EP2525825A1

EP2525825A1 - Combination therapy to treat autoimmune diseases

Info

Publication number: EP2525825A1
Application number: EP11735172A
Authority: EP
Inventors: Matthias Von Herrath; Hideki Garren; Nanette Solvason; Michael Leviten
Original assignee: La Jolla Institute for Allergy and Immunology; Bayhill Therapeutics Inc
Current assignee: La Jolla Institute for Allergy and Immunology; Bayhill Therapeutics Inc
Priority date: 2010-01-20
Filing date: 2011-01-20
Publication date: 2012-11-28
Also published as: WO2011091138A1; JP2013517329A

Abstract

This invention relates to the treatment of autoimmune diseases with a combination of a B cell specific agent and a DNA antigen specific therapeutic agent encoding a self protein associated with the autoimmune disease. More specifically, the present invention provides a combination of a B cell specific agent, such as an antibody with a DNA antigen specific therapeutic agent, such as a DNA plasmid vector encoding an autoantigen associated the autoimmune disease. Pharmaceutical compositions and articles of manufacture such as kits comprising the agents and combinations thereof are also provided.

Description

COMBINATION THERAPY TO TREAT AUTOIMMUNE DISEASES

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Application No. 61/296,839, filed January 20, 2010, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] This invention relates to the treatment of autoimmune diseases with a combination of an immunosuppressive or immunomodulatory agent, for example, one that targets B cells (e.g., by arresting their growth, destroying them, deleting them or altering their differentiation) and a DNA antigen specific therapeutic agent encoding a self protein associated with the autoimmune disease. More specifically, the present invention provides, for example a combination of a immunosuppressive or immunomodulatory agent, such as an antibody and a DNA antigen specific therapeutic agent, such as a DNA plasmid vector encoding an autoantigen associated the autoimmune disease. Pharmaceutical compositions and articles of manufacture such as kits comprising the agents and combinations thereof are also provided.

Background

[0003] Autoimmune disease is a disease caused by adaptive immunity that becomes misdirected at healthy cells and/or tissues of the body. Autoimmune disease affects 3% of the U.S. population and likely a similar percentage of the industrialized world population (Jacobson et al., Clin Immunol Immunopathol, 84:223-43 (1997)). Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, polypeptides, peptides, and/or other self-molecules causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical

manifestations of the disease (Marrack et al., Nat Med, 7:899-905 (2001 )). Autoimmune diseases include diseases that affect specific tissues as well as diseases that can affect multiple tissues. This may, in part, for some diseases depend on whether the autoimmune responses are directed to an antigen confined to a particular tissue or to an antigen that is widely distributed in the body. The characteristic feature of tissue-specific autoimmunity is the selective targeting of a single tissue or individual cell type. Nevertheless, certain autoimmune diseases that target ubiquitous self-proteins can also effect specific tissues. For example, in polymyositis the autoimmune response targets the ubiquitous protein histidyl-tRNA synthetase, yet the clinical manifestations primarily involved are autoimmune destruction of muscle.

[0004] The immune system employs a highly complex mechanism designed to generate responses to protect mammals against a variety of foreign pathogens while at the same time preventing responses against self-antigens. In addition to deciding whether to respond (antigen specificity), the immune system must also choose appropriate effector functions to deal with each pathogen (effector specificity). A cell critical in mediating and regulating these effector functions is the CD4⁺ T cell. Furthermore, it is the elaboration of specific cytokines from CD4⁺ T cells that appears to be the major mechanism by which T cells mediate their functions. Thus, characterizing the types of cytokines made by CD4⁺ T cells as well as how their secretion is controlled is extremely important in understanding how the immune response is regulated.

[0005] The characterization of cytokine production from long-term mouse CD4⁺ T cell clones was first published more than 10 years ago (Mosmann et al., J. Immunol , 136:2348-2357 (1986)). In these studies, it was shown that CD4⁺ T cells produced two distinct patterns of cytokine production, which were designated T helper 1 (Th l ) and T helper 2 (Th2). Thl cells were found to exclusively produce interleukin-2 (IL-2), interferon-γ (IFN-γ) and lymphotoxin (LT), while Th2 clones exclusively produced IL-4, IL-5, IL-6, and IL- 13 (Cherwinski et al., J. Exp. Med., 169: 1229-1244 (1987)). Somewhat later, additional cytokines, IL-9 and IL-10, were isolated from Th2 clones (Van Snick et al., J. Exp. Med., 169:363-368 (1989); Fiorentino et al., J. Exp. Med. , 170:2081 -2095 (1989)). Finally, additional cytokines, such as IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF-a) were found to be secreted by both Th l and Th2 cells.

[0006] Autoimmune disease encompasses a wide spectrum of diseases that can affect many different organs and tissues within the body as outlined in Table 1 . See, e.g., Paul W.E. (ed. 2003) Fundamental Immunology (5th Ed.) Lippincott Williams & Wilkins; ISBN- 10:

0781735149, ISBN-13: 978-0781735148; Rose and Mackay (eds. 2006) The Autoimmune Diseases (4th ed.) Academic Press, ISBN- 10: 0125959613, ISBN-13: 978-0125959612; Erkan, et al. (eds. 2004) The Neurologic Involvement in Systemic Autoimmune Diseases, Volume 3 {Handbook of Systemic Autoimmune Diseases) Elsevier Science, ISBN-10: 0444516514, ISBN- 13: 978-0444516510; and Richter, et al. (eds. 2003) Treatment of Autoimmune Disorders, Springer, ISBN-10: 321 1837728, ISBN-13: 978-321 1837726.

Table 1

Primary Organ(s) Targeted Disease

skin, joints, kidneys, brain, blood cells Systemic lupus erythematosus (SLE)

[0007] Current therapies for human autoimmune disease, include glucocorticoids, cytotoxic agents, and recently developed biological therapeutics. In general, the management of human systemic autoimmune disease is empirical and unsatisfactory. For the most part, broadly immunosuppressive drugs, such as corticosteroids, are used in a wide variety of severe autoimmune and inflammatory disorders. In addition to corticosteroids, other

immunosuppressive agents are used in management of the systemic autoimmune diseases.

Cyclophosphamide is an alkylating agent that causes profound depletion of both T- and B- lymphocytes and impairment of cell-mediated immunity. Cyclosporine, tacrolimus, and mycophenolate mofetil are natural products with specific properties of T-lymphocyte

suppression, and they have been used to treat SLE, RA and, to a limited extent, in vasculitis and myositis. Methotrexate is also used as a "second line" agent in RA, with the goal of reducing disease progression. It is also used in polymyositis and other connective-tissue diseases. Other approaches that have been tried include monoclonal antibodies intended to block the action of cytokines or to deplete lymphocytes. (Fox, D. A., Am. J. Med., 99:82-88 (1995).) Treatments for multiple sclerosis (MS) include interferon β and copolymer 1 , which reduce relapse rate by 20-30% and only have a modest impact on disease progression. MS is also treated with immunosuppressive agents including methylprednisolone, other steroids, methotrexate, cladribine and cyclophosphamide. These immunosuppressive agents, as currently used, have minimal efficacy in treating MS. Current therapy for RA utilizes agents that non-specifically suppress or modulate immune function such as methotrexate, sulfasalazine, hydroxychloroquine, leuflonamide, prednisone, as well as the recently developed TNFa antagonists etanercept and infliximab (Moreland et al., J Rheumatol, 28: 1431 -52 (2001 )).

[0008] In the case of organ-specific autoimmunity, a number of different therapeutic approaches have been tried. Soluble protein antigens have been administered systemically to inhibit the subsequent immune response to that antigen. Such therapies include delivery of myelin basic protein, its dominant peptide, or a mixture of myelin proteins to animals with experimental autoimmune encephalomyelitis and humans with multiple sclerosis (Brocke et al., Nature, 379:343-6 (1996); Critchfield et al., Science, 263: 1 139-43 (1994); Weiner et al., Annu Rev Immunol, 12:809-37 (1994)), administration of type II collagen or a mixture of collagen proteins to animals with collagen-induced arthritis and humans with rheumatoid arthritis

(Gumanovskaya et al, Immunology, 97:466-73 ( 1999); cKown et al., Arthritis Rheum, 42: 1204-8 (1999); Trentham et al., Science, 261 : 1727-30 (1993), delivery of insulin to animals and humans with autoimmune diabetes (Pozzilli and Gisella Cavallo, Diabetes Metab Res Rev, 16:306-7 (2000), and delivery of S-antigen to animals and humans with autoimmune uveitis (Nussenblatt et al., Am J Ophthalmol, 123:583-92 (1997). A problem associated with this approach is T cell unresponsiveness induced by systemic injection of antigen. Another approach is the attempt to design rational therapeutic strategies for the systemic administration of a peptide antigen based on the specific interaction between the T cell receptors and peptides bound to MHC molecules. One study using the peptide approach in an animal model of diabetes, resulted in the development of antibody production to the peptide (Hurtenbach, U. et al., J Exp. Med, 177: 1499 (1993)). Another approach is the administration of T cell receptor (TCR) peptide immunization. See, e.g., Vandenbark, A. A. et al., Nature, 341 :541 (1989). Still another approach is the induction of oral tolerance by ingestion of peptide or protein antigens. See, e.g., Weiner, H. L., Immmunol Today, 18:335 (1997).

[0009] In the case of T1 D, various treatment modalities have been investigated with the goal of inducing long-term tolerance to one or all of the antigens targeted in T1 D. See, e.g., M. von Herrath, S. Sanda, K. Herold, Nat Rev Immunol 7, 988 (Dec, 2007) and T. Staeva-Vieira, M. Peakman, M. von Herrath, Clin Exp Immunol 148, 17 (Apr, 2007). The various modalities can be broadly classified into two categories; antigen-nonspecific and antigen-specific interventions.

[0010] In antigen nonspecific intervention, polyclonal antibodies like anti-thymocyte globulin (ATG or ALS), systemic immuno-modulators like cyclosporine and anti-CD3 have all shown varying degrees of success. ALS selectively depletes Teff cells while sparing or even inducing the generation of CD4⁺ CD25⁺ Foxp3⁺ Tregs See, e.g., M. Lopez, M. R. Clarkson, M. Albin, M. H. Sayegh, N. Najafian, J Am Soc Nephrol 17, 2844 (Oct, 2006) and K. Minamimura, W. Gao, T. Maki, J Immunol 176, 4125 (Apr 1 , 2006) and effectively reverses spontaneous T1 D in the NOD mouse. See, e.g., N. Ogawa, K. Minamimura, T. Kodaka, T. Maki, J Autoimmun 26, 225 (Jun, 2006) and G. Simon et al, Diabetes 57, 405 (Feb, 2008). Similarly, immunosuprression with cyclosporine can halt β-cell destruction See, e.g., J. L. Mahon, J. Dupre, C. R. Stiller, Ann N Y Acad Sci 696, 351 (Nov 30, 1993). but, protection lasts only as long as the drug is present; long-term tolerance to B-cell antigens is not achieved. Similar to ALS, anti-CD3 treatment induces shift from Th l toward Th2 profile in mice (Belghith, 2003 #221 ;von Herrath, 2002 #92) and in humans (B. Bisikirska, J. Colgan, J. Luban, J. A. Bluestone, K. C. Herold, J Clin Invest 115, 2904 (Oct, 2005) and K. C. Herold et al. , J Clin Invest 111, 409 (Feb, 2003)) through a combination of activation induced cell death of Teff cells and selective inactivation of THl -cell subsets (J. A. Smith, Q. Tang, J. A. Bluestone, J Immunol 160, 4841 (May 15, 1998) and J. A. Smith, J. Y. Tso, M. R. Clark, M. S. Cole, J. A. Bluestone, J Exp Med 185, 1413 (Apr 21 , 1997)), while sparing Tregs (M. Belghith et al. , Nat Med 9, 1202 (Sep, 2003)). Clinical studies in recent onset T1D patients indicate that treatment with anti-CD3 halts T1D progression for significant periods of time post-therapy, preserving residual endogenous B-cell mass (K. C.

Herold et al., Diabetes 54, 1763 (Jun, 2005). However, in some cases anti-CD3 mAbs induced reactivation of latent EBV infection (B. eymeulen et al., N Engl J Med 352, 2598 (Jun 23, 2005)), while others developed antibodies to the drug (K. C. Herold et al., N Engl J Med 346, 1692 (May 30, 2002)). Moreover, efficacy of anti-CD3 treatment is very limited because in the long term, tolerance to β cell antigens is lost (L. Chatenoud, J. Primo, J. F. Bach, J Immunol 158, 2947 (Mar 15, 1997)).

[0011] In antigen specific intervention, the variable effects were seen with the use of

DiaPep277 (derived from Hsp60). In one study, during a period of two years, administration of DiaPep277 maintained insulin secretion in patients with new onset T1D (I. Raz et al., Lancet 358, 1749 (Nov 24, 2001)), but this was not seen in all follow-up studies, although the effect on T cells was consistent (Roep et al.). Similarly, administration of insulin or insulin peptides in mice leads to the generation of antigen-specific Treg cells that can prevent T1D (N. R. Martinez et al., J Clin Invest 111, 1365 (May, 2003), D. Homann, T. Dyrberg, J. Petersen, M. B. Oldstone, M. G. von Herrath, J Immunol 163, 1833 (Aug 15, 1999), and M. G. von Herrath, T. Dyrberg, M. B. Oldstone, J Clin Invest 98, 1324 (Sep 15, 1996)). Such Tregs selectively proliferate in the - pancreatic draining lymph nodes (PLN) and are capable of dampening autoaggressive CD8 responses (D. Homann, T. Dyrberg, J. Petersen, M. B. Oldstone, M. G. von Herrath, J Immunol 163, 1833 (Aug 15, 1999)) but incapable of reverting recent-onset T1 D (M. Larche, D. C.

Wraith, Nat Med 11, S69 (Apr, 2005)). However, so far, oral and nasal insulin trials in humans have not produced positive results (Nanto-Salonen, K. et al., Lancet 372(9651 ): 1746-55, 208; Skyler et al., Diabetes Care 28 (5): 1068-76, 2005), likely because induction of Tregs alone is insufficient to revert human T1 D (as it is in animal models) or because the dose and regimen of antigen may have been wrong, which provides a strong rationale for pursuing combination therapies with systemic agents that can facilitate Treg induction and maintenance. T and B Cells in Autoimmunity

[0012] Despite positive and negative selection in the thymus, all individuals still harbor a subset of T cells directed toward self-antigens in peripheral tissues (J. F. Bach, Rev Neurol (Paris) 158, 881 (Oct, 2002)). As an example, in TI D, such auto-reactive T-cells, and in some cases B-cells (H. Bour-Jordan, J. A. Bluestone, J Clin Invest 117, 3642 (Dec, 2007)), infiltrate the pancreas (insulitis). In humans, TI D is characterized by the early appearance of islet-specific autoantibodies (L. Yu et al, Proc Natl Acad Sci USA 97, nO\ (Feb 15, 2000)). While the role of B-cells in TI D has not been entirely elucidated, CD4⁺ and CD8⁺ T-cells are believed to selectively destroy insulin producing β-cells in the islets (M. von Herrath, D. Homann, Pediatr Diabetes 5 Suppl 2, 23 (2004)). Such loss of B -cells is very gradual and the onset of TI D signifies an insufficient insulin production. The major autoantigens in TI D include proinsulin or insulin itself, glutamic acid decarboxylase 65 (GAD65), the islet tyrosine phosphatase (IA-2), and the islet-specific glucose-6-phosphatase catalytic subunit-related protein (M. von Herrath, S. Sanda, K. Herold, Nat Rev Immunol 7, 988 (Dec, 2007)). Current evidence suggests that insulin is the primary antigen leading to islet cell destruction in mice (M. Nakayama et al, Nature 435, 220 (May 12, 2005)), and probably in humans (S. Arif et al. , J Clin Invest 113, 451 (Feb, 2004), P. F. Bougneres et al. , Diabetes 39, 1264 (Oct, 1990), and S. C. Kent et al., Nature 435, 224 (May 12, 2005)).

