embodiments, the CD3 modulating agent and the anti-TNF antagonist are present in the combination in an amount sufficient to produce a synergistic inhibitory effect on one or more biological activities of TNF in the subject. The CD3 modulators and TNF antagonists can be prepared in separate formulations, or alternatively, they can be prepared in the same formulation. In embodiments where the CD3 modulator(s) and TNF antagonist(s) are prepared in separate formulations, the CD3 modulator formulation(s) and TNF antagonist formulation(s) can be administered simultaneously, or at separate times or intervals.

[0023] In some embodiments, the CD3 modulating agent is administered in a pharmaceutical formulation. In some embodiments, the CD3 modulating agent is an anti- CD3 antibody administered in a pharmaceutical formulation. Suitable pharmaceutical formulations are described, for example, in PCT Publication No. WO 07/033230, the contents of which are hereby incorporated by reference in their entirety. [0024] In some embodiments, the CD3 modulating agent is an anti-CD3 antibody that is administered in a pharmaceutical formulation that includes a pH buffering agent in a range of 10 mM to 50 mM and effective in the range of 5.0 to 6.0, wherein said pH buffering agent is sodium acetate; sodium chloride in a range of 100 mM to 140 mM; 0.02% by weight/volume of a surfactant; and a pharmaceutically effective quantity of the anti-CD3 antibody. In some embodiments, the sodium chloride is 125 nM NaCl. In some embodiments, the surfactant is an ionic, anionic or zwitterionic surfactant. In some embodiments, the ionic surfactant is a polysorbate. In some embodiments, the pH buffering agent provides a pH range between 5.2 and 5.8. In some embodiments, the pH buffering agent provides a pH range between 5.4 and 5.6. In some embodiments, the pH buffering range provides a pH of 5.5. In some embodiments, the surfactant is 0.02% by weight/volume and wherein the surfactant is polysorbate 80. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.05 mg to 10 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.1 mg to 5.0 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.5 mg to 3.0 mg of anti-CD3 antibody.

[0025] In some embodiments, the pharmaceutical formulation for the anti-CD3 antibody consists essentially of a pH buffering agent in a range of 10 mM to 50 mM and effective in the range of 5.0 to 6.0, wherein said pH buffering agent is sodium acetate; sodium chloride in a range of 100 mM to 140 mM; 0.02% by weight/volume of a surfactant; and a pharmaceutically effective quantity of the anti-CD3 antibody. In some embodiments, the sodium chloride is 125 nM NaCl. In some embodiments, the surfactant is an ionic, anionic or zwitterionic surfactant. In some embodiments, the ionic surfactant is a polysorbate. In some embodiments, the pH buffering agent provides a pH range between 5.2 and 5.8. In some embodiments, the pH buffering agent provides a pH range between 5.4 and 5.6. In some embodiments, the pH buffering range provides a pH of 5.5. In some embodiments, the surfactant is 0.02% by weight/volume and wherein the surfactant is polysorbate 80. In some embodiments, the pharmaceutically effective quantity of the anti- CD3 antibody is formulated to provide a quantity per dose in the range of 0.05 mg to 10 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.1 mg to 5.0 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.5 mg to 3.0 mg of anti-CD3 antibody.

[0026] In some embodiments, the pharmaceutical formulation for the anti-CD3 antibody consists essentially of a pH buffering agent comprising 25 mM sodium acetate effective in the range of 5.0 to 6.0; 125 rnM sodium chloride; a surfactant comprising a polysorbate; and a pharmaceutically effective quantity of an anti-CD3 antibody. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the pH buffering agent provides a pH range between 5.2 and 5.8. In some embodiments, the pH buffering agent provides a pH range between 5.4 and 5.6. In some embodiments, the pH buffering range provides a pH of 5.5. In some embodiments, the surfactant is 0.02% by weight/volume and wherein the surfactant is polysorbate 80. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.05 mg to 10 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.1 mg to 5.0 mg of anti-CD3 antibody. In some embodiments, the pharmaceutically effective quantity of the anti-CD3 antibody is formulated to provide a quantity per dose in the range of 0.5 mg to 3.0 mg of anti-CD3 antibody.

