WO2007117600A2

WO2007117600A2 - Combination therapy for treating autoimmune diseases

Info

Publication number: WO2007117600A2
Application number: PCT/US2007/008579
Authority: WO
Inventors: Scott Koenig
Original assignee: Macrogenics Inc
Current assignee: Macrogenics Inc
Priority date: 2006-04-07
Filing date: 2007-04-06
Publication date: 2007-10-18
Anticipated expiration: 2008-10-07
Also published as: WO2007117600A3

Abstract

A combination therapy comprising administering to a subject in need of an effective therapeutic treatment for an autoimmune disease, a T cell antibody that is capable of modulating the activation of a T cell response to an antigen presenting cell in combination with a B cell antibody. The combination therapy may provide improved clinical benefits to a subject in need of treatment by reducing the circulating and tissue levels of B cells while providing immunosuppression of T cell activation, thus modulating the immune response to self antigens.

Description

COMBINATION THERAPY FOR TREATING AUTOIMMUNE DISEASES

FIELD OF THE INVENTION [001] The present invention relates to a combination therapy for treating a subject having an autoimmune disease and to methods of treatment that use the combination therapy. In one embodiment, the invention provides a combination therapy for administration to a subject having an autoimmune disease comprising a first therapeutic antibody directed to a T cell and a second antibody directed to an antigen presenting cell where the combination therapy provides improved clinical benefits over therapy comprising administration of a single antibody.

BACKGROUND OF THE INVENTION

[002] The immune system provides host defense mechanisms known as innate immunity that act from the start of an infection without adapting to a particular pathogen. It also provides mechanisms known as adaptive immunity that respond to a specific antigen by developing an immunological memory. The adaptive immune response relies on antigen-specific T and B lymphocytes, both of which respond to antigen. The response of each cell type in adaptive immunity differs significantly. T cells are responsible for cell-mediated immunity. B cells are responsible for making immunoglobulins and antibodies. Both T cell and B cell responses are found in autoimmune disease.

[003] A naive T cell, e.g. a T cell which has not yet encountered its specific antigen, is activated when it first encounters a specific peptide:MHC complex on an antigen presenting cell. The antigen presenting cell may be a B cell, a macrophage or a dendritic cell. When a naive T cell encounters a specific peptide:MHC complex on an antigen presenting cell, a signal is delivered through the T-cell receptor which induces a change in the conformation of the T cell's LFA molecules, and increases their affinity for ICAM present on the surface of the antigen presenting cell. The signal generated by the interaction of the T cell with an antigen presenting cell is necessary, but not sufficient, to activate a naive T cell. A second co-stimulatory signal is required. The naive T cell can be activated only by an antigen-presenting cell carrying both a specific peptide MHC complex and a co-stimulatory molecule on its surface. Antigen recognition by a naive T cell in the absence of co- stimulation results in the T cell becoming anergic. The need for two signals to activate T cells and B cells such that they achieve an adaptive immune response^* may provide a mechanism for avoiding responses to self antigens that may be present on an antigen presenting cell at locations in the system where it can be recognized by a T cell. Where contact of a T cell with an antigen presenting cell results in the generation of only one of two required signals, the T cell does not become activated and an adaptive immune response does not occur. [004] Autoimmune diseases are noninfectious immunological diseases caused by immune responses that are directed to normal components of human cells, tissues and organs. Autoimmune diseases are often chronic diseases that gradually erode targeted tissues and organs. Common diseases now classified as autoimmune diseases due to the presence of inappropriate autoimmune responses include type I insulin-dependent diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) multiple sclerosis (MS), myasthenia gravis, celiac's disease, Sjogren's syndrome, Grave's disease, inflammatory bowel disease, Crohn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis and numerous other diseases involving an inflammatory immune response. [005] Because autoimmune diseases are often chronic, they generally require lifelong treatment and monitoring. Currently, only a few autoimmune diseases can be cured or made to disappear with treatment. Therapies for autoimmune disease are primarily directed to managing the consequences of inflammation caused by the disease. For some autoimmune diseases, administering one of a limited number of immunosuppressive medications may result in periods of remission or disappearance of active disease. Immunosuppressive agents used for adjunct therapy include substances that suppress cytokine production, down regulate or suppress self-antigen expression or mask major histocompatibility (MHC) antigens. Immunosuppressive medications include anti-inflammatory drugs (NSAIDS), cyclophosphamide, bromocryptine cyclosporine A, methotrexate, steroids such as glucocorticosteroids and cytokines or cytokine receptor antagonists. Patients are rarely able to discontinue these immunosuppressive medications as their autoimmune disease usually reappears when medication is discontinued. Autoimmune disease may become refractive to treatment when immunosuppressive medications are continued long term and may require ever increasing doses of immunosuppressive agents. [006] It has been suggested that therapeutic antibodies directed to B cells, particularly B cell depleting antibodies, may be used to treat autoimmune diseases. Therapeutic antibodies directed to antigens present on cells of the immune system may produce fewer long-term side effects than many of the immunosuppressive chemotherapies that are presently available for autoimmune diseases. However, antibody based therapies can be problematic, particularly where repeated administration is desired. Anti-lymphocyte therapies, such as antilymphocyte globulin (ALG), and monoclonal antibodies directed to B cells, such as rituximab (Rituxin^®) and alemtuzumab (CAMPATH^®), reduce circulating and tissue B cell populations in treated subjects. However, these therapies also cause severe immunosuppression, which is undesirable for the long term treatment of a chronic autoimmune disease. The principal complication of severe immunosuppressive therapy is infection. Systemic immunosuppression can also be accompanied by undesirable toxic effects and a reduction in levels of hemopoietic stem cells. In addition, patients receiving antibody therapies often develop significant levels of human anti-mouse antibodies (HAMA), human anti-chimeric antibodies (HACA) and anti-idiotypic responses, which may limit repeated treatments when a remission ends.

[007] Antibodies directed to antigens of the T cell, such as the T-cell receptor complex (TCR), have been suggested as possible therapeutics for autoimmune disease. Anti-T cell antibodies, including anti-CD3, have been used to influence immunological status in a subject by suppressing, enhancing or redirecting T cell responses to an antigen.

[008] Anti-CD3 antibodies induce immunosuppression by reducing pathogenic T cells and inducing regulatory T cells. One well characterized antibody directed to a T-cell antigen is the potent immunosuppressive antibody OKT3. OKT3 is a murine IgG2a monoclonal antibody (mAb) that targets the CD3 complex associated with human TCR.. The therapeutic potential of anti-CD3 antibodies for use as an immunosuppressive agent in humans was recognized as early as 1986, when the mouse anti-CD3 antibody OKT3 was first approved for use as an immunosuppressive agent in the United States. Since that time OKT3 has been available for use an immunosuppressive agent in organ transplantation. [009] Repeated daily administration of OKT3 results in profound immunosuppression and provides effective treatment of rejection following renal transplantation. The in vivo administration of OKT3 results in both T cell activation and suppression of immune responses. However, the use of OKT3 has been hampered by a first toxic dose reaction syndrome that is related to initial T-cell activation events and to the ensuing release of cytokines that occurs before immunosuppression of T cell responses. The reported side effects that follow the first and sometimes the second injection of this mouse monoclonal antibody include flu-like symptoms, respiratory distress, neurological symptoms and acute tubular necrosis. The activating properties of OKT3 have been shown to result from TCR cross-linking mediated by monoclonal antibody bound to T cells and to FcγR- bearing cells, which results in a massive systemic release of cytokines responsible for acute toxicity. (See U.S. Patent No. 6,491,916.)