[0013] Traditionally, B cells mediate autoimmune diseases by producing autoantibodies or by acting as antigen presenting cells (APCs) (F. S. Wong, L. Wen, Rev Diabet Stud 2, 121 (Fall, 2005)). However, in Tl D, while such autoantibodies may facilitate or enhance diabetes development, they do not induce the disease (F. S. Wong, L. Wen, Rev Diabet Stud 2, 121 (Fall, 2005)). Recent evidence suggests that B cells function as islet APCs for T-cells and the autoantibodies expressed on cell surface improve the capture and presentation of autoantigens (M. Falcone, J. Lee, G. Patstone, B. Yeung, N. Sarvetnick, J Immunol 161, 1 163 (Aug 1 , 1998), H. Noorchashm et al. , J Immunol 163, 743 (Jul 15, 1999), and H. Bour-Jordan et al , J Immunol 179, 1004 (Jul 15, 2007)).

[0014] Naturally occurring CD4⁺ CD25⁺ FoxP3⁺ regulatory-T-cells (Tregs) constitute 5-10% of all CD4⁺ T cells in the thymus and lymph nodes. They constitutively express GITR, OX40 (CD134) and CTLA-4 (E. M. Shevach, R. S. McHugh, C. A. Piccirillo, A. M. Thornton, Immunol Rev 182, 58 (Aug, 2001 ), J. Shimizu, S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi, Nat Immunol 3, 135 (Feb, 2002)). CD127 expression is inversely correlated with Foxp3, and together with the other markers, identifies over 95% of the Foxp3⁺ cells in peripheral blood W. Uu et al, J Exp Med 203, 1701 (Jul 10, 2006), N. Seddiki et al. , J Exp Med 203, 1693 (Jul 10, 2006)). Tregs play a central role in peripheral T-cell tolerance through the induction of IL-4, IL-10 or TGF-b (L. Chatenoud, B. Salomon, J. A. Bluestone, Immunol Rev 182, 149 (Aug, 2001)) and can promote infectious tolerance (H. Waldmann, S. Cobbold, Annu Rev Immunol 16, 619 (1998), H. Waldmann, S. Cobbold, Immunity 14, 399 (Apr, 2001)). Hence deficiency or dysfunction of Tregs results in autoimmunity (E. Maggi et al, Autoimmun Rev 4, 579 (Nov, 2005), S. Sakaguchi, Nat Immunol 6, 345 (Apr, 2005)). In addition they can be used for the treatment of Tl D (S. Sakaguchi, Nat Immunol 6, 345 (Apr, 2005), S. Sakaguchi, Nat Immunol 6, 345 (Apr, 2005)).

[0015] The role of B cells may vary depending on the autoimmune disease. In the case of T1D autoantibodies against major T1 D antigens appear early (L. Yu et al , Proc Natl Acad Sci U S A 97, 1701 (Feb 15, 2000)) and are a predictive marker for diabetes progression in humans. Thus B-cells although not essential for the generation of diabetes, are clearly involved in the progression of diabetes, owing to their antigen presenting cell (APC) function to T cells.

[0016] B cell membrane receptors evolve and change throughout the B cell life span. Tumor necrosis factor receptor superfamily member 13B (TACI), tumor necrosis factor receptor superfamily member 17 (BCMA), B cell-activating factor (BAFF-R) (also referred to as B lymphocyte stimulator (BLyS)) are present on both immature B cells and mature B cells. All of the foregoing receptors may be inhibited by an antibody to BLyS (Belimumab). CD22 is a member of the sialic acid binding Ig-like lectins cell surface receptors and is found on B cells as is CD 19.

[0017] The CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35, or B l ) is a four-pass, glycosylated integral membrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al., J. Biol. Chem., 264( 19): 1 1282-1 1287 (1989) and Einfeld et al, EMBO J., 7(3):71 1 -71 7 (1988)). The antigen is also expressed on greater than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al, Blood, 63(6): 1424- 1433 (1984)), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder et al. J. Immunol., 135(2):973- 979 (1985)). CD20 regulates an early step(s) in the activation process for cell- cycle initiation and differentiation (Tedder et al, supra), and possibly functions as a calcium- ion channel.

Tedder et al, J. Cell. Biochem., 14D: 195 (1990). CD20 undergoes phosphorylation in activated B cells (Riley and Sliwkowski, Semin Oncol, 27(12): 17-24 (2000)). CD20 appears on the surface of B-lymphocytes at the pre-B-cell stage and is found on mature and memory B cells, but not plasma cells (Stashenko et al. J. Immunol, 125: 1678-1685 ( 1980); Clark and Ledbetter Adv. Cancer Res., 52:81-149 (1989)). CD20 has calcium-channel activity and may have a role in the development of B cells. The relationship between lysis of peripheral CD20⁺ B cells in vitro and rituximab activity in vivo is unclear. Rituximab displays antibody-dependent cellular cytotoxicity (ADCC) in vitro (Reff et al. Blood, 83:435—445 (1994)). Potent complement- dependent cytotoxic (CDC) activity has also been observed for rituximab on lymphoma cells and cell lines (Reff et al, supra, 1994) and in certain mouse xenograft models (Di Gaetano et al, J. Immunol, 171 : 1581-1587 (2003)). Several anti-CD20 antibodies, including rituximab, have been shown to induce apoptosis in vitro when crosslinked by a secondary antibody or by other means (Ghetie et al. Proc Natl Acad Sci. USA, 94:7509-7514 ( 1997)).

[0018] Given the expression of CD20 on B-cells, this antigen can serve as a candidate for "targeting" of B cells in autoimmune disease. In essence, such targeting can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are administered to a patient. These anti-CD20 antibodies specifically bind to the CD20 antigen of (ostensibly) both normal and autoimmune associated B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of autoimmune associated B cells. Additionally, chemical agents or radioactive labels having the potential to destroy the autoimmune associated B cells can be conjugated to the anti-CD20 antibody such that the agent is specifically "delivered" to the autoimmune associated B cells. Irrespective of the approach, a primary goal is to destroy the autoimmune associated B cells; the specific approach can be determined by the particular anti- CD20 antibody that is utilized, and thus, the available approaches to targeting the CD20 antigen can vary considerably.

[0019] The rituximab (RITUXAN®) antibody is a genetically engineered chimeric

murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody called "C2B8" in US 5736137 (Anderson et al). Rituximab is indicated for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma. In vitro mechanism-of-action studies have demonstrated that rituximab binds human complement and lyses lymphoid B-cell lines through CDC (Reff et al, Blood, 83(2): 435-445 (1994)). Additionally, it has significant activity in assays for ADCC. More recently, rituximab has been shown to have anti-proliferative effects in tritiated thymidine- incorporation assays and to induce apoptosis directly, while other anti-CD19 and anti-CD20 antibodies do not (Maloney et al. Blood, 88(10): 637a (1996)). Synergy between rituximab and chemotherapies and toxins has also been observed experimentally. In particular, rituximab sensitizes drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin, and ricin (Demidem et al, Cancer Chemotherapy & Radiopharmaceuticals, 12(3): 177-186 (1997)). In vivo preclinical studies have shown that rituximab depletes B cells from the peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, presumably through complement- and cell-mediated processes (Reff et al, Blood, 83:435-445 (1994)).

[0020] Rituximab was approved in the United States for the treatment of patients with relapsed or refractory low-grade or follicular CD20⁺ B-cell NHL at a dose of 375 rag/m² weekly for four doses. In April 2001 , the Food and Drug Administration (FDA) approved additional claims for the treatment of low-grade NHL: re-treatment (weekly for four doses) and an additional dosing regimen (weekly for eight doses). Many patients have been exposed to rituximab either as monotherapy or in combination with immunosuppressant or chemotherapeutic drugs. Patients have also been treated with rituximab as maintenance therapy for up to two years. Hainsworth et al, J. Clin. Oncol, 21 : 1746-1751 (2003); Hainsworth et al, J. Clin. Oncol, 20:4261-4267 (2002). Also, rituximab has been used in the treatment of malignant and nonmalignant plasma cell disorders. Treon and Anderson, Semin. Oncol., 27: 79-85 (2000).

[0021] Rituximab has also been approved in the United States in combination with MTX to reduce signs and symptoms in adult patients with moderately- to severely-active RA who have had an inadequate response to at least one TNF antagonist. Many studies address the use of rituximab in a variety of non-malignant autoimmune disorders, including RA, in which B cells and autoantibodies appear to play a role in disease pathophysiology. Edwards et al, Biochem Soc. Trans., 30:824-828 (2002). Rituximab has been reported to potentially relieve signs and symptoms of, for example, RA (Leandro et al, Ann. Rheum. Dis. 61 :883— 888 (2002); Edwards et al, Arthritis Rheum., 46 (Suppl. 9): S46 (2002); Stahl et al, Ann. Rheum. Dis., 62 (Suppl. 1): OP004 (2003); Emery et al, Arthritis Rheum.,4S(9): S439 (2003)), lupus (Eisenberg, Arthritis. Res. Ther. 5: 157-159 (2003); Leandro et al.Arthritis Rheum. 46: 2673-2677 (2002); Gorman et al, Lupus, 13: 312-316 (2004)), immune thrombocytopenic purpura (D'Arena et al, Leuk. Lymphoma, 44:561-562 (2003); Stasi et al.,Blood, 98: 952-957 (2001); Saleh et al, Semin. Oncol, 27 (Supp 12):99-103 (2000); Zaja et al, Haematologica, 87: 189-195 (2002);

Ratanatharathorn et al, Ann. Int. Med., 133:275-279 (2000)), pure red cell aplasia (Auner et al, Br. J. Haematol, 1 16:725-728 (2002)); autoimmune anemia (Zaja et al, supra (erratum appears in Haematologica 87:336 (2002)), cold agglutinin disease (Layios et al, Leukemia, 15: 187-8 (2001); Berentsen et al, Blood, 103: 2925-2928 (2004); Berentsen et al, Br. J. Haematol, 1 15:79-83 (2001 ); Bauduer, Br. J. Haematol, 1 12: 1083-1090 (2001); Zaja et al , Br. J.

Haematol, 1 15:232-233 (2001 )), type B syndrome of severe insulin resistance (Coll et al, N. Engl. J. Med., 350:310-31 1 (2004), mixed cryoglobulinemia (DeVita et al, Arthritis Rheum. 46 Suppl. 9:S206/S469 (2002)), myasthenia gravis (Zaja et al, Neurology, 55: 1062-1063 (2000); Wylam et al, J. Pediatr., 143:674-677 (2003)), Wegener's granulomatosis (Specks et al, Arthritis & Rheumatism 44:2836-2840 (2001 )), refractory pemphigus vulgaris (Dupuy et al, Arch Dermatol, 140:91 -96 (2004)), dermatomyositis (Levine, Arthritis Rheum., 46 (Suppl. 9):S 1299 (2002)), Sjogren's syndrome (Somer et al, Arthritis & Rheumatism, 49:394-398 (2003)), active type-II mixed cryoglobulinemia (Zaja et al, Blood, 101 :3827-3834 (2003)), pemphigus vulgaris (Dupay et al, Arch. Dermatol, 140:91 -95 (2004)), autoimmune neuropathy (Pestronk et al, J. Neurol. Neurosurg. Psychiatry 74:485^-89 (2003)), paraneoplastic opsoclonus-myoclonus syndrome (Pranzatelli et al. Neurology 60(Suppl. 1 ) P05.128:A395 (2003)), and relapsing-remitting multiple sclerosis (RRMS) (Cross et al. (abstract) "Preliminary Results from a Phase II Trial of Rituximab in MS" Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis, 20-21 (2003)).

[0022] A Phase II study (WA 16291 ) has been conducted in patients with RA, providing 48- week follow-up data on safety and efficacy of rituximab (Emery et al, Arthritis Rheum., 48(9):S439 (2003); Szczepanski et al, Arthritis Rheum., 48(9):S 121 (2003)). A total of 161 patients were evenly randomized to four treatment arms: MTX, rituximab alone, rituximab plus MTX, and rituximab plus cyclophosphamide (CTX). The treatment regimen of rituximab was one gram administered intravenously on days 1 and 15. Infusions of rituximab in most patients with RA were well tolerated by most patients, with 36% of patients experiencing at least one adverse event during their first infusion (compared with 30% of patients receiving placebo). Overall, the majority of adverse events was considered to be mild to moderate in severity and was well balanced across all treatment groups. There were a total of 19 serious adverse events across the four arms over the 48 weeks, which were slightly more frequent in the rituximab/CTX group. The incidence of infections was well balanced across all groups. The mean rate of serious infection in this RA patient population was 4.66 per 100 patient-years, which is lower than the rate of infections requiring hospital admission in RA patients (9.57 per 100

patient-years) reported in a community-based epidemiologic study (Doran et al, Arthritis Rheum. 46:2287-2293 (2002)).

[0023] The reported safety profile of rituximab in a small number of patients with neurologic disorders, including autoimmune neuropathy (Pestronk et al, supra), opsoclonus-myoclonus syndrome (Pranzatelli et al, supra), and RRMS (Cross et al, supra), was similar to that reported in oncology or RA. In an investigator-sponsored trial (1ST) of rituximab combined with interferon-beta (IFN-β)) or glatiramer acetate in patients with RRMS (Cross et al, supra), one of ten treated patients was admitted to the hospital for overnight observation after experiencing moderate fever and rigors following the first infusion of rituximab, while the other nine patients completed the four-infusion regimen without any reported adverse events.

[0024] Patents and patent publications concerning CD20 antibodies, CD20-binding molecules, and self-antigen vaccines include U.S. 5,776,456, 5,736,137, 5,843,439, 6,399,061 , and

6,682,734, as well as US 2002/0197255, US 2003/0021781, US 2003/0082172, US

2003/0095963, US 2003/0147885, US 2005/0186205, and WO 1994/1 1026 (Anderson et al); U.S. 6,455,043, US 2003/0026804, US 2003/0206903, and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); US 2004/0213784 and WO 2000/27433 (Grillo- Lopez and Leonard); WO 2000/44788 (Braslawsky et al); WO 2001/10462 (Rastetter, W.); WO 2001/10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US

2001/0018041 , US 2003/0180292, US 2002/0028178, WO 2001/34194, and WO 2002/22212 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665, US 2005/0180975, WO 2001/74388, and U.S. 6,896,885 (Hanna, N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and Hanna); US 2005/0123540 (Hanna et al); US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858; US 2005/01 12060, US 2002/0039557, and U.S. 6,846,476 (White, C); US 2002/0128448 and WO 2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et «/.);WO 2002/096948 (Braslawsky et a/.);WO 2002/079255 (Reff and Davies); U.S. 6,171 ,586 and 6,991 ,790, and WO 1998/56418 (Lam et al); US 2004/0191256 and WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, U.S. 6,194,551 , U.S. 6,242,195, 6,528,624 and 6,538,124 (Idusogie et al); U.S. 7,122,637, US 2005/01 18174, US 2005/0233382, US 2006/0194291 , US 2006/0194290, US 2006/0194957, and WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al); WO 2001/03734 (Grillo-Lopez et al); US 2002/0004587, US 2006/0025576, and WO 2001/77342 (Miller and Presta); US 2002/0197256 and WO 2002/078766 (Grewal, I.); US 2003/0157108 and WO 2003/035835 (Presta, L.); U.S. 5,648,267, 5,733,779, 6,017,733, and 6,159,730, and WO

1994/1 1523 (Reff et al on expression technology); U.S. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721 (Kaminski et al); U.S. 5,500,362, 5,677,180, 5,721 ,108, 6,120,767, 6,652,852, and 6,893,625 as well as WO 1988/04936 (Robinson et al); U.S.