[0027] The invention also provides methods of enhancing or otherwise supplementing anti-TNF therapy in a subject that is receiving or has been administered an anti-TNF antagonist in an amount that is sufficient to produce a desired therapeutic outcome in the subject comprising administering to the subject a CD3 modulating agent. For example, the CD3 modulating agent is administered to the subject in an amount that is sufficient to reduce the dosage of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject. For example, the CD3 modulating agent is administered to the subject in an amount that is sufficient to decrease the frequency of administration of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject.

[0028] In some embodiments, the desired biological outcome is treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease in the subject. In some embodiments, the autoimmune disease is rheumatoid arthritis or Crohn's disease. In some embodiments, the autoimmune disease is selected from the group consisting of ankylosing spondylitis, asthma, Behcet's syndrome, glomerular nephritis, graft-versus-host disease, grave's disease, Hashimoto's thyroiditis, hidradenitis suppurativa, juvenile rheumatoid arthritis, luminal and fϊstulizing Crohn's disease, polyarticular juvenile arthritis, polymyositis/myositis/giant cell myocarditis and dermatomyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, undifferentiated polyarthritis, and uveitis. [0029] In some embodiments, the modulator of CD3 is an anti-CD3 antibody. For example, the anti-CD3 antibody is a monoclonal antibody. Suitable anti-CD3 antibodies for use in these methods include, for example, mouse, chimeric, humanized and/or fully human monoclonal antibodies. In some embodiments, the anti-CD3 antibody is the fully human anti-CD3 monoclonal antibody 28Fl 1 comprising a heavy chain CDRl having the amino acid sequence GYGMH (SEQ ID NO:5), a heavy chain CDR2 having the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:6), a heavy chain CDR3 having the amino acid sequence QMGYWHFDL (SEQ ID NO: 7), a light chain CDRl having the amino acid sequence RASQSVSSYLA (SEQ ID NO:8), a light chain CDR2 having the amino acid sequence DASNRAT (SEQ ID NO: 9), and a light chain CDR3 having the amino acid sequence QQRSNWPPLT (SEQ ID NO: 10). In some embodiments, the 28Fl 1 antibody further comprises a mutation in the heavy chain at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof, and reduces the release of cytokines from a T-cell. In some embodiments, the mutation results in an alanine or glutamic acid residue at the position. In some embodiments, the 28Fl 1 antibody is an IgGl isotype and contains at least a first mutation at position 234 and a second mutation at position 235, wherein the first mutation results in an alanine residue at position 234 and the second mutation results in a glutamic acid residue at position 235. In some embodiments, the 28Fl 1 antibody comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 2 and a variable light chain region comprising the amino acid sequence of SEQ ID NO: 4.

[0030] In some embodiments, the antagonist of TNF is an anti-TNF antibody or a fusion protein that binds to TNF. In some embodiments, the anti-TNF antibody is a monoclonal antibody. Suitable anti-TNF antibodies for use in these methods include, for example, chimeric, humanized or fully human monoclonal antibodies. In some embodiments, the antagonist of TNF is a soluble Fc-fusion receptor protein. [0031] In a preferred embodiment, the subject is a human. In some embodiments, the subject is non-responsive, less responsive or has exhibited a decrease in responding to treatment with an anti-TNF antagonist. [0032] In some embodiments, the CD3 modulating agent is administered in an amount sufficient to produce a synergistic inhibitory effect on one or more biological activities of TNF in the subject.

[0033] Also provided herein are uses of a combination of modulating agents for treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease, wherein the combination of modulating agents includes a modulating agent that binds to CD3 and an antagonist that binds to tumor necrosis factor α (TNF) present in an amount sufficient to treat, delay the progression of, prevent a relapse of, or alleviate the symptom of the autoimmune disease in a subject. Also provided herein are uses of a combination of modulating agents in the manufacture of medicaments for treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease, wherein the combination of modulating agents includes a modulating agent that binds to CD3 and an antagonist that binds to tumor necrosis factor α (TNF) present, and wherein the CD3 modulating agent and the TNFα antagonist are present in the medicament in an amount sufficient to treat, delay the progression of, prevent a relapse of, or alleviate the symptom of the autoimmune disease in a subject.