[0010) The production of an immune response to the mouse monoclonal antibodies is also a major obstacle to the therapeutic use of OKT3. Humanized and primatized chimeric antibodies were developed to address the problems experienced with administering mouse monoclonal antibodies such as OKT3. Chimeric antibodies have reduced the occurrence of antiglobulin responses induced in patients receiving therapeutic antibodies such as OKT3. However, antiglobulin responses to primatized and humanized antibodies continue to be problematic due to their xenogenic nature, particularly for therapies where repeated treatments may be desirable or even required, as is often the case for therapies for patients with autoimmune disease.

[0011] Bluestone et al. (U.S. Patent No. 6,491,916 Bl) produced humanized OKT3 anti-CD3 antibodies that retain the immunosuppressive properties of the OKT3 mouse monoclonal, while reducing the potential for the production of human anti-mouse antibodies (HAMA) in a human anti-mouse immune response. These humanized OKT3 antibodies have Fc receptor binding regions that provide reduced T cell activating properties relative to murine OKT3. One specific humanized OKT3 antibody, which is referred to as hOKT3γl(Ala-Ala), has an IgGl Fc region that contains an alanine at positions 234 and 235. The alanines are believed to prevent or at least reduce the ability of an effector cell to bind to the Fc receptor. [0012] Humanized anti-CD3 antibodies such as the hOKT3γ 1(AIa- Ala) antibody of Bluestone may provide anti-CD3 antibody therapy that is more suitable for treatment of autoimmune disease than the mouse monoclonal OKT3 antibody, due to the potential for adverse reactions and for strong humoral responses induced by the mouse monoclonal antibody. Herold et al. (N. Engl. Med. 346:1692-169% (2002)) have reported preliminary studies of the effects of a Bluestone nonactivating humanized monoclonal antibody on loss of insulin production in patients with type 1 diabetes mellitus. Diabetic patients who received a 14-day course of anti-CD3 monoclonal antibody hOKT3γ 1(AIa- Ala) within six weeks of their diagnosis required less insulin than recently diagnosed patients not treated with antibody. [0013] Bolt et al. (U.S. Patent Nos. 5,585,097 and 6,706,265) have suggested that it is possible to address the "first dose response" by producing aglycosylated CD3 antibodies of the IgG subclass, where the antibodies retain their antigen binding specificity and immunosuppressive properties, but do not induce T cell mitogenesis in vitro. They provide an aglycosylated antibody with a binding affinity for the CD3 antigen complex having an amino acid substitution at position 297 of the Fc chain, which is asserted to produce an aglycosylated antibody that may be useful as an immunosuppressive agent.

[0014] Cole et al. have described humanized antibodies with low affinity for Fc receptors that exhibit diminished mitogenic activity in vitro.. They prepared human IgG2 variants of chimeric OKT3 and showed that they are less mitogenic to T cells in vitro than IgGl variants of the antibody. (J. Immunol. 7JP:3613-21 (1997).) They also have reported the isolation of a mouse monoclonal, M291, which competes with OKT3 for binding to T cells. (Transplantation 68:563-71 (1999).) M291 was humanized and different complementary-determining region versions were prepared. A humanized version of the antibody bearing a IgG2 M3 isotype was shown to be less mitogenic to T cells than OKT3 and yet retain immunosuppressive properties in vitro. [0015] Anti-B cell antibodies have been used as therapy for B cell lymphomas and have been suggested as therapy for autoimmune diseases, such as systemic lupus erythematosus, and multiple sclerosis. (See for example, U.S. Patent Publication Nos. 2003/0219433 Al , 2005/0271658 Al, 2006/024295 Al and 2005/0281817 Al .) However, severe B cell depletion is required to induce significant therapeutic effects with anti-B cell antibodies. Circulating levels of serum autoantibodies are not significantly reduced and significant numbers of patients develop human anti- chimeric antibody (HACA) responses. [0016] B-cell antibodies are known in the art. Antibodies 2H7 and 1F5, which are directed to the 35-kilodalton polypeptide Bp35 (CD20) expressed on the surface of B cells were used to demonstrate that the polypeptide plays a role in B-cell activation. See Clark et al., "Role of the 35 Bp cell surface polypeptide in human B- cell activation," PNAS 82:1166-1770 (1985) and Press et al., "Monoclonal Antibody 1F5 (Anti-CD20) Serotherapy of Human B Cell Lymphomas," Blood 69:584-591 (1987). A chimeric version of antibody 2H7 is described in U.S. Patent No.

5,500,362. Humanized 2H7 CD 20 binding antibodies are described in U.S. Patent Publication Nos. 2006024300 and 2006034835.

[0017] Anderson et al., (U.S. Patent No. 5,736,137) describe mouse monoclonal anti-CD 20 antibody 2B8 and the methods by which a chimeric 2B8 antibody was prepared. Hansen et al. (U.S. Patent Publication No. 2003/0219433 Al) describes the humanization of the anti-CD 20 antibody A20.

[0018] Tedder et al. (WO 2005/000901 A2) describes additional monoclonal antibodies and antigen-binding fragments that specifically bind to CD 20, including the antibodies designated HB20-3, HB20-4, HB20-25 and MB20-11 and modified variants thereof. Telling et al. (WO 2004/035607 A2) describes isolate human monoclonal antibodies which bind to and mediate the killing of B cells expressing CD 20 by a variety of mechanisms.

[00191 Brunetta et al. (U.S. Patent Publication No. 2005/0271658 Al and 2006/024295 Al) have suggested that the anti-B cell antibody rituximab, which is the chimeric anti-CD20 antibody 2B8, may find use in therapies for autoimmune disease, including SLE and rheumatoid arthritis. Rituximab is a chimeric human IgGl with mouse variable regions from a hybridoma directed to human CD20. CD20 is a specific B-cell marker present in all stages of B-cell development except the earliest and latest stages. While the function of CD20 is not known, it is expressed at high levels, it does not shed or endocytose when exposed to antibody and it does not exist in a soluble form. CD20 has proven to be a good target for therapy directed at B cell malignancies, particularly lymphomas.

[0020] Rituximab must be administered in doses that produce severe B cell depletion to induce significant therapeutic effects in patients having autoimmune disease. In most patients, four weekly intravenous doses of rituximab completely deplete normal B cells from the peripheral blood. (See Eisenberg, Arthritis Research & Therapy 5: 157-9 (2003).) However, severe B cell depletion does not significantly reduce the levels of circulating serum antibodies, including the autoantibodies present in serum of patients with autoimmune disease. The required doses induce HACA responses in a significant number of patients, which makes repeated administration of the antibody therapy problematic. Because little is known about the role of CD20, if any, in the pathogenesis of autoimmune disease, it is not possible to predict the long term effectiveness of treatment with B cell depleting anti-CD20 antibodies such as rituximab, particularly where the possibility of repeated administration is uncertain due to the present lack of information on the long term effects of HACA and anti-idiotype antibody responses. [00211 Koenig and Veri (U.S. Patent Publication No. 2004/0185045) describe monoclonal antibodies that engage only the B isoform of the CD32, which is expressed on B lymphocytes and not CD32A, and propose that these antibodies are useful for treating B cell malignanices. These monoclonal antibodies are produced from clones 3H7 and 2B6, with ATCC accession numbers PTA-4591 and PTA- 4592 respectively.

[00221 Hariharan et al. (U.S. Patent Publication No. 2003/0103971) have proposed using an immunoregulatory B cell antibody in combination with a B-cell depleting antibody in a synergistic combination therapy for treating neoplasms, in particular an anti-B7, anti-CD23 or anti-CD40L antibody, in combination with a B cell depleting antibody, such as anti-CD19, anti-CD20, anti-CD22 or anti-CD37. It is asserted that the synergistic effect of the combination of the two antibodies, both of which target B cells, may result from the different mechanisms by which antibodies elicit a therapeutic benefit.