6,410,391 (Zelsacher); U.S. 6,224,866 and WO 2000/20864 (Barbera-Guillem, E.); WO

2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); U.S. 7,074,403 (Goldenberg and Hansen); U.S. 7,151 ,164 (Hansen et al); US 2003/0133930; WO 2000/74718 and US 2005/0191300A1 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 (Hansen et al); WO 2004/058298 (Goldenberg and Hansen); WO 2000/76542 (Golay et al); WO

2001/72333 (Wolin and Rosenblatt); U.S. 6,368,596 (Ghetie et al); U.S. 6,306,393 and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and Hartmann); WO 2002/102312 (Engleman, E.); US 2003/0068664 (Albitar et al); WO 2003/002607 (Leung, S.); WO

2003/049694, US 2002/0009427, and US 2003/0185796 (Wolin et al); WO 2003/061694 (Sing and Siegall); US 2003/0219818 (Bohen et al); US 2003/0219433 and WO 2003/0.68821 (Hansen et al); US 2003/0219818 (Bohen et al); US 2002/0136719 (Shenoy et al); WO 2004/032828 and US 2005/0180972 (Wahl et al); and WO 2002/56910 (Hayden-Ledbetter). See also U.S. 5,849,898 and EP 330,191 (Seed et al); EP332,865A2 (Meyer and Weiss); U.S. 4,861 ,579 (Meyer et al); US 2001/0056066 (Bugelski et al); WO 1995/03770 (Bhat et al); US

2003/0219433 Al (Hansen et al); WO 2004/035607 and US 2004/167319 (Teeling et al); WO 2005/103081 (Teeling et al); US 2006/0034835, US 2006/0024300, and WO 2004/056312 (Lowman et al); US 2004/0093621 (Shitara et al); WO 2004/103404 (Watkins et al); WO 2005/000901 (Tedder et al); US 2005/0025764 (Watkins et al); US 2006/0251652 (Watkins et al); WO 2005/016969 (Carr et al); US 2005/0069545 (Carr et al); WO 2005/014618 (Chang et al); US 2005/0079174 (Barbera-Guillem and Nelson); US 2005/0106108 (Leung and Hansen); US 2005/0123546 (Umana et al); US 2004/0072290 (Umana et al.) ; US 2003/0175884 (Umana et al); and WO 2005/044859 (Umana et al); WO 2005/070963 (Allan et al); US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al); US 2003/1 18592 (Ledbetter et al); US 2003/133939 (Ledbetter and Hayden-Ledbetter); US 2005/0202012 (Ledbetter and Hayden-Ledbetter); US 2005/0175614 (Ledbetter and Hayden-Ledbetter); US 2005/0180970 (Ledbetter and Hayden-Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/0202023 (Hayden-Ledbetter and Ledbetter); WO 2005/017148 (Ledbetter et al); WO 2005/037989 (Ledbetter et al); U.S.

6,183,744 (Goldenberg); U.S. 6,897,044 (Braslawski et al); WO 2006/005477 (Krause et al); US 2006/0029543 (Krause et al); US 2006/0018900 (McCormick et al); US 2006/0051349 (Goldenberg and Hansen); WO 2006/042240 (Iyer and Dunussi-Joannopoulos); US

2006/0121032 (Dahiyat et al); WO 2006/064121 (Teillaud et al); US 2006/0153838 (Watkins), CN 1718587 (Chen et al); WO 2006/084264 (Adams et al); US 2006/0188495 (Barron et al); US 2004/0202658 and WO 2004/091657 (Benynes, K.); US 2005/0095243, US 2005/0163775, WO 2005/00351 , and WO 2006/068867 (Chan, A.); US 2006/0135430 and WO 2005/005462 (Chan et al); US 2005/0032130 and WO 2005/017529 (Beresini et al); US 2005/0053602 and WO 2005/023302 (Brunetta, P.); US 2006/0179501 and WO 2004/060052 (Chan et al); WO 2004/060053 (Chan et al); US 2005/0186206 and WO 2005/060999 (Brunetta, P.); US

2005/0191297 and WO 2005/061542 (Brunetta, P.); US 2006/0002930 and WO 2005/1 15453 (Brunetta et al); US 2006/0099662 and WO 2005/108989 (Chuntharapai et al); CN 1420129A (Zhongxin Guojian Pharmaceutical); US 2005/0276803 and WO 2005/1 13003 (Chan et al); US 2005/0271658 and WO 2005/1 17972 (Brunetta et al); US 2005/0255527 and WO 2005/1 1428 (Yang, J.); US 2006/0024295 and WO 2005/120437 (Brunetta, P.); US 2006/0051345 and WO 2005/1 17978 (Frohna, P.); US 2006/0062787 and WO 2006/012508 (Hitraya, E.); US

2006/0067930 and WO 2006/31370 (Lowman et al); WO 2006/29224 (Ashkenazi, A.); US 2006/01 10387 and WO 2006/41680 (Brunetta, P.); US 2006/01341 1 1 and WO 2006/066086 (Agarwal, S.); WO 2006/069403 (Ernst and Yansura); US 2006/0188495 and WO 2006/076651 (Dummer, W.); WO 2006/084264 (Lowman, H.); WO 2006/093923 (Quan and Sewell); WO 2006/106959 (Numazaki et al); WO 2006/126069 (Morawala); WO 2006/130458 (Gazit- Bornstein et al); US 2006/0275284 (Hanna, G.); US 2007/0014785 (Golay et al); US

2007/0014720 (Gazit-Bornstein et al); and US 2007/0020259 (Hansen et al); US

2007/0020265 (Goldenberg and Hansen); US 2007/0014797 (Hitraya); US 2007/0224189 (Lazar et al); WO 2007/014238 (Bruge and Bruger); and WO 2008/003319 (Parren and Baadsgaard).

[0025] Scientific publications concerning treatment with rituximab include: Perotta and Abuel, "Response of chronic relapsing ITP of 10 years duration to rituximab" Abstract # 3360 Blood, 10(l)(part 1-2):88B (1998); Perotta et al, "Rituxan in the treatment of chronic idiopathic thrombocytopaenic purpura (ITP)", Blood, 94:49 (abstract) (1999); Matthews, R., "Medical Heretics" New Scientist, 7 (April, 2001); Leandro et al, "Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion" Ann Rheum Dis., supra; Leandro et al, "Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficacy and dose response" Arthritis and Rheumatism, 44(9):S370 (2001); Leandro et al, "An open study of B lymphocyte depletion in systemic lupus erythematosus" Arthritis and Rheumatism, 46:2673- 2677 (2002), wherein during a two-week period, each patient received two 500-mg infusions of rituximab, two 750-mg infusions of CTX, and high-dose oral corticosteroids, and wherein two of the patients treated relapsed at seven and eight months, respectively, and have been retreated, although with different protocols; "Successful long-term treatment of systemic lupus

erythematosus with rituximab maintenance therapy" Weide et al, Lupus, 12:779-782 (2003), wherein a patient was treated with rituximab (375 mg/m² x 4, repeated at weekly intervals) and further rituximab applications were delivered every five to six months and then maintenance therapy was received with rituximab 375 mg/m² every three months, and a second patient with refractory SLE was treated successfully with rituximab and is receiving maintenance therapy every three months, with both patients responding well to rituximab therapy; Edwards and Cambridge, "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes" Rheumatology, 40:205-21 1 (2001 ); Cambridge et al., "B lymphocyte depletion in patients with rheumatoid arthritis: serial studies of immunological parameters" Arthritis Rheum., 46 (Suppl. 9): S 1350 (2002); Cambridge et al., "Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis" Arthritis Rheum., 48:2146-2154 (2003); Edwards et al, "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders" Biochem Soc. Trans., supra; Edwards et al, "Efficacy and safety of rituximab, a B- cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis" Arthritis and Rheumatism, 46(9):S 197 (2002); Edwards et al., "Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis" N Engl. J. Med., 350:2572-2582 (2004); Pavelka et al, Ann. Rheum. Dis., 63:(S l):289-290 (2004);

Emery et al, Arthritis Rheum. 50 (S9):S659 (2004); Levine and Pestronk, "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using Rituximab" Neurology, 52: 1701 -1704 (1999); Uchida et al, "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J. Exp. Med., 199: 1659-1669 (2004); Gong et al, "Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy" J. Immunol., 174:817-826 (2005); Hamaguchi et al, "The peritoneal cavity provides a protective niche for B l and conventional B lymphocytes during anti-CD20 immunotherapy in mice" J. Immunol., 174:4389-4399 (2005); Cragg et al. "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun., 8: 140-174 (2005); Eisenberg, "Mechanisms of autoimmunity" Immunol. Res., 27:203-218 (2003); DeVita et al., "Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis" Arthritis & Rheum, 46:2029-2033 (2002); Higashida et al. "Treatment of DMARD- refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology (Abstract #LB 1 1 ), New Orleans, LA (Oct., 2002); Tuscano,

"Successful treatment of infliximab-refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology, New Orleans, LA. (Oct., 2002), published as Tuscano, Arthritis Rheum. 46:3420 (2002); "Pathogenic roles of B cells in human autoimmunity; insights from the clinic" Martin and Chan, Immunity, 20:517-527 (2004);

Silverman and Weisman, "Rituximab therapy and autoimmune disorders, prospects for anti-B cell therapy", Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz and Isenberg, "Anti B cell therapy (rituximab) in the treatment of autoimmune diseases" Current opinion in pharmacology, 4:398-402 (2004); Virgolini and Vanda, "Rituximab in autoimmune diseases" Biomedicine & pharmacotherapy, 58: 299-309 (2004); Klemmer et al., "Treatment of antibody mediated autoimmune disorders with a AntiCD20 monoclonal antibody Rituximab" Arthritis And Rheumatism , 48(9) (SEP):S624-S624 (2003); Kneitz et al, "Effective B cell depletion with rituximab in the treatment of autoimmune diseases" Immunobiology, 206:519-527 (2002); Arzoo et al., "Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab)' nnals of the Rheumatic Diseases, 61 (10):922-924 (2002) Comment in Ann Rheum Dis. 61 :863-866 (2002); Tuture strategies in immunotherapy" by Lake and Dionne, in Burger's Medicinal Chemistry and Drug Discovery (John Wiley & Sons, Inc., 2003) (Chapter 2 "Antibody-Directed Immunotherapy"); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target (2002); Appendix 4A entitled "Monoclonal Antibodies to Human Cell Surface Antigens" by Stockinger et al., eds: Coligan et al., in Current Protocols in Immunology (John Wiley & Sons, Inc., 2003); Penichet and Morrison, "CD Antibodies/molecules: Definition; Antibody Engineering" in Wiley

Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies (2002). [0026] Further, see Looney "B cells as a therapeutic target in autoimmune diseases other than rheumatoid arthritis" Rheumatology, 44 Suppl 2:iil 3-ii l 7 (2005); Chambers and Isenberg, "Anti- B cell therapy (rituximab) in the treatment of autoimmune diseases" Lupus, 14(3):210-214 (2005); Looney et al., "B-cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/I I dose-escalating trial of rituximab" Arthritis Rheum., 50:2580-2589 (2004); Looney, "Treating human autoimmune disease by depleting B cells" Ann Rheum. Dis., 61 :863-866 (2002); Edelbauer et al, "Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression Case report" Pediatr. Nephrol., 20(6): 81 1 -813 (2005); D'Cruz and Hughes, "The treatment of lupus nephritis" BMJ, 330(7488):377-378 (2005); Looney, "B cell-targeted therapy in diseases other than rheumatoid arthritis" J. Rheumatol. Suppl, 73: 25-28- discussion 29-30 (2005); Rastetter et al, "Rituximab: expanding role in therapy for lymphomas and autoimmune diseases" Annu. Rev. Med., 55:477-503 (2004); Silverman, "Anti-CD20 therapy in systemic lupus erythematosus: a step closer to the clinic" Arthritis Rheum., 52(2):371 -377 (2005), Erratum in: Arthritis Rheum. 52(4): 1342 (2005); Ahn et al , "Long-term remission from life-threatening hypercoagulable state associated with lupus anticoagulant (LA) following rituximab therapy" Am. J. He atol, 78(2): 127-129 (2005); Tahir et al , "Humanized anti-CD20 monoclonal antibody in the treatment of severe resistant systemic lupus erythematosus in a patient with antibodies against rituximab" Rheumatology, 44(4):561 -562 (2005), Epub 2005 Jan 1 1 ; Looney et al , "Treatment of SLE with anti-CD20 monoclonal antibody" Curr. Dir.

Autoimmun., 8: 193-205 (2005); Cragg et al, "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun., 8: 140-174 (2005); Gottenberg et al , "Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases" Ann. Rheum. Dis., 64(6):913-920 (2005) Epub 2004 Nov 18. See also Leandro et al, "B cell repopulation occurs mainly from na^'ive B cells in patient with rheumatoid arthritis and systemic lupus erythematosus" Arthritis Rheum., 48 (Suppl 9):S 1 160 (2003).

[0027] Specks et al. "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism, 44(12):2836-2840 (2001 ) disclosed successful use of four infusions of 375 mg/m² of rituximab and high-dose glucocorticoids to treat Wegener's granulomatosis. The therapy was repeated after 1 1 months when the cANCA recurred, but therapy was without glucocorticoids. At eight months after the second course of rituximab, the patients' disease remained in complete remission. In another study rituximab was found to be a well-tolerated, effective remission induction agent for severe ANCA-associated vasculitis, when used in a dose of 375 mg/m² x four along with oral prednisone at 1 mg/kg/day, which was reduced to 40 mg/day by week four, and to total discontinuation over the following 16 weeks. Four patients were re-treated with rituximab alone for recurring/rising ANCA titers. Other than glucocorticoids, no additional immunosuppressive agents seem necessary for remission induction and maintenance of sustained remission (six months or longer). Keogh et al, Kidney Blood Press. Res., 26:293 (2003) reported that eleven patients with refractory ANCA-associated vasculitis went into remission upon treatment with four weekly 375 mg/m² doses of rituximab and high-dose glucocorticoids.