[0034] In some embodiments of these uses, the modulator of CD3 is an anti-CD3 antibody. For example, the anti-CD3 antibody is a monoclonal antibody, such as, e.g. , a mouse, chimeric, humanized, or fully human monoclonal antibody. In some embodiments of these uses, the antagonist of TNF is an anti-TNF antibody or a fusion protein that binds to TNF. For examples, the anti-TNF antibody is a monoclonal antibody such as, e.g., a chimeric, humanized or fully human monoclonal antibody. In some embodiments of these uses, the antagonist of TNF is a soluble Fc-fusion receptor protein. [0035] In some embodiments of these uses, the subject is a human. In some embodiments of these uses, the subject is non-responsive, less responsive or has stopped responding to treatment with an anti-TNF antagonist.

[0036] In some embodiments of these uses, the autoimmune disease is rheumatoid arthritis or Crohn's disease. In some embodiments, the autoimmune disease is selected from the group consisting of ankylosing spondylitis, asthma, Behcet's syndrome, glomerular nephritis, graft-versus-host disease, grave's disease, Hashimoto's thyroiditis, hidradenitis suppurativa, juvenile rheumatoid arthritis, luminal and fistulizing Crohn's disease, polyarticular juvenile arthritis, polymyositis/myositis/giant cell myocarditis and dermatomyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, undifferentiated polyarthritis, and uveitis. [0037] In some embodiments of these uses, the CD3 modulating agent and the anti-

TNF antagonist are present in the combination of modulating agents in an amount sufficient to produce a synergistic inhibitory effect on one or more biological activities of TNF in the subject.

[0038] In some embodiments of these uses, the CD3 modulating agent is a fully human anti-CD3 monoclonal antibody that includes a heavy chain CDRl having the amino acid sequence GYGMH (SEQ ID NO:5), a heavy chain CDR2 having the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:6), a heavy chain CDR3 having the amino acid sequence QMGYWHFDL (SEQ ID NO: 7), a light chain CDRl having the amino acid sequence RASQSVSSYLA (SEQ ID NO:8), a light chain CDR2 having the amino acid sequence DASNRAT (SEQ ID NO: 9), and a light chain CDR3 having the amino acid sequence QQRSNWPPLT (SEQ ID NO: 10). In some embodiments of these uses, the anti-CD3 antibody also includes a variable heavy chain region including the amino acid sequence of SEQ ID NO: 2 and a variable light chain region including the amino acid sequence of SEQ ID NO: 4.

[0039] In some embodiments of these uses, the anti-CD3 antibody also includes a mutation in the heavy chain at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof, and reduces the release of cytokines from a T-cell. For example, the mutation results in an alanine or glutamic acid residue at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof. In some embodiments of these uses, the anti-CD3 antibody antibody is an IgGl isotype and contains at least a first mutation at position 234 and a second mutation at position 235, wherein the first mutation results in an alanine residue at position 234 and the second mutation results in a glutamic acid residue at position 235.

[0040] Also provided herein are uses of an CD3 modulating agent for enhancing or supplementing anti-TNF therapy in a subject that is receiving or has been administered an anti-TNF antagonist in an amount that is sufficient to produce a desired therapeutic outcome in the subject. In these uses, the subject is currently receiving or has received in the past an anti-TNF therapy to achieve a desired therapeutic outcome, e.g. , treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease in the subject. In some embodiments of these uses, the subject is non-responsive, less responsive or otherwise exhibits a decrease in responsiveness to the anti-TNF therapy. In some embodiments of these uses, the CD3 modulating agent is used in an amount that is sufficient to reduce the dosage of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject. In some embodiments of these uses, the CD3 modulating agent is used in an amount that is sufficient to decrease the frequency of administration of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject.