[0023] There is a growing recognition that numerous chronic diseases involve inappropriate autoimmune responses to self antigens. Increasing numbers of patients are developing these diseases every year. Antibodies directed against T cell antigens, such as OKT3, and CAMPATH (alemtuzumab) and polyclonal anti- lymphocyte globulin (ALG) are known to reduce populations of circulating and tissue associated T cells and in some cases B cells when these cells share antigens recognized by antibodies in these preparations. Reduction of these cell populations may offer clinical benefit to patients having an autoimmune disease by counteracting against the inappropriate autoimmune responses. However, these therapeutic antibodies may also cause severe immunosuppression and HAMA, HACA and antiidiotype antibody response, both of which are undesirable in treatments for autoimmune disease. Therapies using B cell depleting antibodies are know to have the same problems. Thus, there is a need for improved, effective therapies for treating the increasing population of patients in need of treatment for the symptoms and the underlying cellular processes of autoimmune disease.

SUMMARY OF THE INVENTION [0024] The present invention provides a combination therapy for administration to a subject in need of treatment for an autoimmune disease. In one embodiment, the combination therapy comprises administration of an antibody directed to a T cell in combination with an antibody directed to an antigen present on an antigen presenting cell, or an antigen that functions to activate or modulate an immune response .

[0025] In one embodiment, the combination therapy comprises administration of an antibody directed to a T cell antigen in combination with an antibody directed to an antigen presenting cell. [0026] In a further embodiment, the combination therapy comprises administration of an antibody directed to a T cell in combination with an antibody directed to a B cell antigen. In one preferred embodiment, the antibody may be involved in eliciting B cell activation. [0027] In a further embodiment, the combination therapy may comprise administration of an antibody directed to a T cell in combination with an antibody directed to an antigen that functions as an immunomodulator, such as an that is expressed or secreted in response to the contact between a T cell and an antigen presenting cell, such as an antibody to a member of the tumor necrosis factor (TNF) family of cytokines.

[0028] In a further embodiment the combination therapy may comprise administration of an antibody directed to a T cell in combination with an antibody directed to a cytokine such as interferon (IFN). Antibodies for the combination therapy may be administered in any order, or they may be administered concurrently.

[0029] In one preferred embodiment, the invention provides a combination therapy wherein a first antibody is directed to a polypeptide chain of the T-cell receptor (TCR), including the TCR α chain, the TCR β chain, or a polypeptide chain of CD3. In an alternative embodiment, the antibody may be directed to T cell surface antigen, such as CD28, CD2 or to an intercellular adhesion molecule (ICAM), which participates in processes that lead to T cell activation following contact with an antigen presenting cell. [0030] In one embodiment, the invention provides a combination therapy in which a nonactivating anti-T cell antibody, or an antibody that provides reduced T cell activation, is administered in combination with a B cell antibody. In a further embodiment, the invention provides a combination therapy in which an anti-T cell antibody is administered in combination with an anti-B cell antibody directed to a B cell surface marker, such as a marker selected from CD19, CD20, CD22, CD23, CD32B, CD40, B7-1 (CD80), B7-2 (CD86), CD79a, CD79b, CD38, CD27, a lymphocyte function-associated antigen (LFA), such as LFA-I or LFA-3, CFA-I, or another accessory molecule involved in the T cell, B cell association that leads to T cell and B cell activation in an adaptive immune response. In a further preferred embodiment, the anti-B cell antibody may be a B cell depleting antibody, such as an antibody directed to a marker selected from CD 19, CD20, CD22, CD23, CD32B, CD40, B7-1 (CD80), B7-2 (CD86), a lymphocyte function-associated antigen (LFA), such as LFA-I or LFA-3, CFA-I, or an accessory molecule involved in the T cell, B cell association.

[0031] In one preferred embodiment, the combination therapy comprises administration of a T cell antibody, for example an anti-CD3 antibody, in combination with an antibody that recognizes an antigen present on an antigen presenting cell. In a still further preferred embodiment, the combination therapy comprises administration of an anti-CD3 antibody, such as humanized kOKT3γ 1(AIa- Ala), in combination with an antibody that recognizes a polypeptide involved in B cell activation either directly or indirectly, or an immunomodulator such as a member of TNF cytokine family, or an interferon.

[0032] In a further preferred embodiment, the combination therapy for an autoimmune disease comprises the anti-T cell antibody hOKT3γl(Ala-Ala) in combination with a B cell depleting antibody, such as an anti-CD20 antibody or an anti-CD 19 antibody. The combination therapy may achieve a clinical benefit in a patient with an autoimmune disease that is greater than the benefit achieved with a single antibody therapy. It may also achieve less severe immunosuppression than the immunosuppression needed to achieve a clinical response using a single antibody therapy. [0033] In one preferred embodiment, the present invention provides a combination therapy where an anti-T cell antibody and/or the B cell antibody may be an antibody fragment such as an F(ab)₂ fragment derived from an antibody directed to a T cell, for example an antibody fragment having specificity for a polypeptide chain of TCR, including an antibody fragment directed to the CD3 chain of the TCR. Another preferred embodiment provides anti-T cell antibody, in which the Fc fragment of the antibody may be modified such that the anti-T cell antibody is not readily bound by Fc receptors present on effector cells such as macrophages, neutrophils, eosinophils, mast cells, and NK cells. In one preferred embodiment, the T cell antibody may be a nonactivating T cell antibody, or a T cell antibody having reduced T cell activating properties. [0034] In a further embodiment, the anti-T cell antibody may be a nonmitogenic antibody or a reduced-mitogenic antibody that inhibits or prevents T cell activation when a T cell comes in contact with its specific antigen on an antigen presenting cell, in particular an antigen presenting B cell. The non-mitogenic or reduced mitogenic antibody may be useful for preventing initial "first dose side effects" seen when an anti- lymphocyte antibody is administered to patient. The non-mitogenic or reduced mitogenic antibody may be an engineered antibody having a modified Fc fragment that prevents or inhibits binding by effector cells. [0035] In a further embodiment, the present invention provides a method for treating an autoimmune disease comprising administering to subject in need of treatment for an autoimmune disease an effective amount of a nonactivating anti-T cell antibody, or an antibody that provides reduced T cell activation, in combination with an anti-B cell antibody, where each antibody is administered in an amount effective to achieve a clinical remission for the autoimmune disease. In a preferred embodiment, the clinical remission is longer than treatment with either antibody administered alone.

DETAILED DESCRIPTION OF THE INVENTION

[0036] In one embodiment, the present invention provides a combination therapy for treating autoimmune disease that comprises administering to a subject a first antibody targeted to a T cell antigen in combination with a second antibody directed to an antigen present on an presenting cell, or otherwise antigen that functions to activate or modulate an immune response .. In one embodiment, the T cell antibody is targeted to a polypeptide chain present on a naive T cell that may be involved in the process that leads to T cell activation. In a further embodiment, the T cell antibody may be directed to a polypeptide chain of the TCR complex, for example the TCR α and β chains or the CD3 proteins or the ζ chain of the T cell receptor complex.