[0028] Patients with refractory ANCA-associated vasculitis were administered rituximab along with immunosuppressive medicaments such as intravenous CTX, mycophenolate mofetil, azathioprine, or leflunomide, with apparent efficacy. Eriksson, "Short-term outcome and safety in 5 patients with ANCA-positive vasculitis treated with rituximab" Kidney and Blood Pressure Research, 26:294 (2003) (five patients with ANCA-associated vasculitis treated with rituximab 375 mg/m² once a week for four weeks responded to the treatment); Jayne et al., "B-cell depletion with rituximab for refractory vasculitis" Kidney and Blood Pressure Research, 26:294- 295 (2003) (six patients with refractory vasculitis receiving four weekly infusions of rituximab at 375 mg/m² with CTX along with background immunosuppression and prednisolone experienced major falls in vasculitic activity). A further report of using rituximab along with intravenous CTX at 375 mg/m² per dose in four doses for administering to patients with refractory systemic vasculitis is provided in Smith and Jayne, "A prospective, open label trial of B-cell depletion with rituximab in refractory systemic vasculitis" poster 998 (1 1 ^th International Vasculitis and ANCA workshop), American Society of Nephrology, J. Am. Soc. Nephrol , 14:755A (2003). See also Eriksson, J. Internal Med., 257:540-548 (2005) regarding nine patients with ANCA- positive vasculitis who were successfully treated with two or four weekly doses of 500 mg of rituximab; and Keogh et al, Arthritis and Rheumatism, 52:262-268 (2005), who reported that in 1 1 patients with refractory ANCA-associated vasculitis, treatment or re-treatment with four weekly 375 mg/m² doses of rituximab induced remission by B-lymphocyte depletion (study conducted from Jan. 2000 to Sept. 2002).

[0029] As to the activity of a humanized anti-CD20 antibody, see, for example, Vugmeyster et al, "Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca

fascicularis" J. Immunother., 28:212-219 (2005). For discussion of a human monoclonal antibody, see Baker et al., "Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator," Arthritis Rheum., 48:3253-3265 (2003). The MINT trial with rituximab was successful in treating aggressive non-Hodgkin's lymphoma in younger patients (Pfreundschuh et al, Lancet Oncology, 7(5):379-391 (2006)).

DNA Antigen Specific Therapy

[0030] DNA antigen specific therapy can be used to modulate autoimmune processes.

Following intramuscular injection, plasmid DNA is taken up by, for example, by muscle cells allowing for the expression of the encoded polypeptide (Wolff et al., Hum Mol Genet..1(6): 363- 9 (1992)). In the case of autoimmune disease, the effect is a shift in an ongoing immune response to suppress autoimmune destruction and is believed to include a shift in self-reactive lymphocytes from a Th l - to a Th2-type response. The modulation of the immune response may not be systemic but occur only locally at the target organ under autoimmune attack.

[0031] Methods for treating autoimmune disease using DNA antigen specific therapy, specifically by administering a nucleic acid encoding one or more autoantigens have been described, for example, in International Patent Application Nos. WO 00/53019,

WO 2003/045316, and WO 2004/047734.

BRIEF SUMMARY OF THE INVENTION

[0032] The present invention provides methods and compositions useful in the treatment of autoimmune diseases. The present invention further relates to the treatment of autoimmune diseases comprising the administration of an immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent) in combination with a DNA antigen specific therapeutic agent encoding a self protein associated with the autoimmune disease. In certain embodiments, the present invention provides, for example a combination of a B cell specific agent, such as an antibody, in combination with a DNA antigen specific therapeutic agent, such as a DNA plasmid vector encoding an autoantigen associated the autoimmune disease. In certain embodiments the B cell specific agent is an antibody that targets a receptor on the B cell administered in combination with a DNA antigen specific therapeutic agent encoding a self-protein associated with the autoimmune disease. [0033] Thus, in one aspect, the method of treating an autoimmune disease comprises the administration of a B cell specific agent that specifically binds to certain B cell surface antigens, such as, CD20, CD 19, CD22, TACI, BCMA, or BAFF-R, in combination with a DNA antigen specific therapeutic agent encoding a self protein associated with the autoimmune disease.

Exemplary antibodies include rituximab, ocrelizumab, ofatumumab, and veltuzumab. In a certain embodiment, the method of treating an autoimmune disease comprises the administration of a B cell specific agent that specifically binds to a certain B cell surface antigens, such as CD20, in combination with a DNA antigen specific therapeutic agent encoding a self protein associated with the autoimmune disease, T1 D, such as proinsulin. In certain embodiments the B cell specific agent is an antibody, (e.g. a chimeric, humanized or human antibody). In certain embodiments the DNA antigen specific therapeutic agent encodes an autoantigen associated with the autoimmune disease. Pharmaceutical compositions comprising the B cell specific agent and DNA antigen specific therapeutic agent and methods of using the B cell specific agent and DNA antigen specific therapeutic agent are also provided.

[0034] In another aspect, the invention provides methods for the treatment of autoimmune diseases, such as multiple sclerosis, insulin dependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris or myasthenia gravis by administering to the subject a therapeutically effective amount of an immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent) in combination with a DNA antigen specific therapeutic agent encoding a self-protein.

[0035] In another aspect, the invention provides for the treatment of insulin dependent diabetes mellitus comprising administering to a patient a therapeutically effective amount of an immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent) in combination with a DNA antigen specific therapeutic agent encoding a self-protein. In one embodiment the B cell specific agent is a CD20 specific agent in combination with a DNA antigen specific therapeutic agent encoding an autoantigen associated with insulin dependent diabetes mellitus, such as insulin, proinsulin, glutamic acid decarboxylase 65 or islet cell antigen. In one embodiment the CD20 specific agent is a monoclonal antibody such as Rituxan and the DNA antigen specific therapeutic agent encodes proinsulin. Pharmaceutical compositions comprising the B cell specific agent, such as a CD20 specific agent, and DNA antigen specific therapeutic agent encoding an autoantigen associated with insulin dependent diabetes mellitus and methods of using the B cell specific agent, such as a CD20 specific agent, and DNA antigen specific therapeutic agent encoding an autoantigen associated with insulin dependent diabetes mellitus are also provided.

[0036] In another aspect, the invention provides for the treatment of multiple sclerosis comprising administering to a patient a therapeutically effective amount of an

immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent) in combination with a DNA antigen specific therapeutic agent encoding a self-protein. In one embodiment the B cell specific agent is a CD20 specific agent in combination with a DNA antigen specific therapeutic agent encoding an autoantigen associated with multiple sclerosis, such as myelin basic protein, proteolipid protein, myelin associated glycoprotein, cyclic nucleotide

phosphodiesterase, myelin-associated glycoprotein, myelin-associated oligodendrocytic basic protein; alpha-B-crystalin or myelin oligodendrocyte glycoprotein. In one embodiment the CD20 specific agent is a monoclonal antibody such as Rituxan and the DNA antigen specific therapeutic agent encodes myelin basic protein Pharmaceutical compositions comprising the B cell specific agent, such as a CD20 specific agent, and DNA antigen specific therapeutic agent encoding an autoantigen associated with multiple sclerosis and methods of using the B cell specific agent, such as a CD20 specific agent, and DNA antigen specific therapeutic agent encoding an autoantigen associated with multiple sclerosis are also provided.

[0037] In another aspect, the invention provides for the treatment of an autoimmune disease comprising the administration to a subject of an immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent) followed by administration of the DNA antigen specific therapeutic agent. In one embodiment administration of the DNA antigen specific therapeutic agent is one to several days after administration of the immunosuppressive or

immunomodulatory agent. In another embodiment administration of the DNA antigen specific therapeutic agent is one week after administration of the B cell specific agent. In yet another embodiment, a single administration of the immunosuppressive or immunomodulatory agent is followed by weekly administration of the DNA antigen specific therapeutic agent.

[0038] The invention provides pharmaceutical compositions comprising various combinations of the agents useful in the methods of treatment described herein. The compositions may comprise, in an amount effective for the treatment of an autoimmune disease comprising: (a) an immunosuppressive or immunomodulatory agent (e.g., a B cell specific agent); (b) a DNA antigen specific therapeutic agent; and, (c) a pharmaceutically acceptable carrier. According to certain aspects the pharmaceutical compositions comprise an antibody that binds to CD20 and a DNA antigen specific therapeutic agent encoding a self-protein together with pharmaceutically acceptable carriers.

[0039] In certain embodiments of each of the aforementioned aspects or embodiments, as well as other aspects and/or embodiments described elsewhere herein, the immunosuppressive or immunomodulatory agent is an antibody. In certain embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is isolated. In still further embodiments, the antibody is substantially pure.

[0040] In certain embodiments of each of the aforementioned aspects or embodiments, as well as other aspects and/or embodiments described elsewhere herein, the DNA antigen specific therapeutic agent is a DNA plasmid vector. In certain embodiments, the DNA plasmid vector is isolated. In still further embodiments, the DNA plasmid vector is substantially pure.

[0041] The invention includes kits and articles of manufacture. Kits and articles of

manufacture preferably include:

(a) one or more containers;

(b) a label on each said container; and

(c) a first and second composition comprising an active agent contained within said containers;

wherein the compositions are effective for treating an autoimmune disease, the label on said container may indicate that the composition can be used for treating an autoimmune disease, and the active agent in said first composition comprises an immunosuppressive or immunomodulatory agent and the active agent in said second composition comprises a DNA antigen specific therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Anti-CD20 administration alone can induce protection from diabetes in NOD mice. Prediabetic 7-10 week-old NOD mice were injected with 5, 10, 50, 100 or 250 μg of anti- CD20 antibody (5D2) either only on day 1 (Fig. 1 A) or on days 1 , 2, 3, and 4 (Fig. I B). Progression of NOD mice to diabetes was monitored by measuring blood glucose values every week, and the mice were considered diabetic when two consecutive BG values were >200 mg/dL. While the highest dose of lx anti-CD20 (250 μg) appears to mediate 50% protection, all the mice that received 4x anti-CD20 injections appear to be protected from diabetes onset.

[0043] Figure 2: Combining anti-CD20 administration with DNA self-vector encoding proinsulin administration protects NOD mice from diabetes. Prediabetic 7-10 week-old NOD mice were injected with (Fig. 2A) 10, (Fig. 2B) 50, or (Fig. 2C) 100 μg of anti-CD20 antibody (5D2) either alone or in combination with a plasmid expressing proinsulin, either l x (dl ) or 4x (dl , 8, 15, 22). Once a week, blood glucose values were determined. While administration of 10μg anti-CD20 in combination with proinsulin plasmid did not offer protection, 50 μg and 100 μg anti-CD20 antibody combined with either l x or 4x proinsulin plasmid did protect these mice from diabetes onset, in a dose dependent manner.

[0044] Figure 3: Prediabetic 7-10 week-old NOD mice were injected with either (A) anti- CD20 alone, or (B) in combination with 50 μg Proinsulin plasmid, at indicated doses. Untreated NOD mice were used as controls to monitor diabetes development. Once a week, blood glucose values were determined.

[0045] Figure 4: Prediabetic 7-10 week-old NOD mice were injected with 25^g plasmid in each thigh (total 50^g) at weekly intervals, in combination with anti-CD20 administration (doses indicated in Figure legend) as follows; (A) single dose of anti-CD20 and Proinsulin plasmid, or (B) four doses of anti-CD20 (d l , 2, 3, 4) and single dose of Proinsulin plasmid, or (C) single dose of anti-CD20 and four doses of Proinsulin plasmid. Untreated NOD mice were used as controls to monitor diabetes development. Once a week, blood glucose values were determined.

[0046] Figure 5. Rats receiving rBHT-3034/Solu-medrol combination therapy show a statistically significant reduction in disease scores relative to the PBS treated control group and the rBHT-3034 and Solu-Medrol monotherapy groups.

DETAILED DESCRIPTION OF THE INVENTION A UTOIMMUNE DISEASES

[0047] Several examples of autoimmune diseases associated autoantigens are set forth 2, and particular examples are described in further detail hereinbelow.

Table 2. Exemplary Autoimmune Diseases and Associated Autoantigens

Autoimmune Tissue Targeted Autoantigen(s) Associated with the

Disease Autoimmune Disease

Multiple central nervous system myelin basic protein, proteolipid protein, sclerosis myelin associated glycoprotein, cyclic

nucleotide phosphodiesterase, myelin- associated glycoprotein, myelin-associated oligodendrocytic basic protein, myelin oligodendrocyte glycoprotein, alpha-B- cry stalin

Guillian Barre peripheral nervous peripheral myelin protein I and others

Syndrome system

Type 1 Diabetes Dependent β cells in tyrosine phosphatase IA2, ΙΑ-2β; glutamic acid islets of pancreas decarboxylase (65 and 67 kDa forms),

carboxypeptidase H, insulin, proinsulin, pre- proinsulin, heat shock proteins, glima 38, islet cell antigen 69 KDa, p52, islet cell glucose transporter GLUT-2

Rheumatoid synovial joints Immunoglobulin, fibrin, filaggrin, type I, II, III, Arthritis IV, V, IX, and XI collagens, GP-39, hnRNPs

Autoimmune eye, uvea S-antigen, interphotoreceptor retinoid binding Uveitis protein (IRBP), rhodopsin, recoverin

Primary Biliary biliary tree of 1 pyruvate dehydrogenase complexes (2-oxoacid Cirrhosis dehydrogenase)

Autoimmune Liver Hepatocyte antigens, cytochrome P450 Hepatitis

Pemphigus Skin Desmoglein-1 , -3, and others

vulgaris

Myasthenia nerve-muscle junct. acetylcholine receptor

Gravis

Autoimmune stomach/parietal cells H⁺/K⁺ ATPase, intrinsic factor

gastritis

Pernicious Stomach intrinsic factor

Anemia

Polymyositis Muscle histidyl tRNA synthetase, other synthetases, Autoimmune Tissue Targeted Autoantigen(s) Associated with the Disease Autoimmune Disease

other nuclear antigens

Autoimmune Thyroid Thyroglobulin, thyroid peroxidase

Thyroiditis

Graves's Disease Thyroid Thyroid-stimulating hormone receptor

Psoriasis Skin Unknown

Vitiligo Skin Tyrosinase, tyrosinase-related protein-2

Systemic Lupus Systemic nuclear antigens: DNA, histones,

Eryth. ribonucleoproteins

Celiac Disease Small bowel Transglutaminase

[0048] Type 1 Diabetes. Type 1 diabetes (T1D) (sometimes also referred to as human type I or insulin-dependent diabetes mellitus (IDDM)) is characterized by autoimmune destruction of the β cells in the pancreatic islets of Langerhans. The depletion of β cells results in an inability to regulate levels of glucose in the blood. Overt diabetes occurs when the level of glucose in the blood rises above a specific level, usually about 250 mg/dl. In humans a long presymptomatic period precedes the onset of diabetes. During this period there is a gradual loss of pancreatic beta cell function. The development of disease is implicated by the presence of autoantibodies against insulin, glutamic acid decarboxylase, and the tyrosine phosphatase IA2 (IA2).

[0049] Markers that may be evaluated during the presymptomatic stage are the presence of insulitis in the pancreas, the level and frequency of islet cell antibodies, islet cell surface antibodies, aberrant expression of Class II MHC molecules on pancreatic beta cells, glucose concentration in the blood, and the plasma concentration of insulin. An increase in the number of T lymphocytes in the pancreas, islet cell antibodies and blood glucose is indicative of the disease, as is a decrease in insulin concentration.

[0050] The Non-Obese Diabetic (NOD) mouse is an animal model with many clinical, immunological, and histopathological features in common with human T1 D. NOD mice spontaneously develop inflammation of the islets and destruction of the β cells, which leads to hyperglycemia and overt diabetes. Both CD4⁺ and CD8⁺ T cells are required for diabetes to develop, although the roles of each remain unclear. It has been shown that administration of insulin or GAD, as proteins, under tolerizing conditions to NOD mice prevents disease and down-regulates responses to the other autoantigens.