[0041] Also provided are uses of an CD3 modulating agent in the manufacture of a medicament for enhancing or supplementing anti-TNF therapy in a subject that is receiving or has been administered an anti-TNF antagonist in an amount that is sufficient to produce a desired therapeutic outcome in the subject. In these uses, the subject is currently receiving or has received in the past an anti-TNF therapy to achieve a desired therapeutic outcome, e.g., treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease in the subject. In some embodiments of these uses, the subject is non-responsive, less responsive or otherwise exhibits a decrease in responsiveness to the anti-TNF therapy. In some embodiments of these uses, the CD3 modulating agent is present in the medicament in an amount that is sufficient to reduce the dosage of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject. In some embodiments of these uses, the CD3 modulating agent is present in the medicament in an amount that is sufficient to decrease the frequency of administration of anti-TNF antagonist that is needed to produce the desired therapeutic outcome in the subject. [0042] In some embodiments of these uses, the modulator of CD3 is an anti-CD3 antibody. For example, the anti-CD3 antibody is a monoclonal antibody, such as, e.g. , a mouse, chimeric, humanized, or fully human monoclonal antibody. In some embodiments of these uses, the antagonist of TNF is an anti-TNF antibody or a fusion protein that binds to TNF. For examples, the anti-TNF antibody is a monoclonal antibody such as, e.g., a chimeric, humanized or fully human monoclonal antibody. In some embodiments of these uses, the antagonist of TNF is a soluble Fc-fusion receptor protein. [0043] In some embodiments of these uses, the subject is a human. In some embodiments of these uses, the subject is non-responsive, less responsive or has stopped responding to treatment with an anti-TNF antagonist.

[0044] In some embodiments of these uses, the subject is currently receiving or has received in the past an anti-TNF therapy to achieve a desired level of treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease in the subject. In some embodiments of these uses, the autoimmune disease is rheumatoid arthritis or Crohn's disease. In some embodiments, the autoimmune disease is selected from the group consisting of ankylosing spondylitis, asthma, Behcet's syndrome, glomerular nephritis, graft-versus-host disease, grave's disease, Hashimoto's thyroiditis, hidradenitis suppurativa, juvenile rheumatoid arthritis, luminal and fϊstulizing Crohn's disease, polyarticular juvenile arthritis, polymyositis/myositis/giant cell myocarditis and dermatomyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, undifferentiated polyarthritis, and uveitis. [0045] In some embodiments of these uses, the CD3 modulating agent and the anti-

TNF antagonist are used and/or are present in the combination of modulating agents in an amount sufficient to produce a synergistic inhibitory effect on one or more biological activities of TNF in the subject.

[0046] In some embodiments of these uses, the CD3 modulating agent is a fully human anti-CD3 monoclonal antibody that includes a heavy chain CDRl having the amino acid sequence GYGMH (SEQ ID NO:5), a heavy chain CDR2 having the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:6), a heavy chain CDR3 having the amino acid sequence QMGYWHFDL (SEQ ID NO: 7), a light chain CDRl having the amino acid sequence RASQSVSSYLA (SEQ ID NO:8), a light chain CDR2 having the amino acid sequence DASNRAT (SEQ ID NO: 9), and a light chain CDR3 having the amino acid sequence QQRSNWPPLT (SEQ ID NO: 10). In some embodiments of these uses, the anti-CD3 antibody also includes a variable heavy chain region including the amino acid sequence of SEQ ID NO: 2 and a variable light chain region including the amino acid sequence of SEQ ID NO: 4.

[0047] In some embodiments of these uses, the anti-CD3 antibody also includes a mutation in the heavy chain at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof, and reduces the release of cytokines from a T-cell. For example, the mutation results in an alanine or glutamic acid residue at an amino acid residue at position 234, 235, 265, or 297 or combinations thereof. In some embodiments of these uses, the anti-CD3 antibody is an IgGl isotype and contains at least a first mutation at position 234 and a second mutation at position 235, wherein the first mutation results in an alanine residue at position 234 and the second mutation results in a glutamic acid residue at position 235. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a graph depicting that a single i.p. injection of Vlq-Novi demonstrates a dose dependent effect on clinical score evolution when using three different doses in CIA mice.

[0049] Figure 2 is a graph depicting that five doses of 50 μg of 2Cl 1-Novi does not ameliorate arthritis in CIA mice.

[0050] Figure 3 is a graph depicting that the combitherapy (i.e., combination therapy) controls arthritis in CIA mice for prolonged time.

[0051] Figures 4 A and 4B are a series of graphs depicting that anti-CD3/anti-TNF combination therapy impairs T cell proliferation capacity and completely abolishes the production of the pro-inflammatory cytokines IL- 17 and IFNγ.

DETAILED DESCRIPTION

[0052] The present invention provides combination therapies and methods that use modulators of CD3 and antagonists of TNF to treat, delay the progression of, prevent relapse of or alleviate a symptom of an autoimmune disease. The combination therapies are particularly useful in patients who do not respond to TNF antagonists at all or in patients who receive an anti-TNF therapy and may even initially have responded favorably to this therapy, but in time, see the use of the anti-TNF reagent become less effective. [0053] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. [0054] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: [0055] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F _ab , F _ab' and F _(ab')2 fragments, and an F _ab expression library. By "specifically bind" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (K _d > 10 ^~6 ) with other polypeptides.