[00371 Preferred antibodies may be directed to antigens present on antigen presenting cells that interact with T cell surface molecules when a T cell recognizes and attempts to interact with an antigen presenting cell in the processes which result in T cell activation. Such antigens may include the B7 antigen which is expressed on monocytes and macrophages, or CD27, which is expressed on T cells. These antigens may also include CD 19 and CD20, which are highly specific for B cells. The combination therapy is administered to provide improved clinical benefits to a subject in need of treatment for an autoimmune disease by reducing the levels of antibody-producing and antigen-presenting B cells while rebalancing the T cell response toward a regulated state of T cell activation. [0038] In one embodiment, the present invention provides a combination therapy for treating autoimmune disease that comprises administering to a subject an antibody to a T cell antigen in combination with an antibody to tumor necrosis factor (TNF). In a preferred embodiment, the combination therapy provides an antibody directed to TNF wherein the antibody is selected form anti-TNF-α, TNF-β, or soluble TNFR. In additional preferred embodiments, the combination is administered to a subject in need of treatment for inflammatory bowel disease (IBD), systemic lupus erythematosus (SLD) or rheumatoid arthritis (RA). (0039] In another embodiment, the present invention provides a combination therapy for treating autoimmune disease that comprises administering to a subject having active autoimmune disease an antibody to a T cell antigen in combination with an antibody to an interferon. As is understood by those of skill in the art, the cytokines INF-α, -β and -γ are involved in the regulation of proteins that work together in antigen processing and presentation. These cytokines stimulate cells to increase their expression of HLA class I heavy chains. In one preferred embodiment, the combination therapy comprises administering to a subject having active autoimmune disease an antibody to a T cell antigen in combination with an antibody to INF-β. In a further preferred embodiment, the combination therapy comprises administering to a subject an antibody targeted to a T cell antigen in combination with an antibody selected from INF-β antibodies Avonex^®, Betaseron^® and Rebif^®. In a further embodiment, the combination therapy comprises administering to a subject an antibody targeted to a T cell antigen in combination with an antibody targeted to INF-β for treatment of a subject having multiple sclerosis.

(0040 J In one aspect, embodiments of the present invention provide a combination therapy in which treated subjects achieve and maintain clinical remissions for longer periods than remissions achieved by subjects treated with a single antibody therapy. For example, where a single antibody therapy achieves a remission of symptoms of an autoimmune disease for three months, the combination therapy may provide a complete remission of symptoms of up to six months, up to 12 months and in some cases up to one to two years or longer. It is contemplated that for certain autoimmune diseases it may be possible to provide a complete remission that does not relapse, particularly where therapy begins shortly after the autoimmune disease is diagnosed.

[0041] The clinical remission achieved with the combination therapy may be a complete remission, or it may be a partial remission in which significant reductions in disease symptoms are maintained for an extended period. For example, a subject receiving the combination therapy of the present invention may have reduced autoimmune responses as determined by reduced levels of detectable autoantibodies in body fluids and tissues, for example in cerebrospinal fluid (CSF), serum, urine or in body tissues. A subject receiving the combination therapy also may have reduced T cell responses to autoantigens as detected by in vitro by proliferation or cytokine production assays using peripherial blood mononuclear cells (PBMCs) or purified T cells when compared with subjects treated with a T cell specific or a B cell specific antibody alone.

[0042] In another aspect, embodiments of the present invention provide a combination therapy in which the administration of a T cell antibody and a B cell antibody in combination, may induce fewer or less frequent HAMA, HACA and anti-idiotype responses to the therapeutic antibodies when compared to subjects treated with either a T cell specific or B cell specific antibody administered singlely. In the event patients treated with the combination therapy do develop a HAMA, HACA and/or anti-idiotype response, the levels of antibodies produced will be less than the levels of antibodies produced in a subject treated with only a single T cell or B cell therapeutic antibody.

[0043] In one aspect, embodiments of the present invention provide a combination therapy where administration of a first therapeutic T cell antibody and a second B cell antibody provides a synergistic therapeutic effect by interrupting, modulating or otherwise adjusting the signaling events that lead to T cell activation and the subsequent proliferation and differentiation of the T cell into effector T cells. Likewise, the combination therapy may provide a synergistic therapeutic benefit by interrupting, modulating or otherwise adjusting the signaling events that result in differentiation of an antigen presenting B cell into an antibody producing cell. The immunomodulation provided by the combination therapy also may provide a reduction of serum antibodies directed to autoantigens in serum of the treated subject.

[0044] DEFINITIONS

[0045] The following definitions are provided to further describe features of embodiments of the invention as discussed herein.

[0046] As used herein, the term "antibody" is used in its broadest sense to include antigen binding molecules produced by a B-cell in response to the antigen, and fragments thereof. An antibody may be a monoclonal antibody, an antibody present in or isolated from a polyclonal serum human antibody, or an antibody produced using the techniques of molecular biology known to those of skill in the art relating to the art of antibody engineering, including chimeric antibodies, bispecifϊc antibodies, primatized or humanized antibodies, and fragments thereof.

Embodiments of the present invention generally relate to therapeutic antibodies, which are intended to be biological drugs or pharmaceuticals. [0047] The term "antibody fragment" as used herein means a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Antibody fragments include Fab, Fab', F(ab)2 and Fv fragments, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments may be prepared by methods known to those of skill in the art. For example F(ab)2 fragments may be prepared by treating antibodies with pepsin. The resulting F(ab)2 fragment may be treated to reduce the disulfide bridges of the antibody fragment to produce F(ab') fragments.

[0048] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" as used herein refers to the killing of antibody coated target cells by NK cells having the FcγR receptors (CD 16, CD 32 or CD64) that recognizes the Fc region of the bound antibody. [0049] As used herein, the term "antigen presenting cell" refers to cells that express either MHC class I and/or MHC class II molecules, and thus may display complexes of a MHC molecule and peptide antigen on their surfaces. Three kinds of antigen presenting cells are dendritic cells, the macrophage and the B cell. All three types are found in secondary lymphoid tissues, but at different locations. [0050] An "autoantibody" as used herein means an antibody produced by a subject that binds to a self-antigen also produced by the subject. A "self antigen" is a normal constituent of the body to which the immune system would respond were it not for the mechanisms of tolerance that destroy or inactivate self-reactive B and T cells.

[0051] As used herein, "autoimmune disease" refers to a disease, in which the disease pathology is caused by an immune response to normal components of tissue. Examples of autoimmune diseases or disorders include, but are not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, Sjogren's syndrome, Guillain-Barre syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, Goodpasture's syndrome, pemphigus, dermatomyositis, Wegener's granulomatosis, termporal arterites,

Takayasu's arteritis, small vessel vasculitis, idiopathic thrombocytopenia purpura, autoimmune thyroiditis, glomerulonephritis, primary biliary cirrhosis, membranous nephropathy, autoimmune hepatitis, celiac disease, Addison's disease, polymyositis/dermatomyositis, monoclonal gammopathy, Factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, anti-phospholipid antibody syndrome, Hashimoto's thyroiditis asthma and allergic rhinitis.

[0052] A "B-cell" is a lymphocyte that is dedicated to making immunoglobulin and antibodies. [0053] As used herein, the term "B cell depleting antibody" means an antibody or antibody fragment that upon administration to a subject results in demonstrable B cell depletion. Typically, a B cell depleting antibody will bind to a B cell antigen or B cell marker expressed on the surface of a B cell. Preferably administration of the B cell depleting antibody will result in depletion of B cell numbers in a patient to a level of 50% or less than the numbers of B cells present at the time of administration. [0054] As used herein, the term "B cell surface marker" is an antigen expressed on the surface of a B cell that can be targeted with an antibody. Known B cell surface markers include CDlO, CD19, CD20, CD21, CD22, CD23, CD24, CD32B, CD37, CD40, CD53, CD72, CD 73, CD74, CDw75, CDw76, CD77, CD80 (B7.1) CD 86 (B7.2) CDw84, CD85 and CD86. CDlO, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers (see for example, The Leukocyte Antigen Facts Book, 2nd Edition. 1997, Barclay et al., ends, Academic Press, Harcourt Brace & Co., New York). Other B cell surface markers include RP 105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, NAG 14, SLGC 16270, FcRHl , IRTA2, ATWD578, FcRH3, IRTAl, FcRH6, BCMA, and 239287. Antibodies directed to LFAs, including LFA-I and LFA-3, CFAs and other B cell accessory proteins may also be used. The B cell surface marker of interest is preferentially expressed on B cells compared to other non-B cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells.