[0051] The presence of combinations of autoantibodies with various specificities in serum are highly sensitive and specific for human type I diabetes mellitus. For example, the presence of autoantibodies against GAD and/or IA-2 is approximately 98% sensitive and 99% specific for identifying type I diabetes mellitus from control serum. In non-diabetic first degree relatives of type I diabetes patients, the presence of autoantibodies specific for two of the three autoantigens including GAD, insulin and IA-2 conveys a positive predictive value of >90% for development of type IDM within 5 years.

[0052] Autoantigens targeted in human insulin dependent diabetes mellitus may include, for example, tyrosine phosphatase IA-2; ΙΑ-2β; glutamic acid decarboxylase (GAD) both the 65 kDa and 67 kDa forms; carboxypeptidase H; insulin; proinsulin (e.g., SEQ ID NOs: 1 and 2) ; heat shock proteins (HSP); glima 38; islet cell antigen 69 KDa (ICA69); p52; two ganglioside antigens (GT3 and GM2-1 ); islet-specific glucose-6-phosphatase-related protein (IGRP); and an islet cell glucose transporter (GLUT 2).

[0053] Human T1 D is currently treated by monitoring blood glucose levels to guide injection, or pump-based delivery, of recombinant insulin. Diet and exercise regimens contribute to achieving adequate blood glucose control.

[0054] Multiple Sclerosis. Multiple sclerosis (MS) is the most common demyelinating disorder of the CNS and affects 350,000 Americans and one million people worldwide. Onset of symptoms typically occurs between 20 and 40 years of age and manifests as an acute or subacute attack of unilateral visual impairment, muscle weakness, paresthesias, ataxia, vertigo, urinary incontinence, dysarthria, or mental disturbance (in order of decreasing frequency). Such symptoms result from focal lesions of demyelination which cause both negative conduction abnormalities due to slowed axonal conduction, and positive conduction abnormalities due to ectopic impulse generation (e.g. , Lhermitte's symptom). Diagnosis of MS is based upon a history including at least two distinct attacks of neurologic dysfunction that are separated in time, produce objective clinical evidence of neurologic dysfunction, and involve separate areas of the CNS white matter. Laboratory studies providing additional objective evidence supporting the diagnosis of MS include magnetic resonance imaging (MRI) of CNS white matter lesions, cerebral spinal fluid (CSF) oligoclonal banding of IgG, and abnormal evoked responses. Although most patients experience a gradually progressive relapsing remitting disease course, the clinical course of MS varies greatly between individuals and can range from being limited to several mild attacks over a lifetime to fulminant chronic progressive disease. A quantitative increase in myelin-autoreactive T cells with the capacity to secrete IFN-gamma is associated with the pathogenesis of MS and EAE.

[0055] The autoantigen targets of the autoimmune response in autoimmune demyelinating diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), may comprise epitopes from proteolipid protein (PLP); myelin basic protein (MBP); myelin oligodendrocyte glycoprotein (MOG); cyclic nucleotide phosphodiesterase (CNPase); myelin- associated glycoprotein (MAG), and myelin-associated oligodendrocytic basic protein (MBOP); alpha-B-crystalin (a heat shock protein); viral and bacterial mimicry peptides, e.g., influenza, herpes viruses, hepatitis B virus, etc.; OSP (oligodendrocyte specific-protein); citrulline- modified MBP (the C8 isoform of MBP in which 6 arginines have been de-imminated to citrulline), etc. The integral membrane protein PLP is a dominant autoantigen of myelin.

Determinants of PLP antigenicity have been identified in several mouse strains, and include residues 139-151 , 103-1 16, 215-232, 43-64 and 178-191 . At least 26 MBP epitopes have been reported (Meinl et al., J Clin Invest 92, 2633-43, 1993). Notable are residues 1 -1 1 , 59-76 and 87-99. Immunodominant MOG epitopes that have been identified in several mouse strains include residues 1 -22, 35-55, 64-96.

[0056] In human MS patients the following myelin proteins and epitopes were identified as targets of the autoimmune T and B cell response. Antibody eluted from MS brain plaques recognized myelin basic protein (MBP) peptide 83-97 (Wucherpfennig et al., J Clin Invest 100: 1 1 14-1 122, 1997). Another study found approximately 50% of MS patients having peripheral blood lymphocyte (PBL) T cell reactivity against myelin oligodendrocyte

glycoprotein (MOG) (6-10% control), 20% reactive against MBP (8-12% control), 8% reactive against PLP (0% control), 0% reactive MAG (0% control). In this study 7 of 10 MOG reactive patients had T cell proliferative responses focused on one of 3 peptide epitopes, including MOG 1 -22, MOG 34-56, MOG 64-96 ( erlero de Rosbo et al , Eur J Immunol 27, 3059-69, 1997). T and B cell (brain lesion-eluted Ab) response focused on MBP 87-99 (Oksenberg et al, Nature 362, 68-70, 1993). In MBP 87-99, the amino acid motif HFFK is a dominant target of both the T and B cell response (Wucherpfennig et al., J Clin Invest 100, 1 1 14-22, 1997). Another study observed lymphocyte reactivity against myelin-associated oligodendrocytic basic protein (MOBP), including residues MOBP 21 -39 and MOBP 37-60 (Holz et al , J Immunol 164, 1 103- 9, 2000). Using immunogold conjugates of MOG and MBP peptides to stain MS and control brains both MBP and MOG peptides were recognized by MS plaque-bound Abs (Genain and Hauser, Methods 10, 420-34, 1996).

[0057] Rheumatoid Arthritis. Rheumatoid arthritis (RA) is a chronic autoimmune

inflammatory synovitis affecting 0.8% of the world population. It is characterized by chronic inflammatory synovitis that causes erosive joint destruction. RA is mediated by T cells, B cells and macrophages.

[0058] Evidence that T cells play a critical role in RA includes the (1) predominance of CD4⁺. T cells infiltrating the synovium, (2) clinical improvement associated with suppression of T cell function with drugs such as cyclosporine, and (3) the association of RA with certain HLA-DR alleles. The HLA-DR alleles associated with RA contain a similar sequence of amino acids at positions 67-74 in the third hypervariable region of the β chain that are involved in peptide binding and presentation to T cells. RA is mediated by autoreactive T cells that recognize a self- protein, or modified self-protein, present in synovial joints. Autoantigens that are targeted in RA comprise, e.g., epitopes from type II collagen; hnRNP; A2/RA33; Sa; filaggrin; keratin;

citrulline; cartilage proteins including gp39; collagens type I, III, IV, V, IX, XI; HSP-65/60; IgM (rheumatoid factor); RNA polymerase; hnRNP-B l ; hnRNP-D; cardiolipin; aldolase A; citrulline- modified filaggrin and fibrin. Autoantibodies that recognize filaggrin peptides containing a modified arginine residue (de-iminated to form citrulline) have been identified in the serum of a high proportion of RA patients. Autoreactive T and B cell responses are both directed against the same immunodominant type II collagen (CII) peptide 257-270 in some patients.

[0059] Autoimmune Uveitis. Autoimmune uveitis is an autoimmune disease of the eye that is estimated to affect 400,000 people, with an incidence of 43,000 new cases per year in the U.S. Autoimmune uveitis is currently treated with steroids, immunosuppressive agents such as methotrexate and cyclosporin, intravenous immunoglobulin, and TNFa-antagonists.

Experimental autoimmune uveitis (EAU) is a T cell-mediated autoimmune disease that targets neural retina, uvea, and related tissues in the eye. EAU shares many clinical and immunological features with human autoimmune uveitis, and is induced by peripheral administration of uveitogenic peptide emulsified in Complete Freund's Adjuvant (CFA). Autoantigens targeted by the autoimmune response in human autoimmune uveitis may include S-antigen, interphotoreceptor retinoid binding protein (IRBP), rhodopsin, and recoverin.

[0060] Primary Billiary Cirrhosis. Primary Biliary Cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women between 40-60 years of age. The prevalence reported among this group approaches 1 per 1 ,000. PBC is characterized by progressive destruction of intrahepatic biliary epithelial cells (IBEC) lining the small intrahepatic bile ducts. This leads to obstruction and interference with bile secretion, causing eventual cirrhosis. Association with other autoimmune diseases characterized by epithelium lining /secretory system damage has been reported, including Sjogren's Syndrome, CREST Syndrome, Autoimmune Thyroid Disease and Rheumatoid Arthritis. Attention regarding the driving antigen(s) has focused on the mitochondria for over 50 years, leading to the discovery of the antimitochondrial antibody (AM A) (Gershwin et al, Immunol Rev 174:210-225, 2000); (Mackay et al, Immunol Rev 174:226-237, 2000). AMA soon became a cornerstone for laboratory diagnosis of PBC, present in serum of 90-95% patients long before clinical symptoms appear. Autoantigenic reactivities in the mitochondria were designated as Ml and M2. M2 reactivity is directed against a family of components of 48-74 kDa. M2 represents multiple autoantigenic subunits of enzymes of the 2-oxoacid dehydrogenase complex (2-OADC) and is another example of the self-protein, -polypeptide, or -peptide of the instant invention. Studies identifying the role of pyruvate dehydrogenase complex (PDC) antigens in the etiopathogenesis of PBC support the concept that PDC plays a central role in the induction of the disease (Gershwin et al, Immunol Rev 174:210-225, 2000); (Mackay et al, Immunol Rev 174:226-237, 2000). The most frequent reactivity in 95% of cases of PBC is the E2 74 kDa subunit, belonging to the PDC-E2. There exist related but distinct complexes including: 2-oxoglutarate dehydrogenase complex (OGDC) and branched-chain (BC) 2-OADC. Three constituent enzymes (El , 2,3) contribute to the catalytic function which is to transform the 2-oxoacid substrate to acyl co-enzyme A (CoA), with reduction of NAD⁺ to NADH. Mammalian PDC contains-an additional component, termed protein X or E-3 Binding protein: (E3BP). In PBC patients, the, major antigenic response is directed against PDC-E2 and E3BP. The E2 polypeptide contains two tandemly repeated lipoyl domains, while E3BP has a single lipoyl domain. The lipoyl domain is found in a number of autoantigen targets of PBC and is referred to herein as the "PBC lipoyl domain." PBC is treated with glucocorticoids and immunosuppressive agents including methotrexate and cyclosporin A. [0061] A murine model of experimental autoimmune cholangitis (EAC) uses intraperitoneal (i.p.) sensitization with mammalian PDC in female SJL/J mice, inducing non-suppurative destructive cholangitis (NSDC) and production of AMA (Jones, J Clin Pathol 53:813-21 , 2000).

[0062] Other A utoimmune Diseases And Associated A utoantigens. Autoanti gens for myasthenia gravis may include epitopes within the acetylcholine receptor. Autoantigens targeted in pemphigus vulgaris may include desmoglein-3. Sjogren's syndrome antigens may include SSA (Ro); SSB (La); and fodrin. The dominant autoantigen for pemphigus vulgaris may include desmoglein-3. Panels for myositis may include tRNA synthetases {e.g., threonyl, histidyl, alanyl, isoleucyl, and glycyl); Ku; Scl; SSA; Ul Sn ribonuclear protein; Mi-1 ; Mi- 1 ; Jo-1 ; Ku; and SRP. Panels for scleroderma may include Scl-70; centromere; U l ribonuclear proteins; and fibrillarin. Panels for pernicious anemia may include intrinsic factor; and glycoprotein beta subunit of gastric H/K ATPase. Epitope Antigens for systemic lupus erythematosus (SLE) may include DNA; phospholipids; nuclear antigens; Ro; La; Ul ribonucleoprotein; Ro60 (SS-A); Ro52 (SS-A); La (SS-B); calreticulin; Grp78; Scl-70; histone; Sm protein; and chromatin, etc. For Grave's disease epitopes may include the Na+/I- symporter; thyrotropin receptor; Tg; and TPO.

[0063] Graft Versus Host Disease. One of the greatest limitations of tissue and organ transplantation in humans is rejection of the tissue transplant by the recipient's immune system. It is well established that the greater the matching of the MHC class I and II (HLA-A, HLA-B, and HLA-DR) alleles between donor and recipient the better the graft survival. Graft versus host disease (GVHD) causes significant morbidity and mortality in patients receiving transplants containing allogeneic hematopoietic cells. Hematopoietic cells are present in bone-marrow transplants, stem cell transplants, and other transplants. Approximately 50% of patients receiving a transplant from a HLA-matched sibling will develop moderate to severe GVHD, and the incidence is much higher in non-HLA -matched grafts. One-third of patients that develop moderate to severe GVHD will die as a result. T lymphocytes and other immune cell in the donor graft attack the recipients' cells that express polypeptides variations in their amino acid sequences, particularly variations in proteins encoded in the major histocompatibility complex (MHC) gene complex on chromosome 6 in humans. The most influential proteins for GVHD in transplants involving allogeneic hematopoietic cells are the highly polymorphic (extensive amino acid variation between people) class I proteins (HLA-A, -B, and -C) and the class II proteins (DRB 1 , DQB 1 , and DPB 1 ) (Appelbaum, Nature 41 1 :385-389, 2001 ). Even when the MHC class I alleles are serologically 'matched' between donor and recipient, DNA sequencing reveals there are allele-level mismatches in 30% of cases providing a basis for class I-directed GVHD even in matched donor-recipient pairs (Appelbaum, Nature 41 1 , 385-389, 2001). The minor histocompatibility self-antigens GVHD frequently causes damage to the skin, intestine, liver, lung, and pancreas. GVHD is treated with glucocorticoids, cyclosporine, methotrexate, fludarabine, and OKT3.

[0064] Tissue Transplant Rejection. Immune rejection of tissue transplants, including lung, heart, liver, kidney, pancreas, and other organs and tissues, is mediated by immune responses in the transplant recipient directed against the transplanted organ. Allogeneic transplanted organs contain proteins with variations in their amino acid sequences when compared to the amino acid sequences of the transplant recipient. Because the amino acid sequences of the transplanted organ differ from those of the transplant recipient they frequently elicit an immune response in the recipient against the transplanted organ. Rejection of transplanted organs is a major complication and limitation of tissue transplant, and can cause failure of the transplanted organ in the recipient. The chronic inflammation that results from rejection frequently leads to dysfunction in the transplanted organ. Transplant recipients are currently treated with a variety of immunosuppressive agents to prevent and suppress rejection. These agents include glucocorticoids, cyclosporin A, Cellcept, FK-506, and OKT3.

IMMUNOSUPPRESSIVE OR IMMUNOMODULATORY AGENTS

[0065] As used herein an "immunsuppressive or immunomodulatory agent" is an agent that generally or specifically suppresses or modulates a mammalian immune response. Such agents may be, for example, a small molecule or a biological therapeutic {e.g. an inhibitory nucleic acid or a protein, such as a soluble receptor or antibody). Exemplary immunosuppressive agents include glucocorticoids {e.g., methylprednisolone), cytotoxic agents, corticosteroids {e.g., prednisone), cyclophosphamide, cyclosporine, tacrolimus, methotrexate, cladribine, mycophenolate mofetil, and the like. Immunomodulatory agents include antibodies or other proteins that affect the action of lymphocytes (B cells or T cells) or cytokines, such as TNFa {e.g., etanercept and infliximab). As explained below, in a typical embodiment, the

immunomodulatory agent is a B cell specific agent. B Cell Specific Agents

[0066] A "B cell" is a lymphocyte that matures within the bone marrow, and includes a naive B cell, memory B cell, or effector B cell (plasma cells). The B cell herein is a normal or non- malignant B cell.