[0056] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site.

[0057] The term "monoclonal antibody" (mAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the mAb are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0058] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG ₁ , IgG ₂ , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0059] The term "antigen-binding site" or "binding portion" refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs." The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia &Lesk J. MoI. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878- 883 (1989).

[0060] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T- cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 μM; preferably < 100 nM and most preferably < 10 nM.

[0061] As used herein, the terms "immunological binding," and "immunological binding properties" refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (IQ) of the interaction, wherein a smaller IQ represents a greater affinity. Immunological binding properties of selected polypeptides are quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K _0n ) and the "off rate constant" (K _o ff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361 :186-87 (1993)). The ratio of K _off /K _0n enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant IQ. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is said to specifically bind to a CD3 epitope when the equilibrium binding constant (K _d ) is ≤l μM, preferably ≤ 100 nM, more preferably ≤ 10 nM, and most preferably ≤ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0062] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human mAb has the same specificity as a human mAb of the invention (e.g., mAb 28Fl 1) by ascertaining whether the former prevents the latter from binding to a CD3 antigen polypeptide. If the human mAb being tested competes with a human mAb of the invention, as shown by a decrease in binding by the human mAb of the invention, then the two mAbs bind to the same, or a closely related, epitope. Another way to determine whether a human mAb has the specificity of a human mAb of the invention is to pre-incubate the human mAb of the invention with the CD3 antigen polypeptide with which it is normally reactive, and then add the human mAb being tested to determine if the human mAb being tested is inhibited in its ability to bind the CD3 antigen polypeptide. If the human mAb being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the mAb of the invention. [0063] Various procedures known within the art are used for the production of the mAbs directed against a protein such as a CD3 protein, or against derivatives, fragments, analogs homo logs or orthologs thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies" or "fully human antibodies" herein. Human mAbs are prepared, for example, using the procedures described in PCT Publication No. WO 05/118635. Human mAbs can be also prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human mAbs (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human mAbs may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0064] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffϊnity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28). [0065] It is desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating immune-related diseases. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol, 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). [0066] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the modulating agents, antagonists and antibodies used in the combination therapies and methods provided herein. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0067] Coupling is accomplished by any chemical reaction that will bind the two molecules so long as the modulating agent, antagonist or the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding is achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents.

[0068] The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0069] The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature. [0070] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules and the human light chain immunoglobulin molecules shown in SEQ ID NOs: 2 and 4, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. [0071] The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0072] The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. [0073] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0074] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E. S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland7 Mass. (1991)). Stereoisomers (e.g., D- amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α- disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N- trimethyllysine, ε -N-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0075] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0076] Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0077] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine- arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine. [0078] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0079] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long' more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to CD3, under suitable binding conditions, (2) ability to block appropriate CD3 binding, or (3) ability to inhibit CD3 -expressing cell growth in vitro or in vivo. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally- occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide. [0080] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0081] As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin {e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ 1, ¹³¹ I), fluorescent labels {e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0082] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

[0083] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[0084] Generally, a substantially pure composition will comprise more than about

80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0085] The term patient includes human and veterinary subjects.

Therapeutic Administration and Formulations

[0086] It will be appreciated that administration of combinations of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2) :210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1- 2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery- some emerging concepts." J Pharm Sci.89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists. [0087] Combination therapies of the invention, which include a CD3 modulating agent and an anti-TNF antagonist, are used to treat or alleviate a symptom associated with an immune-related disorder, such as, for example, an autoimmune disease. [0088] Autoimmune diseases include, for example, Acquired Immunodeficiency

Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospho lipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue- dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis- juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff- man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis. [0089] Symptoms associated with immune-related disorders include, for example, inflammation, fever, loss of appetite, weight loss, abdominal symptoms such as, for example, abdominal pain, diarrhea or constipation, joint pain or aches (arthralgia), fatigue, rash, anemia, extreme sensitivity to cold (Raynaud's phenomenon), muscle weakness, muscle fatigue, changes in skin or tissue tone, shortness of breath or other abnormal breathing patterns, chest pain or constriction of the chest muscles, abnormal heart rate (e.g., elevated or lowered), light sensitivity, blurry or otherwise abnormal vision, and reduced organ function. For example, symptoms of RA include joint pain, joint tenderness, joint swelling, fatigue, loss of appetite, joint stiffness including morning stiffness lasting more than 1 hour, widespread muscle aches, weakness, anemia (e.g., due to failure of the bone marrow to produce sufficient new red blood cells), eye burning, itching, and other discharge, deformities in the hands and feet, limited range of motion, low-grade fever, lung inflammation (pleurisy), nodules under the skin, numbness or tingling sensation, skin redness, paleness, warmth or inflammation, and swollen glands. Symptoms of CD include abdominal cramps and pain, fever, fatigue, loss of appetite, pain associated with passing stool (tenesmus), persistent, watery diarrhea, unintentional weight loss, constipation, eye inflammation, fistulas, joint pain, liver inflammation, mouth ulcers, rectal bleeding, skin rash and swollen gums.

[0090] The therapeutic combinations of CD3 modulators and TNF antagonists are administered to a subject suffering from an immune-related disorder, such as an autoimmune disease or an inflammatory disorder, such as, for example, RA and CD. A subject suffering from an autoimmune disease or an inflammatory disorder is identified by methods known in the art. For example, subjects suffering from an autoimmune disease such as RA or CD, are identified using any of a variety of clinical and/or laboratory tests such as, physical examination, radiologic examination and blood, urine and stool analysis to evaluate immune status. Patients suffering from CD are identified, e.g., using an upper gastrointestinal (GI) series and/or a colonoscopy to evaluate the small and large intestines, respectively. Patients suffering from RA are identified, e.g. , using blood tests to distinguish RA from other types of arthritis, e.g., the anti-CCP antibody test, a complete blood count, a C-reactive protein test, evaluation of erythrocyte sedimentation rate, joint x-rays, ultrasound or MRI, rheumatoid factor test, and synovial fluid analysis.

[0091] Administration of the therapeutic combinations of CD3 modulators and TNF antagonists to a patient suffering from an autoimmune disease or an inflammatory disorder is considered successful if any of a variety of laboratory or clinical results is achieved. For example, administration of the therapeutic combinations of CD3 modulators and TNF antagonists to a patient suffering from an immune-related disorder such as an autoimmune disease or an inflammatory disorder, such as, for example, RA or CD, is considered successful one or more of the symptoms associated with the disorder is alleviated, reduced, inhibited or does not progress to a further, i.e., worse, state. Administration of the therapeutic combinations of CD3 modulators and TNF antagonists to a patient suffering from an immune-related disorder such as an autoimmune disease or an inflammatory disorder is considered successful if the disorder, e.g., an autoimmune disorder, enters remission or does not progress to a further, i.e., worse, state. [0092] In some embodiments, the combination therapies used to treat an autoimmune disease are administered in combination with any of a variety of known antiinflammatory and/or immunosuppressive compounds. Suitable known compounds include, but are not limited to methotrexate, cyclosporin A (including, for example, cyclosporin microemulsion), tacrolimus, corticosteroids, statins, interferon beta, non-steroidal antiinflammatory agents, 6-MP (Mercaptopurine, also called 6-Mercaptopurine, or Purinethol). For example, subjects with RA are also administered a disease modifying anti-rheumatic drug (DMARD) such as methotrexate or leflunomide; an anti-inflammatory medication such as aspirin or a nonsteroidal anti-inflammatory drug (NSAID), an anti-malarial medication such as hydroxychloroquine or sulfasalazine, alone or in further combination with methotrexate; a corticosteroid, a cyclooxygenase-2 (COX-2) inhibitor, a specific white blood cell modulating biological agent to control inflammation such as, e.g. , abatacept or rituximab, and combinations thereof. For example, subjects with CD are also administered an anti-diarrheal drug such as loperamide or other over the counter medications, an aminosalicylate (5 -ASA) to control inflammation, a corticosteroid such as prednisone or methylprednisolone, an immunomodulator such as azathioprine or 6-mercaptopurine, an antibiotic, or combinations thereof.