[0055] As used herein, the term "bispecific antibodies" refers to antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a T cell or a B cell surface marker. Alternatively, the bispecific antibody may bind two different T cell or B cell surface markers, or it may bind an epitope on a T cell and an epitope on a B cell. Techniques for producing bispecific antibodies from antibody fragments are known to those of skill in the art. [0056] As used herein, the term "chimeric antibody" refers to a genetically engineered antibody produced by fusing the variable regions of a rodent antibody, generally a mouse monoclonal antibody with constant regions of a human antibody. [0057] As used herein, the term "epitope" means the portion of an antigenic molecule that is bound by an antibody or that gives rise to the MHC-binding peptide that is recognized by a T-cell receptor. An epitope is also referred to as an antigenic determinant. [0058] As used herein, the term "effective amount" refers to an amount of an antibody which is effective for inducing a therapeutic state in a subject when used according one or more embodiments of the combination therapy described herein. [0059] As used herein, the terms "Fc receptor" or "FcR" mean the receptor that binds to the Fc region of an antibody.

[0060] As used herein, the term "immunoregulatory antibody" or "immunomodulatory antibody" refers to an antibody that elicits an effect on the immune system by exerting an effect on T cell immunity or B cell immunity, for example by inhibiting the TCR signaling required for T cell activation or by inhibiting the co-stimulatory signal that is also required for T cell activation. [0061] As used herein, the term "humanized antibody" refers to a genetically engineered antibody in which only the complementarity determining regions from rodent antibody V-regions are combined with framework regions from human V- regions. Humanized antibodies are more human like than chimeric antibodies and may be less antigenic when administered to a human patient. Humanized antibodies may also be referred to as CDR grafted or reshaped antibodies. [0062] As used herein, the term "immunosuppressive agent" means an agent that inhibits an immune response in a subject, including an adaptive immune response and/or a natural immune response. [0063] As used herein, the term "immunomodulating agent" means an agent that is capable of modifying, regulating or adjusting one or more functions of the immune system.

[0064] As used herein, the term "lymphocyte function associated antigen" (LFA) refers to one or more of the antigens present on B cells that mediates the adhesion of lymphocytes, particularly antigen presenting cells such as B.cells to T cells. LFA-I is one of the leukocyte integrins that mediates the adhesion of lymphocytes to endothelial cells and antigen presenting cells. LFA-3 is a cell adhesion molecule of the immunoglobulin superfamily that is expressed on antigen presenting cells. [0065] As used herein, the term "non-mitogenic T cell antibody" means an antibody that is engineered by altering the Fc receptor of the antibody such that it does not trigger the initial activation events and ensuing release of cytokines that are seen when a T cell is activated. A "reduced mitogenic T cell antibody" is an antibody specific for a T cell antigen that reduces the initial activation events and release of cytokines that occur when a T cell is activated.

[0066] As used herein, the term "T cell" refers to lymphocytes that develop in the thymus and are responsible for cell-mediated immunity. Their cell surface antigen receptor is called the T-cell receptor.

[0067] As used herein, the term "T-cell receptor" refers to the highly variable antigen receptor of T lymphocytes. On most T cells it is composed of a variable α chain and β chain and is known as the α-β T-cell receptor. On a minority of T cells, the variable chains are γ and δ chains, and the receptor is known as the γ-δ T-cell receptor. Both types of receptors are present at the cell surface in association with the complex of invariant CD3 chains and ζ chains, which provide a signaling function.

[0068] The term "T-cell receptor complex" refers to the complex of T-cell receptor α and β and the invariant CD3 and ζ chains that make up a functional antigen receptor on the T cell surface.

[0069] As used herein, the term "tolerance" refers to a state in which a T cell no longer responds to antigen. Thus, tolerance is achieved when the immune system of a subject does not or cannot respond to an antigen, particularly a self antigen. "Partial tolerance" refers to a reduced response to antigen. [0070] Antibodies of the Combination Therapy

[0071] The antibodies to be used in the embodiments of combination therapy may be prepared using techniques available in the art for generating antibodies, examples of which are described in more detail below. The antibodies may be directed to lymphocyte antigens, in particular a first antigen present on a T cell and a second antigen present on a B cell. In one preferred embodiment, the first antibody targets a T cell and prevents or inhibits the T cell from interacting with an antigen MHC complex present on an antigen presenting cell, particularly an antigen presenting a B cell. By blocking the association of the T cell with the antigen-MHC complex of the antigen presenting cell, the first antibody blocks or alters T cell signal transduction events that lead to T cell activation.

[0072] The second antibody of the combination therapy is targeted to a B cell antigen. In a preferred embodiment, the antibody is a B cell depleting antibody. The second antibody may be targeted to any cell surface protein of a B cell that participates in or acts as an accessory protein for the interaction between a T cell and an antigen presenting B cell that leads to T cell activation and differentiation and to B cell activation and antibody production. [0073] Antibody Preparation: Methods for preparing antibodies to cell surface molecules, particularly cell surface molecules present on T cells and B cells are described in numerous publications and are known to those of skill in art. For example, fragments of transmembrane molecules, such as chains of the molecules that make up the T-cell receptor complex present on T-cells, or transmembrane polypeptides present on antigen presenting cells such as B cells can be used as immunogens for generating antibodies. Alternatively, T cells and B cells expressing the transmembrane molecules can be used. Such fragments or cells can be derived from a natural source, such as an appropriate cell line, or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule of interest.

[0074] Polyclonal antibodies are generally prepared by injecting animals multiple times with the antigen of interest, together with an adjuvant either subcutaneously or intraperitoneally using methods known to those of skill in the art of immunology. Approximately one month after the initial injection animals may be boosted with antigen at an appropriate dose. Seven to fourteen days later the animals are bled and the serum is assayed for antibody titer. Animals may be boosted with additional doses of antigen until their antibody titer plateaus. It may helpful or necessary to conjugate the antigen to a protein that is immunogenic in the species to be immunized, for example, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using an appropriate conjugating agent.

[0075] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature 256 (1975) p. 495, or by recombinant DNA methods, that are well known to those of skill in the art. In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies, which will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro.

[0076J Lymphocytes are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (See for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59- 103 (Academic Press, 1986)). The hybridoma cells are then seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0077] Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). [0078] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). If needed, the binding affinity of the monoclonal antibody can be determined by the Scatchard analysis. See for example, Munson et al., Anal. Biochem., 107:220 (1980). [0079] Humanized Antibodies: A humanized antibody is a genetically engineered chimeric antibody which is produced by replacing the CDR loops in a human antibody with the corresponding CDR sequences of a mouse antibody with a desired specificity. The non-human amino acid residues, which may be referred to as "import" residues, are typically taken from an "import" variable domain of the antibody having a desired specificity. Both light and heavy human variable domains are used for making a humanized antibody to reduce antigenicity. Methods for humanizing antibodies are generally known in the art. For example antibodies can be humanized according the methods described by Winter et al. (Jones et al., Nature 321:522-525 (1986): Reichmann et al, Nature 332:323-327 (1988); and Verboeyen et al., Science 239:1534-1536 (1988) Queen et al, U.S. Patent No. 5,530,101. Also see Oliphant et al., Nature Medicine 11:522-30 (2005).