[0067] A "B-cell surface marker" or "B-cell surface antigen" herein is an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds thereto. Exemplary B- cell surface markers include the CD 10, CD 19, 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 (for descriptions, see The Leukocyte Antigen Facts Book, 2^nd Edition. 1997, ed. Barclay et al. 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, BAFF, BLyS, Btig, NAG 14, SLGC 16270, FcRHl , IRTA2, ATWD578, FcRH3, IRTA 1 , FcRH6, BCMA, and 239287. The B-cell surface marker of particular 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. The preferred B-cell surface markers herein are CD20, CD22, CD23, CD40, BR3, BLyS, and BAFF.

[0068] The "CD20" antigen, or "CD20," is an about 35-kDa, non-glycosylated phosphoprotein found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on normal B cells and is not expressed on stem cells. Other names for CD20 in the literature include "B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is described in Clark et al., Proc. Natl. Acad. Sci. (USA), 82: 1766 (1985), for example.

[0069] The "CD22" antigen, or "CD22," also known as BL-CAM or Lyb8, is a type 1 integral membrane glycoprotein with molecular weight of about 130 (reduced) to 140kD (unreduced). It is expressed in both the cytoplasm and cell membrane of B-lymphocytes. CD22 antigen appears early in B-cell lymphocyte differentiation at approximately the same stage as the CD19 antigen. Unlike other B-cell markers, CD22 membrane expression is limited to the late differentiation stages comprised between mature B cells (CD22+) and plasma cells (CD22-). The CD22 antigen is described, for example, in Wilson et al, J. Exp. Med., 173: 137 (1991) and Wilson et al, J. Immunol. 150:5013 (1993).

[0070] As used herein, the terms "B cell specific agent" and "an agent that targets B cells" refer to an immunomodulatory agent that specifically targets a B cell by modifying, altering, reducing or inhibiting B cell function, e.g. by reducing or preventing a humoral response elicited by the B cell. The B cell specific agent preferably is able to deplete B cells {i.e. reduce circulating B-cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such as ADCC and/or CDC, inhibition of B-cell proliferation and/or induction of B-cell death {e.g. via apoptosis) or inhibiting or interfering with B cell

differentiation. Thus, an agent of the invention may be described as "an agent that arrests the growth of B cells" "a B cell growth inhibitory agent," "an agent that arrests B cell

differentiation" or " a B cell differentiation inhibitory agent." The nature of the agent used is not critical to the invention and can be, for example, a polypeptide {e.g., antibodies or antibody fragments), a nucleic acid {e.g., inhibitory nucleic acids such as siRNA) or a small molecule. In some embodiments, the B cells targeted by the invention are B cells expressing CD20. B cell specific agent(s) can be screened by various methods known in the art for apoptosis and other measurements for the depletion, and retardation or stopping of proliferation and growth of B cells or survival of B cells. Exemplary antibodies include rituximab, ocrelizumab, ofatumumab, and veltuzumab, all of which are described in the prior art.

[0071] For example, a method of screening can be employed as described in Sundberg et al, Cancer Research 66, 1775-1782 (2006) wherein a compound was screened for inhibition of B- cell proliferation by targeting c-myc protein for rapid and specific degradation. See also Mackay et al., Annual Review of Immunology, 21 : 231 -264 (2003) regarding BAFF, APRIL, and a tutorial on B-cell survival and screening, and Thangarajh et al., Scandinavian J. Immunol, 65(1 ):92 (2007) on B-cell proliferation and APRIL. In addition, Sakurai et al, European J. Immunol, 37(1 ): 1 10 (2007) discloses that TACI attenuates antibody production co-stimulated by BAFF-R and CD40. Further, Acosta-Rodriguez et al, European J. Immunol, 37(4):990 (2007) discloses that BAFF and LPS cooperate to induce B cells to become susceptible to CD95/F as- mediated cell death. Further screening methods can be found in Martin and Chan, "B Cell Immunobiology in Disease: Evolving Concepts from the Clinic Annual Review of

Immunology," 24:467-496 (2006), Pillai et al, "Marginal Zone B Cells" Annual Review of Immunology, 23: 161 - 196 (2005), and Hardy and Hayakawa, "B Cell Development Pathways," Annual Review of Immunology, 19:595-621 (2001 ). From these and other references the skilled artisan can screen for the appropriate antagonists. Microarrays can be used for this purpose (Hagmann, Science, 290:82-83 (2000)), as well as RNA interference (RNAi) (Ngo et al, Nature, 441 : 106- 1 10 (2006)). [0072] B cell specific agents included within the scope of the present invention include antibodies, synthetic or native-sequence peptides, immunoadhesins, and small-molecule antagonists that bind to a B-cell surface marker or a B-cell specific survival or proliferation factor, optionally conjugated with or fused to another molecule. The preferred B cell specific agent comprises an antibody or immunoadhesin. It includes BLyS antagonists such as immunoadhesins, and is preferably anti-CD23 (e.g., lumiliximab), anti-CD20, anti-CD22, or anti-BR3 antibodies, APRIL antagonists, and/or BLyS immunoadhesins. The BLyS

immunoadhesin preferably is selected from the group consisting of BR3 immunoadhesin comprising the extracellular domain of BR3, TACI immunoadhesin comprising the extracellular domain of TACI, and BCMA immunoadhesin comprising the extracellular domain of BCMA. The most preferred BR3 immunoadhesin is hBR3-Fc of SEQ ID NO:2 of WO 2005/00351 and US 2005/0095243. See also US 2005/0163775 and WO 2006/068867. Another preferred BLyS B cell specific agent is an anti-BLyS antibody, more preferably wherein the anti-BLyS antibody binds BLyS within a region of BLyS comprising residues 162-275, or an anti-BR3 antibody, more preferably wherein the anti-BR3 antibody binds BR3 in a region comprising residues 23-38 of human BR3. Especially preferred immunoadhesins herein are TACI-Ig, or atacicept, and BR3-Ig. A preferred set of B cell specific agents are to CD20, CD22, BAFF, or APRIL. The B cell specific agent may be, in one aspect, an antibody or TACI-Ig.

[0073] The term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0074] An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1 ) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0075] "Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0076] An "antibody B cell specific agent" or "antibody antagonist" herein is an antibody that, upon binding to a B-cell surface marker on B cells, destroys or depletes B cells in a mammal and/or interferes with one or more B-cell functions, e.g., by reducing or preventing a humoral response elicited by the B cell. The antibody antagonist preferably is able to deplete B cells (i.e., reduce circulating B-cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such as ADCC and/or CDC, inhibition of B-cell proliferation and/or induction of B-cell death (e.g., via apoptosis).

[0077] An "antibody that binds to a B-cell surface marker" or "antibody to a B-cell surface marker" is a molecule that, upon binding to a B-cell surface marker, destroys or depletes B cells in a mammal and/or interferes with one or more B-cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The antibody preferably is able to deplete B cells (i.e. reduce circulating B-cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), inhibition of B-cell proliferation, and/or induction of B-cell death (e.g. via apoptosis). The antibody that binds to a B-cell surface marker may be designated as follows: an antibody that binds to CD20 or CD22 is an "anti-CD20 antibody" or "anti-CD22 antibody," respectively. In one embodiment, the antibody is an anti-CD20, anti- CD22, anti-CD23, or anti-BR3 antibody. Another embodiment typically used is an antibody that is an anti-CD20, anti-CD22, or anti-BR3 antibody. Another typically used embodiment is an anti-CD20 or anti-CD22 antibody, and more, frequently used embodiment is the antibody that is an anti-CD20 antibody.

[0078] Examples of anti-CD20 antibodies include: "C2B8," which is now called "rituximab" ("RITUXAN®/MABTHERA®") (U.S. 5,736,137); the yttrium-[90]-labelled 2B8 murine antibody designated "Y2B8" or "Ibritumomab Tiuxetan" (ZEVALIN®) commercially available from Biogen Idee, Inc. (e.g., U.S. 5,736,137; 2B8 deposited with ATCC under accession no. HB1 1388 on June 22, 1993); murine IgG2a "Bl ," also called "Tositumomab," optionally labelled with ¹³¹1 to generate the "131 I-B1 " or "iodine 1131 tositumomab" antibody

(BEXXAR™) commercially available from Corixa (see, also, e.g., U.S. 5,595,721); murine monoclonal antibody "1F5" (e.g., Press et al. Blood 69(2):584-591 (1987) and variants thereof including "framework patched" or humanized 1F5 (e.g., WO 2003/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (e.g., U.S. 5,677,180); a 2H7 antibody (e.g., WO 2004/056312 (Lowman et al.) and as set forth below); HUMAX-CD20™ (ofatumumab) fully human, high-affinity antibody targeted at the CD20 molecule in the cell membrane of B-cells (Genmab, Denmark; see, for example, Glennie and van de Winkel, Drug Discovery Today 8: 503-510 (2003) and Cragg et al., Blood 101 : 1045-1052 (2003)); the human monoclonal antibodies set forth in WO 2004/035607 and WO 2005/103081 (Teeling et al, GenMab/Medarex); the antibodies having complex N-glycoside-linked sugar chains bound to the Fc region described in US 2004/0093621 (Shitara et al.); a chimerized or humanized monoclonal antibody having a high binding affinity to an extracellular epitope of a CD20 antigen described in WO 2006/106959 (Numazaki et al, Biomedics Inc.); monoclonal antibodies and antigen- binding fragments binding to CD20 (e.g., WO 2005/000901 , Tedder et al.) such as HB20-3, HB20-4, HB20-25, and MB20-1 1 ; single-chain proteins binding to CD20 including, but not limited to, TRU-015 (e.g., US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US

2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al); US 2005/0202023 (Hayden-Ledbetter and Ledbetter) - Trubion Pharm Inc.); CD20-binding molecules such as the A E series of antibodies, e.g., AME-133™ antibodies as set forth, for example, in WO 2004/103404; US 2005/0025764; and US 2006/0251652 (Watkins et al, Applied Molecular Evolution, Inc.) and the anti-CD20 antibodies with Fc mutations as set forth, for example, in WO 2005/070963 (Allan et al, Applied Molecular Evolution, Inc.); CD20-binding molecules such as those described in WO 2005/016969 and US 2005/0069545 (Carr et al); bispecific antibodies as set forth, for example, in WO 2005/014618 (Chang et al.); humanized LL2 monoclonal antibodies and other anti-CD20 antibodies as described, for example, in U.S. 7, 151 ,164 (Hansen et al.,

Immunomedics; US 2005/0106108 (Leung and Hansen; Immunomedics); fully human antibodies against CD20 as described, e.g., in WO 2006/130458; Gazit et al,

Amgen/AstraZeneca); antibodies against CD20 as described, for example, in WO 2006/126069 (Morawala, Avestha Gengraine Technologies Pvt Ltd.); chimeric or humanized B-Lyl antibodies to CD20 {e.g., GA-101 ) as described, for example, in WO 2005/044859; US 2005/0123546; US 2004/0072290; and US 2003/0175884 (Umana et al; GlycArt Biotechnology AG); A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) and IMMUN-106 {e.g., US 2003/0219433, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1 B3, B-C l or NU-B2 available from the International Leukocyte Typing Workshop {e.g., Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)). The preferred anti-CD20 antibodies herein are chimeric, humanized, or human anti-CD20 antibodies, more preferably rituximab, a 2H7 antibody, chimeric or humanized A20 antibody (Immunomedics), and HUMAX-CD20™ human anti- CD20 antibody (Genmab).

[0079] Examples of anti-CD22 antibodies include those described in EP 1 ,476,120 (Tedder and Tuscano), EP 1 ,485,130 (Tedder), and EP 1 ,504,035 (Popplewell et al), as well as those described in US 2004/0258682 (Leung et al), U.S. 5,484,892 (Dana-Farber), U.S. 6, 183,744 (Immunomedics, epratuzumab), and U.S. 7,074,403 (Goldenberg and Hansen).

[0080] Preferred specific examples of antibodies to B-cell surface markers include rituximab, a 2H7 antibody and variants thereof as defined herein, 2F2 (HUMAX-CD20™) (ofatumumab) human anti-CD20 antibody (an IgG l κ human MAb that binds to a different CD20 epitope than rituximab), humanized A20 antibody veltuzumab (IMMUN-106™ or hA20), a humanized engineered antibody with complementarity-determining regions (CDRs) of murine origin and with 90% of the human framework regions identical to epratuzumab (a humanized anti-CD22 IgG l antibody); a small, modular immunopharmaceutical (SMIP) (herein called

immunopharmaceutical), and anti-CD22 antibody such as epratuzumab, ABIOGEN™ anti-CD22 antibody, and IMPHERON™ anti-B cell antibody. [0081] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen- binding sites.

[0082] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., N1H Publ. No.91 -3242, Vol. I, 647-669 [1991 ]). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity.

[0083] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0084] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG l , IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0085] It may be desirable to modify the antibody of the invention with respect to effector function, e.g. so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may 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: 1 191 -1 195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 ( 1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design

3:219-230 (1989).

[0086] To identify antibodies within the context of the present invention directed against CD20 such as growth inhibitory or differentiation inhibitory antibodies, one may screen for antibodies which inhibit the differentiation of B cells which express CD20.

[0087] In a preferred embodiment the agents which arrests the growth of, destroys or causes the deletion of cells expressing CD20 are antibodies. A description follows as to exemplary techniques for the production of the preferred antibodies used in accordance with the present invention.

[0088] The CD20 antigen used for production of antibodies may be, e.g., a soluble form of the extracellular domain of CD20 or a portion thereof, containing the desired epitope. Alternatively, cells expressing CD20 at their cell surface can be used to generate antibodies. Other forms of antigen useful for generating antibodies will be apparent to those skilled in the art.

(Tj Polyclonal antibodies

[0089] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl

sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, S0C12, or R1N=C=NR, where R and Rl are different alkyl groups.

[0090] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1 /10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

(ii) Monoclonal antibodies

[0091] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et a!., J. Mol. Biol, 222:581 -597 (1991 ), for example. [0092] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14(3):253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2^nd ed. 1988); Hammerling et al , in: Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991 ); Marks et al, J. Mol. Biol, 222:581 -597 (1992); Sidhu et al, J. Mol. Biol, 338(2):299-310 (2004); Lee et al, J. Mol. Biol, 340(5): 1073- 1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA, 101 (34): 12467- 12472 (2004); and Lee et al, J. Immunol Methods, 284(1 -2): 1 19-132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g. , WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741 ; Jakobovits et al. , Proc. Natl. Acad. Sci. USA, 90: 2551 ( 1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggemann et al, Year in Immunol, 7:33 (1993); U.S. 5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; and 5,661 ,016; Marks et al, Bio/Technology, 10: 779-783 (1992); Lonberg et al, Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al, Nature Biotechnol, 14:845-851 (1996); Neuberger, Nature Biotechnol, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol, 13 :65-93 (1995).

[0093] The monoclonal antibodies herein specifically include "chimeric" antibodies

(immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855 [ 1984]).

[0094] For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 ( 1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567). [0095] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)).

[0096] The hybridoma cells thus prepared are 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 substances prevent the growth of HGPRT-deficient cells.

[0097] 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 medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland 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); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987)).