[0093] In some embodiments, the combination therapies used to treat an autoimmune disease are used in conjunction with a surgical method of treating or otherwise alleviating the autoimmune disease. For example, subjects with RA may require surgery to correct severely affected joints, relieve joint pain, correct deformities, and improve joint function. Subjects with CD may require surgery such as a bowel resection or other surgical methods to reduce bleeding or other hemorrhage, to remove fistulas, to treat infections and abscesses, or to correct intestinal narrowing and strictures.

[0094] The combinations of modulating agents, e.g., CD3 modulators and TNF antagonists, are administered to a subject in an amount sufficient to have a desired modulation effect due to binding with the respective targets. In some embodiments, administration of the combinations will abrogate or inhibit or otherwise interfere with at least one biological property and/or biological activity of that target, such as e.g., a signaling function of the target, binding of the target with an endogenous ligand to which it naturally binds, etc. [0095] A therapeutically effective amount of a combination described herein relates generally to the amount needed to achieve a therapeutic objective such as, for example, treating, delaying the progression of, preventing a relapse of, or alleviating a symptom of an autoimmune disease. As noted above, this may be a binding interaction between the antibody and its target antigen(s) that, in certain cases, interferes with the functioning of the target(s). The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen(s), and will also depend on the rate at which an administered antibody is depleted from the free volume of the subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody, antibody combination or antibody fragment described herein may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week. [0096] The CD3 modulators and TNF antagonists can be prepared in separate formulations, or alternatively, they can be prepared in the same formulation. In embodiments where the CD3 modulator(s) and TNF antagonist(s) are prepared in separate formulations, the CD3 modulator formulation(s) and TNF antagonist formulation(s) can be administered simultaneously, or at separate times or intervals.

[0097] Combination therapies of the invention are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0098] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.

EXAMPLES

[0099] The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.

EXAMPLE 1: Generation and in vitro characterization of anti-TNF and anti-CD3 mAbs

[00100] Anti-mouse TNF mAb: A neutralizing anti-mouse TNF mAb, VIq, has been described. (Echtenacher B, FaIk W, Mannel DN, Krammer PH. Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis. (1990) J. Immunol. 145: 3762-3766). However, this rat mAb has an IgD isotype, which does not bind mouse Fc-gamma-receptors (FcγRs). As the Fc portion of anti-TNF antibodies has been shown to play a role in their mechanism of action (Scallon B, Cai A, Radewonuk J, Naso M. Addition of an extra immunoglobulin domain to two anti-rodent TNF monoclonal antibodies substantially increased their potency. (2004) MoI. Immunol. 41 : 73-80), a chimeric variant of VIq was generated by engineering the rat VIq variable region onto a mouse Fcγ portion. The new Fc region is from mouse IgG2a, which binds to mouse FcγRs. The resulting anti-mouse TNF mAb is designated Vlq-Novi. [00101] Anti-mouse CD3 mAb: A hamster mAb, 145-2C11, directed against the epsilon chain of the mouse CD3/TCR complex, has been described. (Leo O, Foo M, Sachs DH, Samelson L. E, Bluestone J.A. Identification of a monoclonal antibody specific for a murine T3 polypeptide. (1987) PNAS. 84: 1374-1378). However, for these studies, the Fc portion of the mAb was engineered to minimize the toxic cytokine storm associated with anti-CD3 mAbs. (Hirsch. R, Bluestone J.A, Bare CV, and Gress R.E. Advantages of F(ab') ₂ Fragments of Anti-CD3 monoclonal Antibody as Compared to whole Antibody as Immunosuppressive agents in Mice. (1991) Transplant. Proceedings. 23: 270-271). For this, a chimeric variant of 145 -2Cl 1 was generated by grafting the hamster 145 -2Cl 1 variable region onto a mouse Fcγ portion. This new Fc region is from mouse IgGl . This Fc region was rendered incapable of binding to FcγRs by introducing a single point mutation. This mutation replaces an aspartic acid by an alanine at position 265. (Horiuchi K, Ravetch JV. FCγRIV: A Novel FcR with Distinct IgG Subclass Specificity. (2005) Immunity. 23: 41-51). The resulting chimeric anti-mouse CD3ε mAb is designated 2Cl 1-Novi. [00102] In Vitro Assays for assessing the function of the antibodies: Both mAbs were characterized using relevant functional assays. 2Cl 1-Novi modulated the CD3/TCR complex from the plasma membrane of mouse T cells and V Iq-No vi neutralized the biological activity of TNF in a WEHI cell death assay. A dose-dependent effect was observed in both assays.