[0080] The humanized antibody should be engineered to retain or improve the affinity of the antibody to be humanized and to retain other favorable biological properties. In some embodiments this may involve making specific changes to framework or other sequences to produce a desired effect. For example, it may be desirable to modify the framework sequence for the Fc region of the human sequence to produce an aglycosylated antibody and/or a non mitogenic or reduced mitogenic antibody that lacks effector function. Methods for manipulating the DNA sequences used to produce the humanized antibody gene such. as site directed mutagenesis are known to those of skill in the art. See also the methods described in Oliphant et al., Nature Medicine 11 :522-30 (2005), the disclosure of which, including the cited references are incorporated herein in its entirety. [0081] The choice of human light and heavy chain variable domains to be used in making the humanized antibodies is very important to reduce antigenicity. One method that may be used is the "best-fit" method, in which the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. MoI. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light- or heavy-chain variable regions. The same framework may be used for different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 757:2623(1993)). Sequences of framework and CDR regions may be identified in available computer protein sequence databases and in Kabat et al., Sequences of Proteins of Immunological Interest, (1991) U.S. National Institutes of Health, Bethesda, Maryland. Hypervariable region sequences and possibly some framework sequences from the T cell antibody and/or the B cell antibody to be humanized are substituted for corresponding sequences of the human antibody. [0082] To achieve a antibody that retains a high affinity for antigen and other favorable biological properties, humanized antibodies may be prepared based on an analysis of the parental sequences and various conceptual humanized antibodies using three-dimensional models of the parental and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. [0083] Human antibodies can be derived from libraries displayed on phage or yeast as described in Oliphant et al., Nature Medicine 77:522-30 (2005) and Shusta et al., Nature Biotechnology 75:784-59 (2002).

[0084] Administration of the Combination Therapy: The present invention provides a method of treating an autoimmune disease in a subject eligible for treatment, comprising administering an effective amount of a first antibody that binds to a T cell surface marker and a second antibody that binds to a B cell surface marker to the subject in need of an effective treatment for an autoimmune disease. As one of skill in the art will understand the exact mode and method of administration of the present combination therapy may vary depending on the characteristics of the particular therapeutic T cell antibody and B cell antibody used for a particular patient and for treatment of a particular autoimmune disease. [0085] In one embodiment, an initial antibody exposure of about 0.05 (50 mg) to 4 grams, preferably about 0.1 to 3.5 grams, and more preferably about 0.5 to 2.5 grams of antibody is administered to the subject followed by a second antibody exposure of about 0.05 to 4 grams, preferably about 0.1 to 3.5 grams, and more preferably about 0.5 to 2.5 grams of antibody. The first and second antibody may be administered in any order, or they may be administered concurrently. In addition the amount of each antibody needed for an effective treatment may be the same or may be different depending upon the respective affinities and other biological characteristics of the antibody to be administered. [0086] In one embodiment, it may be advantageous to administer the first antibody on day 1 to 4 and to administer the second antibody on days 5 to 10. For a particular antibody it may be desirable to administer one or both of the antibodies as an initial low dose on day one of treatment and to gradually increase the dose on following days such that the maximum dose of antibody is administered on day 10. [0087] In a further embodiment, it may be advantageous to provide a second exposure to the T cell antibody or to the B cell antibody from about 16 to 54 weeks, preferably from about 20 to 30 weeks and more preferably from about 46 to 54 weeks from the initial exposure. Each antibody treatment is provided to the subject as a single dose or as two to ten separate doses of antibody. (0088 ] Any one or more of the antibody exposures herein may be provided to the subject as a single dose of a T cell antibody, or as two or three separate doses of the T cell antibody (i.e., constituting a first and second dose or a first, second, and third dose). The particular number of doses (whether one, two, or three) employed for each antibody exposure is dependent, for example, on the type of autoimmune disease treated, the type of antibody employed, and the method and frequency of administration. Where separate doses are administered, the second dose or third dose is preferably administered from about 1 to 20 days, more preferably from about 6 to 16 days, and most preferably from about 14 to 16 days from the time the previous dose was administered. The separate doses may be administered within a total period of between about 1 day and 4 weeks, more preferably between about 1 and 20 days (e.g., within a period of 6-18 days).

[0089] In one embodiment, the subject is provided at least about three exposures of the antibody, for example, from about 3 to 60 exposures, and more preferably about 3 to 40 exposures, most preferably, about 3 to 20 exposures. Preferably, such exposures are administered at intervals each of about 24 weeks. In one embodiment, each antibody exposure is provided as a single dose of the antibody. In an alternative embodiment, each antibody exposure is provided as separate doses of the antibody. However, not every antibody exposure need be provided as a single dose or as separate doses.

[0090] One preferred embodiment, of the combination therapy the T cell antibody HuOKT3γl (AIaAIa) is administered with an anti-CD 20 antibody such as rituximab, humanized 2H7, or HUMAX-CD20 ™ antibody (Genmab). [0091] In one embodiment, the subject has never been previously treated with drug(s), such as immunosuppressive agent(s), to treat his autoimmune disease and/or has never been previously treated with an antibody to a T cell surface marker or a B- cell surface marker. In another embodiment, the subject has been previously treated with drug(s) to treat the autoimmune disease and/or has been previously treated with a single antibody therapy, which may have been an anti-T cell therapeutic antibody or an anti-B cell therapeutic antibody.

[0092] The antibody is administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody may suitably be administered by pulse infusion, e.g., with increasing doses of the antibody. Preferably, the dosing is given intravenously or subcutaneously, and more preferably by intravenous infusion(s). Each exposure may be provided using the same or a different administration means. In one embodiment, each exposure is by intravenous administration. In another embodiment, each exposure is given by subcutaneous administration. In yet another embodiment, the exposures are given by both intravenous and subcutaneous administration. [00931 In one embodiment, the therapeutic antibodies are administered as a slow intravenous infusion which may commence at a rate of about 50 mg/hour. This may be escalated, e.g., at a rate of about 50 mg/hour increments every about 30 minutes to a maximum of about 500 mg/hour. However, if the subject is experiencing an infusion-related reaction, the infusion rate is preferably reduced, e.g., to half the current rate, e.g., from 100 mg/hour to 50 mg/hour. Preferably, the infusion of such dose of an antibody (e.g., an about 1000-mg total dose) is completed at about 255 minutes (4 hours 15 min.). The treated subjects may receive a prophylactic treatment of acetaminophen/paracetamol (e.g., about 1 g) and diphenhydramine HCl (e.g., about 50 mg or equivalent dose of similar agent) by mouth about 30 to 60 minutes prior to the start of an infusion.

[0094] If more than one infusion (dose) of an antibody is given to achieve the total exposure, the second or subsequent antibody infusions in this infusion embodiment are preferably commenced at a higher rate than the initial infusion, e.g., at about 100 mg/hour. This rate may be escalated, e.g., at a rate of about 100 mg/hour increments every about 30 minutes to a maximum of about 400 mg/hour. Subjects who experience an infusion-related reaction preferably have the infusion rate reduced to half that rate, e.g., from 100 mg/hour to 50 mg/hour. Preferably, the infusion of such second or subsequent dose of antibody (e.g., an about 1000-mg total dose) is completed by about 195 minutes (3 hours 15 minutes).

[0095] The combination therapy may be administered to a subject by providing an initial dose of the first antibody that is less than the amount of antibody needed to achieve a clinical response in therapy for an autoimmune disease when administered as a single antibody therapy. A dose of a therapeutic anti-T cell antibody that is less than the dose needed to achieve depletion of T cells that are able to recognize and respond to autoantigens in a therapy providing a single antibody may be sufficient to provide a desired clinical response. Methods for determining the dosage of a therapeutic antibody needed to achieve a clinical response are known to those of skill in the art. For example, a clinical response in the subject may be measured as time to disease progression, reduction of clinical symptoms, reduction in levels of laboratory markers, reduction in the need for retreatment or by any other clinical means recognized as a useful indicator of improvement in status of the autoimmune disease.