[0098] 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).

[0099] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980). [0100] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPM1-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

[0101] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs., 130: 151 - 188 (1992).

[0103] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., J. Mol. Biol, 222:581 -597 (1991 ) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology,

10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. [0104] The DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0105] Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one

antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

(iii) Humanized antibodies

[0106] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric

immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human

immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 :522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 ( 1992). The humanized antibody includes a PRIMATIZED^® antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. [0107] Methods for humanizing non-human antibodies have been described in the art.

Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0108] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, 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 which 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. Mol. 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 chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993)).

[0109] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products 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 which 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, 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.

[0110] Various forms of the humanized or affinity matured antibody are contemplated. For example, the humanized or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized or affinity matured antibody may be an intact antibody, such as an intact IgG l antibody.

(iv) Human antibodies

[0111] A "human antibody" is one which possesses an amino-acid sequence which

corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591 ,669, 5,589,369 and 5,545,807.

[0112] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M l 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991 ) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581 -597 (1991 ), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905.

(v) Antibody fragments

[0113] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.

8(10): 1057-1062 [ 1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0114] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al. , Journal of Biochemical and Biophysical Methods 24: 107-1 17 ( 1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al.,

Bio/Technology 10: 163- 167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571 ,894; and U.S. Patent No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific. [0115] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0116] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI ) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH I domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0117] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0118] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0119] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). (vi) Bispecific antibodies

[0120] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of CD40 or CD20. Other such antibodies may combine a CD20 binding site with with a binding site which binds to an autoantigen associated with an autoimmune disease of interest. Alternatively, an anti-CD20 or anti-CD40 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the CD20-expressing cell.

[0121] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 ( 1991 ).

[0122] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g. , an antibody) and its binding partner (e.g. , an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant ( d). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0123] In one embodiment, the "Kd" or "Kd value" according to this invention is measured by a radiolabeled antigen-binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution-binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al, J. Mol. Biol, 293:865-881 (1999)). To establish conditions for the assay, microtiter plates (DYNEX Technologies, Inc.) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-anti gen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab- 12, in Presta et al., Cancer Res., 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% TWEEN-20™ surfactant in PBS. When the plates have dried, 150 μΐ/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a

TOPCOU T™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[0124] According to another embodiment, the Kd or Kd value is measured by using surface- plasmon resonance assays using a BIACORE^®-2000 or a BIACORE^®-3000 instrument

(BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (-0.2 μΜ) before injection at a flow rate of 5 μΐ/minute to achieve approximately ten response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN 20™ surfactant (PBST) at 25°C at a flow rate of approximately 25 μΐ/min. Association rates (k_on) and dissociation rates (k_otf) are calculated using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k_0ff/k_on. See, e.g., Chen et al, J. Mol. Biol, 293:865-881 (1999). If the on-rate exceeds 10⁶ M^"'s^" ¹ by the surface-plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence-emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti- antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow-equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0125] An "on-rate," "rate of association," "association rate," or "k_on" according to this invention can also be determined as described above using a BIACORE^®-2000 or a BIACORE^®- 3000 system (BIAcore, Inc., Piscataway, NJ).

[0126] The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody of the invention and the other associated with a reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

[0127] The phrase "substantially reduced," or "substantially different," as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

(vii) Inhibitory Nucleic Acids

[0128] In some embodiments, the immunosuppressive or immunomodulatory agent is an inhibitory nucleic acid that inhibits expression of a target gene in B cells or other lymphocytes and includes, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)). [0129] As used herein, the term "siRNA" refers to a double-stranded RNA molecule that prevents translation of a target B cell mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a part of sense nucleic acid sequence of the target gene (also referred to as "sense strand"), a part of antisense nucleic acid sequence of the target gene (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.

[0130] As used herein, the term "dsRNA" refers to a construct of two RNA molecules composed of complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.

[0131] The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, and the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single- strand".

(viii) Administration of immunosuppressive or immunomodulatory agents

[0132] One of skill will recognize that the immunosuppressive or immunomodulatory agents (e.g., B cell specific agents) can be administered in a variety of ways, depending upon the agent, the particular disease being treated, the patient, and the like. The agents or compositions of the present invention can be used to treat and/or prevent autoimmune disease in subjects or patients including human and any other mammal including, but not limited to, mouse, rat, guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, and chimpanzee, particularly a commercially important animal or a domesticated animal. [0133] The immunosuppressive or immunomodulatory agents are conveniently formulated in pharmaceutical compositions which can optionally include other therapeutic substances, in particular, the DNA antigen specific therapy agents described, below. The formulations may also include other agents such as, anti-inflammatory agents, pain killers, and the like.

[0134] The immunosuppressive or immunomodulatory agents and DNA antigen specific therapy agents of the invention can be administered sequentially or simultaneously with the one or more other pharmacologic agents. The amounts of medicament and pharmacologic agent depend, for example, on what type of pharmacologic agent(s) is/are used, the disease being treated, and the scheduling and routes of administration.

[0135] It should be understood that in addition to the ingredients particularly mentioned herein, the pharmaceutical agents or compositions of this invention can include other agents

conventional in the art having regard to the type of formulation in question.

[0136] In some embodiments the immunosuppressive or immunomodulatory agents and/or DNA antigen specific therapy agents can be included in articles of manufacture and kits containing materials useful for treating autoimmune diseases. The article of manufacture can include a container of any of the present pharmaceutical compositions with a label. Suitable containers include bottles, vials, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. The label on the container should indicate the agent is used for treating or prevention of one or more conditions of the disease. The label can also indicate directions for administration and so on.

[0137] The immunosuppressive or immunomodulatory agents of the invention can be administered directly as a pharmaceutical composition, or if necessary, that has been formulated by conventional formulation methods. Thus, pharmaceutical compositions of the invention will include pharmaceutically acceptable, carriers, excipients, and such that are ordinarily used for drugs can be included as appropriate without particular limitations. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such. Furthermore, the pharmaceutical agents or compositions can contain as necessary, stabilizers, suspensions, preservatives, surfactants and such. The pharmaceutical agents or compositions of this invention can be used for anticancer purposes. [0138] A "medicament" is an active drug to treat an autoimmune disease or the signs or symptoms or side effects of an autoimmune disease.

[0139] The term "pharmaceutical formulation" refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0140] A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0141] A "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products or medicaments, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products or medicaments, etc.

[0142] A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g., a medicament for treatment of an autoimmune disease, or a probe for specifically detecting a biomarker gene or protein of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.

[0143] A "target audience" is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.

[0144] The term "sample" shall generally mean any biological sample obtained from an individual, body fluid, body tissue, cell line, tissue culture, or other source. Body fluids are, e.g., lymph, sera, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid and spinal fluid. Samples also include synovial tissue, skin, hair follicle, and bone marrow. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the term "sample" is used alone, it shall still mean that the "sample" is a "biological sample", i.e., the terms are used interchangeably. [0145] The term "serum sample" shall generally mean any serum sample obtained from an individual. Methods for obtaining sera from mammals are well known in the art.

[0146] The expression "not responsive to," as it relates to the reaction of subjects or patients to one or more of the medicaments that were previously administered to them, describes those subjects or patients who, upon administration of such medicament(s), did not exhibit any or adequate signs of treatment of the disorder for which they were being treated, or they exhibited a clinically unacceptably high degree of toxicity to the medicament(s), or they did not maintain the signs of treatment after first being administered such medicament(s), with the word treatment being used in this context as defined herein. The phrase "not responsive" includes a description of those subjects who are resistant and/or refractory to the previously administered

medication(s), and includes the situations in which a subject or patient has progressed while receiving the medicament(s) that he or she is being given, and in which a subject or patient has progressed within 12 months (for example, within six months) after completing a regimen involving the medicament(s) to which he or she is no longer responsive. The non-responsiveness to one or more medicaments thus includes subjects who continue to have active disease following previous or current treatment therewith. For instance, a patient may have active disease activity after about one to three months of therapy with the medicament(s) to which they are non-responsive. Such responsiveness may be assessed by a clinician skilled in treating the autoimmune disorder in question.

[0147] For purposes of non-response to medicament(s), a subject who experiences "a clinically unacceptably high level of toxicity" from previous or current treatment with one or more medicaments experiences one or more negative side-effects or adverse events associated therewith that are considered by an experienced clinician to be significant, such as, for example, serious infections, congestive heart failure, demyelination (leading to multiple sclerosis), significant hypersensitivity, neuropathological events, high degrees of autoimmunity, a cancer such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, or melanoma, tuberculosis (TB), etc.

[0148] By "reducing the risk of a negative side effect" is meant reducing the risk of a side effect resulting from treatment with the antagonist herein to a lower extent than the risk observed resulting from treatment of the same patient or another patient with a previously administered medicament. Such side effects include those set forth above regarding toxicity, and are preferably infection, cancer, heart failure, or demyelination.

[0149] By "correlate" or "correlating" is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to various

embodiments herein, one may use the results of an analytical assay to determine whether a specific therapeutic regimen using a B-cell specific agent in combination with a DNA antigen specific therapeutic agent should be performed.

[0150] It will be appreciated by one of skill in the medical arts that the exact manner of administering to the patient a therapeutically effective amount of a immunosuppressive or immunomodulatory agent in combination with a DNA antigen specific therapeutic agent will be at the discretion of the attending physician. The mode of administration, including dosage, combination with other autoimmune agents, timing and frequency of administration, and the like, may be affected by the extent of the diagnosis of the patient's likely responsiveness to such combination therapy as well as the patient's condition and history.

[0151] Each agent comprising the combination therapy will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular type of autoimmune disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the autoimmune disease, the site of delivery of the agent, possible side-effects, the type of agent, i.e.

immunosuppressive or immunomodulatory agent or DNA antigen specific therapeutic agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The effective amount of the agent to be administered will be governed by such considerations.

[0152] The term "therapeutically effective amount" is used to refer to an amount of an active immunosuppressive or immunomodulatory agent, such as a B cell specific agent having a growth arrest effect, differentition inhibitory effect or causes the deletion of the B cell. In particular aspects, the therapeutically effective amount refers to a target serum concentration that has been shown to be effective in, for example, slowing disease progression. Efficacy can be measured in conventional ways, depending on the condition to be treated. A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition of each agent required, depending on such factors as the particular agent, i.e. B cell specific agent or DNA antigen specific therapeutic agent, and safety profile. For example, the physician could start with doses of B cell specific agent, such as an anti-CD20 or anti-CD22 antibody or immunoadhesin, employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect to assess safety, and gradually increase the dosage until the desired effect (without compromising safety) is achieved with subsequent administration of the DNA antigen specific therapeutic agent. The effectiveness of a given dose or treatment regimen of the agent can be determined, for example, by assessing signs and symptoms in the patient using the standard measures of efficacy for a particular autoimmune disease.

[0153] As a general proposition, the effective amount of the B cell specific agent administered parenterally per dose will be in the range of about 20 mg to about 5000 mg, by one or more dosages. Exemplary dosage regimens for intact antibodies such as anti-CD20 antibodies and anti-CD22 antibodies, and BAFF and APRIL antagonists, include 375 mg/m² weekly x 4 (e.g., on days 1 , 8, 15, and 22); or 500 mg x 2 (e.g., on days 1 and 15), or 1000 mg x 2 (e.g., on days 1 and 15); or 1 gram x 3 (e.g., on days 1 , 15, and 21 ); or 200 mg x 1 -4; or 300 mg x 1 -4, or 400 mg x 1 -4; or 500 mg x 3-4; or 1 gram x 4.

[0154] Preferably, the B cell specific agent is administered in a dose of about 0.2 to 4 grams, more preferably about 0.2 to 3.5 grams, more preferably about 0.4 to 2.5 grams, more preferably about 0.5 to 1.5 grams, and even more preferably about 0.7 to 1 .1 gram. More preferably, such doses apply to antagonists that are antibodies or immunoadhesins.

[0155] Alternatively, the B cell specific agent is an anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment. In another alternative preferred embodiment, the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 1.2 g, more preferably about 200 mg to 1.1 g, and still more preferably about 200 mg to 900 mg.

[0156] In another preferred embodiment the B-cell specific agent is an anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 1 5 intravenously at the start of the treatment. Preferably the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 600 mg.

[0157] In a preferred aspect, the B cell specific agent is administered at a frequency of one to four doses within a period of about one month. The antagonist is preferably administered in two to three doses. In addition, the antagonist is preferably administered within a period of about two to three weeks.

[0158] As noted above, however, these suggested amounts of antagonist and frequency of dosing are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and schedule is the result obtained, as indicated above. For example, relatively higher doses may be needed initially for the treatment of an ongoing and acute autoimmune flare. To obtain the most efficacious results, once combination therapy is predicted the combination regimen should be initiated as close to the first sign, diagnosis, appearance, or occurrence of the autoimmune flare as possible or during remissions of the autoimmune disease.

[0159] The term "intravenous infusion" refers to introduction of an agent into the vein of an animal or human patient over a period of time greater than approximately 15 minutes, generally between approximately 30 to 90 minutes.

[0160] The term "intravenous bolus" or "intravenous push" refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, generally 5 minutes or less.

[0161] The term "subcutaneous administration" refers to introduction of an agent under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle. The pocket may be created by pinching or drawing the skin up and away from underlying tissue.

[0162] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats and cows to name but a few.

[0163] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. [0164] The term "subcutaneous infusion" refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less. Optionally, the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen.

[0165] The term "subcutaneous bolus" refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is preferably less than approximately 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds.

Administration is preferably within a pocket between the skin and underlying tissue, where the pocket.

DNA ANTIGEN SPECIFIC THERAPEUTIC AGENT

[0166] The terms "DNA antigen specific therapeutic agent" refers to a polynucleotide that is administered to a subject for the purpose of modulating an immune response. For the purpose of the present invention, "DNA tolerization", "DNA vaccination", "DNA immunization", or "polynucleotide therapy" refers to the administration of polynucleotides encoding one or more self-polypeptides that include one or more autoantigenic epitopes associated with a disease. The "DNA tolerization" or "DNA vaccination" serves the purpose of modulating an ongoing immune response to suppress autoimmune destruction for the treatment or prevention of an autoimmune disease. Modulation of an immune response in reaction to "DNA vaccination" may include shifting self-reactive lymphocytes from a Th l - to a Th2-type response. The modulation of the immune response may occur systemically or only locally at the target organ under autoimmune attack.

[0167] The terms "polynucleotide" and "nucleic acid" refer to a polymer composed of a multiplicity of nucleotide units (ribonucleotide or deoxyribonucleotide or related structural variants) linked via phosphodiester bonds. A polynucleotide or nucleic acid can be of substantially any length, typically from about six (6) nucleotides to about 109 nucleotides to about 4000 nucleotides or larger. Polynucleotides and nucleic acids include RNA, DNA, synthetic forms, and mixed polymers, both sense and antisense strands, double- or single- stranded, and can also be chemically or biochemically modified or can contain non-natural or derivatized nucleotide bases, as will be readily appreciated by the skilled artisan. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[0168] The term "promoter" is used here to refer to the polynucleotide region recognized by RNA polymerases for the initiation of RNA synthesis, or "transcription". Promoters are one of the functional elements of self-vectors that regulate the efficiency of transcription and thus the level of protein expression of a self-polypeptide encoded by a self-vector. Promoters can be "constitutive", allowing for continual transcription of the associated gene, or "inducible", and thus regulated by the presence or absence of different substances in the environment.