EXAMPLE 2. Establishing a suboptimal dose for anti-TNF treatment in collagen-induced arthritis (CIA)

[00103] Collagen induced arthritis (CIA): CIA was induced in male DBA/1 mice, by immunization with 100 μg of bovine type II collagen emulsified in Freund's complete adjuvant (CFA). Three weeks later, a booster injection consisting of 100 μg of collagen in Freund's incomplete adjuvant (IFA) was performed. The mice developed a classic course of disease characterized by chronic inflammation in the limbs and joints commencing a few days after the antigenic boost (Figure 1, isotype control).

[00104] To address the effect of anti-TNF therapy on disease progression in arthritis,

CIA mice were treated with a single injection of Vlq-Novi at onset of disease. As shown in Figure 1, a dose dependent amelioration of the clinical score was observed. Using this data, 100 μg was considered to be a suboptimal dose.

EXAMPLE 3. The effect of anti-CD3 treatment in CIA

[00105] To address the effect of anti-CD3 therapy in arthritis, CIA mice were treated with 2Cl 1-Novi at onset of disease using a protocol that induces remission in the NOD mouse (i.e., 5 daily doses of 50 μg intraperitoneally). The data shown in Figure 2 demonstrate that other forces are dominating the inflammation, masking the expected effect of an anti-CD3 treatment. EXAMPLE 4. Targeting T cells with an anti-CD3 mAb while also neutralizing TNF provides a highly effective improvement of disease in arthritic animals

[00106] As treatment of CIA with 2C 11 -Novi alone did not alter clinical score severity, we hypothesized that in the pathogenesis of CIA, TNF, which plays a dominant role in the effector phase of the disease, masks the importance of pathogenic T cells. To study the contribution of T cells in the chronic phase of CIA, the suboptimal dose of the anti-TNF mAb, V Iq-No vi (i.e. 100 μg), was combined with the administration of the classical treatment of the anti-CD3 mAb, 2Cl 1 -Novi. The results, shown in Figure 3, demonstrate that the combination approach is significantly more effective in preventing disease evolution as well as prolonging time to relapse than occurs with anti-TNF mAb therapy alone. These experiments establish, for the first time, that TNF and T cell responses cooperate on a long term basis during the chronic phase of an autoimmune disease.

EXAMPLE 5. Functional properties of the T cell subpopulations

[00107] To investigate the mechanism behind disease amelioration, cells were isolated from the draining lymph nodes of arthritic mice then activated in vitro with ConA or Collagen. As shown in Figure 4A, the capacity of cells to proliferate (as assessed by thymidine incorporation) was significantly decreased only in the group of mice treated with anti-CD3 and anti-TNF mAbs in combination. Furthermore, to investigate the effect on two cytokines established as key inducers of RA and CD (Dardalhon V, Korn T, Kuchroo VK and Anderson AC. Role of ThI and ThI 7 cells in organ-specific autoimmunity. (2008) J Autoimmun. 31 :252-6), IL- 17 and IFNγ were measured in the supernatants from these lymph node cell cultures following in vitro activation with Con A. While the anti-TNF mAb alone reduced levels of the pro-inflammatory cytokine IL- 17, the combination therapy completely inhibited production of both IL- 17 and IFNγ production (Figure 4B). Therefore, a strategy aimed at targeting both TNF and T cells via the CD3/TCR complex reduces auto- aggressive T cell proliferation capacity and a parallel reduction in pathogenic Th 17 and ThI cells providing long term disease remission.

EXAMPLE 6. Combination Therapies using anti-CD3 mAb and anti-TNF mAbs

[00108] Modifying components of both innate and acquired immunity leads to disease amelioration in a model of autoimmunity. Combination therapy with two mAbs that target CD3 on T cells and neutralize TNF produces a potent synergy that reduces disease severity and prevents disease relapse. This data thus provides the basis to support using such a combination strategy to obtain an effective long-term treatment for RA, CD and other autoimmune diseases where TNF therapies are ultimately found limiting.

OTHER EMBODIMENTS

[00109] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.