[0096] The second antibody of the combination therapy may also be administered to a subject in need of treatment as an initial dose that is significantly less than an effective dose for achieving a clinical response when the antibody is administered alone. For example, doses of a depleting anti-B cell antibody that achieve less than 100% B-cell depletion, less than 50% B cell depletion, less that 30% depletion or even no B cell depletion may be administered together with a first anti-T cell antibody to achieve a clinical response that provides suppression of an immune response to an autoantigen equal to, or better than the clinical response achieved by administering an amount of a B cell depleting antibody that provides 100% depletion of B cells in the subject when administered alone. [0097] In some instances, clinical response may be a response that neither the first nor the second antibody achieves when administered alone. In other instances, the clinical response may be equivalent to that achieved by administration of a single antibody therapy, where the combination therapy provides less immunosuppression of a treated subject's immune system than a single antibody therapy. In one preferred embodiment, the synergistic response provided by the combination therapy reduces or eliminates a subject's response to an autoantigen while providing lower levels of immunosuppression. General immunosuppression is a significant problem for presently available antibody therapies.

[0098] Pharmaceutical Formulations: Therapeutic formulations of the antibodies used in embodiments of the present invention are prepared for storage, shipment and administration by mixing an antagonist having a desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers recognized in the pharmaceutical art in the form of lyophilized formulations or aqueous solutions. Pharmaceutical compositions suitable for injection include sterile aqueous solutions where the active agents are water soluble, or dispersions or sterile powders for extemporaneous preparation of sterile injectable solutions. Compositions for use in the combination therapy may be prepared by incorporating the active antagonist or antibody in the required amount with appropriate carriers, for example water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride may be included in the composition. [0099] In one embodiment, the present invention provides an article of manufacture containing antibodies to be used for the combined therapy for treatment of autoimmune disease. The article of manufacture comprises a container comprising a first antibody that binds an antigen present on a T cell and a pharmaceutically acceptable carrier or diluent within the container. The article of manufacture further comprises a second container comprising a second antibody directed to a B cell surface marker and a pharmaceutically acceptable carrier or diluent and instructions for administering the composition to a subject in need of treatment for autoimmune disease. Where the first and second antibodies are determined to be complementary and to not adversely affect each other, the first and the second antibody may be provided in a single container containing the first and second antibody in appropriate concentrations for administration together with a package insert and instructions for administration.

[00100] Containers of the article of manufacture may be of any suitable material that will not react with or otherwise affect the preparation. The article of manufacture may further comprise a second or a third container comprising a pharmaceutically-acceptable diluent buffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. The article of manufacture may also include other material that may be desired from a commercial and user standpoint including other buffers, diluents, filters, needles and syringes.

EXAMPLE I

[00101] A subject suffering from rheumatoid arthritis and experiencing an inadequate response to conventional chemotherapy is treated with the combination therapy comprised of a therapeutic antibody directed to a T cell administered with a therapeutic antibody directed to a B cell. A physical examination of the patient is conducted prior to treatment to assess the number of tender, swollen joints and at 30 and 90 days after administration of the therapy. The patient is asked to assess her level of pain on an analog scale at the time of treatment and on days 30 and 90 following treatment. The patient is administered an initial dose of 500 mg of a genetically engineered murine/human monoclonal antibody against the CD 20 antigen as an IV infusion on day 1. On day 3, an initial dose of 0.005 mg/day of humanized antibody huOKT3γ 1(AIa- Ala) is administered as an IV infusion. On subsequent days the dose of huOKT3γ 1(AIa- Ala) is increased to a maximum does of 4.0mg/day after six days. Treatment at 4.0 mg/day is continued for an additional 10 days. On day 15, a second dose of the CD20 antibody is administered. [00102] The subject is examined 30 days after the second dose of B cell depleting antibody is administered and found to fewer tender joints and less swelling in her affected joints. Assays for anti-HAMA, anti-HACA and anti-idiotype antibodies are carried out at two weeks, four weeks and six weeks after last day of administration of the OKT3 antibody and no evidence of an anti-HAMA, anti-HACA or antiidiotype antibody response is seen. The subject also indicates that the overall pain of her affected joints is significantly reduced. At 90 day, after the second dose of B cell depleting antibody the patient continues to have fewer tender and swollen joints and her pain continues to be reduced.

EXAMPLE II [00103] A patient receiving chemotherapy, such as corticosteroid and/or methotrexate therapy for management of systemic lupus erythematosus, experiences a lupus flare and becomes refractive to the chemotherapy. As a result of the flare, the patient experiences additional or more severe physical problems associated with lupus, such as increased skin problems and rashes, development or worsening of arthritis, and/or indications of increased organ involvement, such as inflammation of the lungs or heart. The patient may also develop kidney problems, such as those that are often associated with systemic lupus. The patient is determined to be a candidate for combination antibody therapy to stabilize and induce remission of the autoimmune disease following assessment of the status of his condition. [00104] An initial dose of 500 mg of a humanized antibody directed against a B cell antigen, such as CD 20, is administered as an IV infusion on day 1. On day 3, an initial dose of 0.005 mg of a humanized T-cell antibody such as huOKT3γl (Ala- Ala) is administered as an IV infusion. On subsequent days the dose of huOKT3γ 1(AIa- Ala) is increased incrementally to a maximum dose of 4.0mg/day after six days. Treatment is continued at 4.0 mg/day for an additional 10 days. On day 10, a second dose of the CD20 antibody is administered.

[00105] The patient is monitored by determining complete blood cell counts, levels of serum creatinine and albumin and levels of anti-double stranded DNA antibodies. Indications of involvement of heart, lungs and kidneys are also monitored. After three months a normalization of levels of auto-antibodies are observed and a long- lasting remission of up to three years is obtained. [00106] While the combination therapy and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the relevant art that variations may applied to the combination and methods and to the steps and sequence of the steps of the methods described herein without departing from the concept, spirit and scope of the invention. Specifically, it will be apparent that agents that are chemically, physiologically and structurally related may be substituted for the specific agents described herein to achieve similar results. All similar substitutes and modifications that are apparent to those of skill in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. The disclosures of all citations in the present specification are expressly incorporated by reference.

Claims

We claim: 1. A therapeutic combination for achieving remission in a subject having an active autoimmune disease comprising; a) a first antibody that binds a T-cell antigen and b) a second antibody that binds an antigen that functions to activate or modulate an immune response, wherein the first antibody and the second antibody may be administered separately in any order or together to provide a therapeutically effective treatment for the autoimmune disease.

2. The therapeutic combination of claim 1, wherein the first antibody binds a T- cell receptor (TCR) complex.

3. The therapeutic combination of claim 1, wherein the first antibody binds an α chain of a T-cell receptor.

4. The therapeutic combination of claim 1, wherein the first antibody targets a β chain of a T-cell receptor.

5. The therapeutic combination of claim 1, wherein the first antibody binds CD3.

6. The therapeutic combination of claim 1, wherein the first antibody is a non- mitogenic anti-CD3 antibody.

7. The therapeutic combination of claim 1, wherein the first antibody is a reduced-mitogenic anti-CD3 antibody.

8. The therapeutic combination of claim 1, wherein the first antibody is a non- activating anti-CD3 antibody.

9. The therapeutic combination of claim 1, wherein the first antibody is hOKT3γl(Ala-Ala).

10. The therapeutic combination of claim 1, wherein the first antibody is administered in an amount of from about 0.1 to about 100 mg per day.

11. The therapeutic combination of claim 1 , wherein the first antibody is administered in an amount of from about 1 to about 10 mg per day.

12. The therapeutic combination of claim 1, wherein the first antibody is administered in an amount from about 2 to about 4 mg per day.

13. The therapeutic combination of claim 1, wherein the first antibody is administered in an amount of about 4 mg per day.

14. The therapeutic combination of claim 1, wherein the first antibody is administered in an amount of about 2 mg per day.

15. The therapeutic combination of claim 1, wherein the second antibody binds an antigen present on an antigen presenting cell.

16. The therapeutic combination of claim 15, wherein the antigen-presenting cell is selected from the groups consisting of a B-cell, a macrophage, and a dendritic cell.

17. The therapeutic combination of claim 1, wherein the second antibody is directed to an antigen present on a B-cell.

18. The therapeutic combination of claim 1, wherein the second antibody is a B- cell depleting antibody.

19. The therapeutic combination of claim 16, wherein the second antibody targets a cell surface marker selected from the group consisting of CDlO, CD 19, CD20, CD21, CD22, CD23, CD24, CD32B, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86, B-7.1 (CD80) and B-7.2 (CD86).

20. The therapeutic combination of claim 17, wherein the second antibody targets the cell surface marker CD20.

21. The therapeutic combination of claim 17, wherein the second antibody targets the cell surface marker CD32B.

22. The therapeutic combination of claim 17, wherein the second antibody targets the cell surface marker CD 19.

23. The therapeutic combination of claim 17, wherein the second antibody targets the cell surface marker B-7.1(CD80).

24. The therapeutic combination of claim 17, wherein the second antibody targets the cell surface marker B-7.2(CD86).

25. The therapeutic combination of claim 1, wherein the second antibody binds a TNF cytokine.

26. The therapeutic combination of claim 25, wherein the TNF cytokine is selected from the group consisting of TNF-α, TNF-β and a soluble TNFR.

27. The therapeutic combination of claim 1, wherein the second antibody binds interferon.

28. The therapeutic combination of claim 27, wherein the second antibody binds interferon-β.

29. The therapeutic combination of claim 1, wherein the second antibody is administered in an amount from about 0.1 to 1000 mg per day.

30. The therapeutic combination of claim 1, wherein the second antibody is administered in an amount from about 10 to 500 mg per day.

31. The therapeutic combination of claim 1 , wherein the second antibody is administered in an amount of about 250 mg per day.

32. The therapeutic combination of claim 1, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease (IBD), Sjogren's syndrome, Guillain-Barre syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, Goodpasture's syndrome, pemphigus, dermatomyositis, Wegener's granulomatosis, termporal arterites, Takayasu's arteritis, small vessel vasculitis, idiopathic thrombocytopenia purpura, autoimmune thyroiditis, glomerulonephritis, primary biliary cirrhosis, membranous nephropathy, autoimmune hepatitis, celiac disease, Addison's disease, polymyositis/dermatomyositis, monoclonal gammopathy, Factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, anti-phospholipid antibody syndrome, Hashimoto's thyroiditis asthma and allergic rhinitis.

33. The therapeutic combination of claim 26, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and inflammatory bowel disease (IBD.

34. The therapeutic combination of claim 28, wherein the autoimmune disease is multiple sclerosis.

35. A method for treating a subject having an autoimmune disease comprising administering to the subject a therapeutically effective amount of a first non- activating anti-T cell antibody in combination with a second antibody that functions to activate or modulate an immune response, wherein each antibody is administered in an amount effective to achieve a remission of the autoimmune disease.

36. The method of claim 29, wherein the second antibody binds an antigen on an antigen presenting cell.

37. The method of claim 30, wherein the second antibody is a B cell antigen.

38. The method of claim 35, wherein the second antibody binds a TNF cytokine.

39. The method of claim 38, wherein the TNF cytokine is selected from the group consisting of TNF-α, TNF-β and a soluble TNFR.

40. The method of claim 35, wherein the second antibody binds an interferon.

41. The method of claim 40, wherein the interferon is interferon-β.

42. The method of claim 35, wherein the combination therapy provides remission in the subject for a time longer than a remission achieved in a patient administered a single antibody therapy.

43. A method for treating an autoimmune disease comprising administering to a subject having active autoimmune disease a nonactivating anti-human CD3 antibody in combination with an anti-CD20 in amounts that achieve clinical remission for a period of time longer than treatment with either antibody alone.

44. A method for treating an autoimmune disease comprising administering to a subject having active autoimmune disease a nonactivating anti-human CD3 antibody in combination with an anti-CD32B in amounts that achieve clinical remission for a period of time longer than treatment with either antibody alone.

45. A method for treating a subject having an autoimmune disease comprising administering to the subject an amount hOKT3γl(Ala-Ala) sufficient to provide clinical remission of the autoimmune disease when administered in combination with an antt-CD20 antibody.

46. The method of claim 28 wherein the autoimmune disease is selected from the group selected from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, Sjogren's syndrome, Guillain-Barre syndrome, myasthenia gravis, large vessel vasculitis, medium vessel vasculitis, polyarteritis nodosa, pemphigus, scleroderma, Goodpasture's syndrome, pemphigus, dermatomyositis, Wegener's granulomatosis, temporal arterites, Takayasu's arteritis, small vessel vasculitis, idiopathic thrombocytopenia purpura, autoimmune thyroiditis, glomerulonephritis, primary biliary cirrhosis, membranous nephropathy, autoimmune hepatitis, celiac disease, Addison's disease, polymyositis/dermatomyositis, monoclonal gammopathy, Factor VIII deficiency, cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, anti-phospholipid antibody syndrome, Hashimoto's thyroiditis asthma and allergic rhinitis.

47. A method of suppressing autoimmune response-triggered inflammation in a subject having an autoimmune disease comprising the step of administering to a subject in need of treatment for an autoimmune disease an anti-CD3 antibody in combination with a anti-CD20 antibody where each antibody is administered in an amount effective to suppress immune response triggered inflammation for at least 12 months.

48. A method of suppressing autoimmune response-triggered inflammation in a subject having an autoimmune disease comprising the step of administering to a subject in need of treatment for an autoimmune disease an anti-CD3 antibody in combination with a anti-CD32B antibody wherein each antibody is administered in an amount effective to suppress immune response triggered inflammation for at least 12 months.

49. The method of claim 31, wherein the anti-CD3 antibody is administered in an amount of from about 0.1 to 100 mg per day.

50. The method of claim 31, wherein the anti-CD20 antibody is administered in an amount of from about 0.1 to 1000 mg per day.

51. The method of claim 31 , wherein the anti-CD32B antibody is administered in an amount of from about 0.1 to 1000 mg per day.

52. A method for treating a subject in need of treatment for an autoimmune disease comprising administering to the subject a non-activating anti-T cell antibody in combination with a B cell depleting antibody, wherein the effective amount of each individual antibody needed to achieve remission of symptoms of the autoimmune disease when administered in combination is less than the effective amount of one antibody when either antibody is administered as a single antibody therapy.

53. A method for treating a subject in need of treatment for an autoimmune disease comprising administering to the subject a reduced mitogenic anti-T cell antibody in combination with a B cell depleting antibody, wherein the effective amount of each individual antibody needed to achieve remission of symptoms of the autoimmune disease when administered in combination is less than the effective amount of one antibody when either antibody is administered as a single antibody therapy.

54. A method for treating a subject in need of treatment for an autoimmune disease comprising administering to the subject a reduced mitogenic anti-T cell antibody in combination with a B cell depleting antibody, wherein the effective amount of each individual antibody needed to achieve remission of symptoms of the autoimmune disease when administered in combination is less than the effective amount of one antibody when either antibody is administered as a single antibody therapy.

55. ' An article of manufacture comprising a nonactivating T-cell antibody in a pharmaceutically acceptable carrier, a B-cell depleting antibody in a pharmaceutically acceptable carrier and instructions for administering the nonactivating T-cell antibody and the second antibody in combination to a subject in need of treatment for an autoimmune disease to induce a clinical remission of the autoimmune disease.