Additionally, promoters can also either be general, for expression in a broad range of different cell types, or cell-type specific, and thus only active or inducible in a particular cell type, such as a muscle cell. Promoters controlling transcription from vectors may be obtained from various sources, for example, the genomes of viruses such as: polyoma, simian virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus and preferably cytomegalovirus, or from heterologous mammalian promoters, e.g., b-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as is the immediate early promoter of the human cytomegalovirus.

[0169] "Enhancer" refers to cis-acting polynucleotide regions of about from 10-300 basepairs that act on a promoter to enhance transcription from that promoter. Enhancers are relatively orientation and position independent and can be placed 5' or 3' to the transcription unit, within introns, or within the coding sequence itself.

[0170] The terms "intron" or "intronic sequence" as used herein refers to intervening polynucleotide sequences within a gene or portion of a gene present in a self-vector that is situated upstream of or between "exons", polynucleotide sequences that are retained during RNA processing and most often code for a polypeptide. Introns do not function in coding for protein synthesis and are spliced out of a RNA before it is translated into a polypeptide.

[0171] "Splicing" refers to the mechanism by which a single functional RNA molecule is generated by the removal of introns and juxtaposition of exons during processing of the primary transcript, or preRNA. Consensus sequences are present at intron-exon junctions that define the 5' end, or donor site, of an intron and the 3' end, or acceptor site, and at a branchpoint site located approximately 20-50 basepairs upstream of the acceptor site within the intron sequence. Most introns start from the sequence GU and end with the sequence AG (in the 5' to 3' direction) with a branchpoint site approximating CU(A/G)A(C/U), where A is conserved in all genes. These sequences signal for the looping out of the intron and its subsequent removal.

[0172] A "terminator sequence" as used herein means a polynucleotide sequence that signals the end of DNA transcription to the RNA polymerase. Often the 3' end of a RNA generated by the terminator sequence is then processed considerably upstream by polyadenylation.

"Polyadenylation" is used to refer to the non-templated addition of about 50 to about 200 nucleotide chain of polyadenylic acid (polyA) to the 3' end of a transcribed messenger RNA. The "polyadenylation signal" (AAUAAA) is found within the 3' untranslated region (UTR) of a mRNA and specifies the site for cleavage of the transcript and addition of the polyA tail.

Transcription termination and polyadenylation are functionally linked and sequences required for efficient cleavage/polyadenylation also constitute important elements of termination sequences (Connelly and Manley, 1988).

[0173] "Self-vector," "self-vector specific agent" means one or more vector(s) which taken together comprise a polynucleotide either DNA or RNA encoding one or more self-protein(s), -polypeptide(s), -peptide(s) (referred to herein as "DNA tolerizing vector", "DNA tolerizing plasm id" "DNA self-vector" or "RNA self-vector" respectively). A DNA tolerizing vector or DNA tolerizing plasmid when used in the context of a particular autoimmune disease may include the particular autoantigen in the description, for example in the case of Tl D the DNA tolerizing plasmid may be referred to as "Proinsulin tolerizing plasmid (PTP)" when proinsulin is the autoantigen encoded by the self-vector. In the case of multiple sclerosis, the DNA tolerizing plasmid may be referred to as "Myelin basic protein tolerizing plasmid (MTP)" when myelin basic protein is the autoantigen encoded by the self-vector. Polynucleotide, as used herein is a series of either deoxyribonucleic acids including DNA or ribonucleic acids including RNA, and their derivatives, encoding a self-protein, -polypeptide, or -peptide of this invention. The self- protein, -polypeptide or -peptide coding sequence is inserted into an appropriate plasmid expression self-cassette. Once the polynucleotide encoding the self-protein, -polypeptide, or -peptide is inserted into the expression self-cassette the vector is then referred to as a "self- vector." In the case where polynucleotide encoding more than one self-protein(s),

-polypeptide(s), or -peptide(s) is to be administered, a single self-vector may encode multiple separate self -protein(s), -polypeptide(s) or -peptide(s). In one embodiment, DNA encoding several self-protein(s), -polypeptide(s), or -peptide(s) are encoded sequentially in a single self- plasmid utilizing internal ribosomal re-entry sequences (IRES) or other methods to express multiple proteins from a single DNA molecule. The DNA expression self-vectors encoding the self-protein(s), -polypeptide(s), or -peptide(s) are prepared and isolated using commonly available techniques for isolation of plasmid DNA such as those commercially available from Qiagen Corporation. The DNA is purified free of bacterial endotoxin for delivery to humans as a therapeutic agent. Alternatively, each self-protein, -polypeptide or -peptide is encoded on a separate DNA expression vector.

[0174] A "high expression self-vector" or "HESV" refers herein to a modified self-vector that is altered to increase expression of an encoded self-protein(s), -polypeptide(s), or -peptide(s) relative to an unmodified self-vector encoding the same self-protein(s), -polypeptide(s), or - peptide(s). A HESV comprises a polynucleotide encoding and capable of expressing a self- polypeptide associated with an autoimmune disease and a modification to generate increased expression of the self-polypeptide relative to the same self-vector unmodified. A HESV further comprises in operative combination: a promoter; a polynucleotide encoding a self-polypeptide that includes at least one autoantigenic epitope associated with the autoimmune disease; a transcription terminator; and at least one modification for generating increased expression of the self-polypeptide in a host cell, in which the increased expression is relative to an unmodified self-vector comprising the promoter, polynucleotide, and transcription terminator. Modifications of a self-vector to generate a HESV with increased expression of a self-polypeptide are selected from alterations that increase: transcription initiation, transcription termination, mRNA stability, translation efficiency, and/or protein stability. More specifically, modifications of a self-vector to increase expression of a self-polypeptide are selected from the group consisting of: using a stronger promoter region, addition of enhancer regions, using a more efficient transcription terminator sequence, addition of polyadenylation signals, using a more ideal consensus kozak sequence, optimizing codon usage, inclusion of introns or combinations of the foregoing modifications. Single or multiple modifications may be incorporated into a self-vector to generate a HESV. In one preferred embodiment the modification is the inclusion of an intron downstream of the promoter region and upstream of the start codon of a polynucleotide encoding one or more self-polypeptides. More particularly the preferred intron is intron A of the human cytomegalovirus (CMV) or a β-globin/Ig chimeric intron and most preferably the preferred intron is the β-globin/Ig chimeric intron. An HESV is generated that expresses increased amounts of a self-protein(s), -polypeptide(s), or peptide(s) associated with an autoimmune disease, such as insulin-dependent diabetes mellitus (T1 D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), or rheumatoid arthritis (RA) compared to an unmodified self-vector encoding the same self-protein(s), -polypeptide(s), or peptide(s). In the case of T I D, a HESV is generated that expresses increased amounts of the self-polypeptide preproinsulin compared to the unmodified self-vector. An HESV is generated that expresses increased amounts of a self- polypeptide associated with the autoimmune disease multiple sclerosis (MS) compared to an unmodified self-vector encoding the same self-polypeptide. More particularly the HESV is generated that expresses increased amounts of the self-polypeptide myelin basic protein (MBP) compared to the unmodified self-vector. In preferred embodiments a HESV contains a β- globin/Ig chimeric intron 5' to the start codon of the self-polypeptide MBP.

[0175] A "non-secreted self-vector" or "N-SSV" or "non-secreted self-vector specific agent" refers herein to a modified self-vector that contains a polynucleotide encoding for an intracellular or non-secreted self-polypeptide version of a extracellular or secreted autoantigen (e.g., a transmembrane protein or secreted soluble factor) associated with an autoimmune disease. A N- SSV comprises a polynucleotide encoding and capable of expressing a secreted self-polypeptide associated with an autoimmune disease and a modification to express a non-secreted or non- membrane bound self-polypeptide from a host cell. A N-SSV further comprises in operative combination: a promoter; a polynucleotide encoding an extracellular or secreted self-polypeptide that includes at least one autoantigenic epitope associated with the autoimmune disease; a transcription terminator; and at least one modification to prevent secretion of the self-polypeptide from a host cell relative to an unmodified self-vector comprising the promoter, polynucleotide, and transcription terminator. Modifications to a self-vector to generate a N-SSV encoding and expressing a non-secreted or non-membrane bound version of a secreted or membrane bound self-polypeptide include but are not limited to eliminating the signal sequence, mutating the signal sequence, and adding alternative protein localization (ER retention, plasma membrane attachment, etc.) protein degradation signals or modifying or deleting, transmembrane domains or hydrophobic regions of the self-polypeptide.

[0176] A "non-secreted high expression self-vector" or "N-SHESV" or "non-secreted high expression self-vector specific agent" refers to a modified self-vector that is altered to increase expression of an encoded intracellular or non-secreted version of an extracellular or secreted self-polypeptide or non-membrane bound version of a membrane bound self-polypeptide in which expression and secretion is relative to an unmodified self-vector. A N-SHESV specific agent comprises a polynucleotide encoding and capable of expressing a secreted or membrane bound self-polypeptide associated with an autoimmune disease and a modification to generate increased expression of the self-polypeptide in a non-secreted or non-membrane bound form relative to the unmodified self-vector. A N-SHESV further comprises in operative combination: a promoter; a polynucleotide encoding a extracellular or secreted self-polypeptide that includes at least one autoantigenic epitope associated with the autoimmune disease; a transcription terminator; and at least one modification for generating increased expression of the self- polypeptide and at least one modification to express the non-secreted or non-membrane bound self-polypeptide from a host cell where both modifications are relative to an unmodified self- vector comprising the promoter, polynucleotide, and transcription terminator.

[0177] A "secreted self-vector" or "SSV" or "secreted self-vector specific agent" refers herein to a modified self-vector that contains a polynucleotide encoding a secreted self-polypeptide version of a membrane associated or intracellular autoantigen associated with an autoimmune disease. A SSV comprises a polynucleotide encoding and capable of expressing a membrane associated or intracellular self-polypeptide associated with an autoimmune disease and a modification to allow secretion of the self-polypeptide from a host cell. A SSV comprises a polynucleotide encoding and capable of expressing a membrane associated or intracellular self- polypeptide associated with an autoimmune disease and a modification to allow secretion of the self-polypeptide from a host cell. A SSV further comprises in operative combination: a promoter; a polynucleotide encoding a membrane associated or intracellular self-polypeptide that includes at least one autoantigenic epitope associated with the autoimmune disease; a

transcription terminator; and at least one modification to permit secretion of the self-polypeptide from a host cell compared to an unmodified self-vector comprising the promoter, polynucleotide, and transcription terminator. Modifications to a self-vector to generate a SSV encoding and expressing a secreted version of an intracellular self-polypeptide include, but are not limited to, addition of a signal sequence. Additionally, the modification may further include signals for membrane association including, for example, a transmembrane domain or a GPI anchor so that intracellular epitope(s) are presented extracellularly. Modifications to a self-vector to generate a SSV encoding and expressing a secreted version of a membrane associated self-polypeptide include but are not limited to: removal of a transmembrane domain; removal of a GPI linkage, removal of an extracellular and transmembrane domain with addition of a signal sequence to an intracellular domain; and removal of a transmembrane domain and intracellular domain.

[0178] A "secreted high expression self-vector" or "SHESV" or "secreted high expression self- vector specific agent" as used herein refers to a modified self-vector that is altered to increase expression of an encoded secreted version of a membrane associated or intracellular self- polypeptide in which expression and secretion is relative to an unmodified self-vector. A SHESV comprises a polynucleotide encoding and capable of expressing a membrane associated or intracellular self-polypeptide associated with an autoimmune disease and a modification to generate increased expression of the self-polypeptide in a secreted or extracellular membrane associated form relative to the same self-vector unmodified. A SHESV further comprises in operative combination: a promoter; a polynucleotide encoding a membrane associate or intracellular self-polypeptide that includes at least one autoantigenic epitope associated with the autoimmune disease; a transcription terminator; and at least one modification for generating increased expression of the self-polypeptide and at least one modification to allow secretion of the self-polypeptide from a host cell where both modifications are relative to an unmodified self- vector comprising the promoter, polynucleotide, and transcription terminator.

[0179] The term "vector backbone" refers to the portion of a plasmid vector other than the sequence encoding a self-antigen, -protein, -polypeptide, or -peptide.

[0180] An "immunosuppressive vector backbone" refers to a vector backbone that either (i) elicits a reduced immune response in comparison to a parent vector backbone, or (ii) prevents or inhibits an immune response. The immune response can be measured using in vitro or in vivo assays known in the art. For example, the immune response can be determined by measuring proliferation of lymphocytes exposed to the vector backbone, or by measuring production of cytokines (in cell culture media, in serum, etc.) indicative of immune stimulation {e.g., IL-2, lFN-γ, IL-6). In some embodiments, an immunosuppressive vector backbone contains fewer immunostimulatory sequences (e.g., CpG sequences) in comparison to a parent vector backbone. In some embodiments, an immunosuppressive vector backbone contains one or more

immunoinhibitory sequences (IIS), for example, as described herein and known in the art. In some embodiments, an immunosuppressive vector backbone promotes a Th2 immune response and inhibits a Thl immune response.

[0181] In certain variations, the method for treating autoimmune disease further includes the administration of a polynucleotide comprising an inhibitory immune modulatory sequence or an immune inhibitory sequence (IIS). The IISs useful in accordance with the present invention comprise the following core hexamer:

5'-purine-pyrimidine-[X]-[Y]-pyrimidine-pyrimidine-3' or

5'-purine-purine-[X]-[Y]-pyrimidine-pyrimidine-3';

[0182] wherein X and Y are any naturally occurring or synthetic nucleotide, except that X and Y cannot be cytosine-guanine.

[0183] The core hexamer of IMSs can be flanked 5' and/or 3' by any composition or number of nucleotides or nucleosides. Preferably, IMSs range between 6 and 100 base pairs in length, and most preferably 16-50 base pairs in length. IMSs can also be delivered as part of larger pieces of DNA, ranging from 100 to 100,000 base pairs. IMSs can be incorporated in, or already occur in, DNA plasmids, viral vectors and genomic DNA. Most preferably IMSs can also range from 6 (no flanking sequences) to 10,000 base pairs, or larger, in size. Sequences present which flank the hexamer core can be constructed to substantially match flanking sequences present in any known immunoinhibitory sequences (IIS). For example, the flanking sequences

TTGACTGTG -Pu-Pyr-X-Y-Pyr-Pyr-AGAGATGA, where TTGACTGTG and AGAGATGA are flanking sequences. Another preferred flanking sequence incorporates a series of pyrimidines (C, T, and U), either as an individual pyrimidine repeated two or more times, or a mixture of different pyrimidines two or more in length. Different flanking sequences have been used in testing inhibitory modulatory sequences. Further examples of flanking sequences for inhibitory oligonucleotides are contained in the following references: U.S. Patent Nos.

6,225,292 and 6,339,068; and Zeuner et al., Arthritis and Rheumatism, 46:2219-24 (2002). [0184] An IIS contains the core hexamer region having the sequence AAGGTT. That sequence is referred to herein as an immune modulatory sequence or IMS. Other related IISs with a similar motif included within the IMSs are:

1. 5'-purine-purine-[X]-[Y]-pyrimidine-pyrimidine-3' IMSs containing GG

dinucleotide cores: GGGGTT, AGGGTT, GAGGTT, AAGGTT, GGGGCT, AGGGCT, GAGGCT, AAGGCT, GGGGTC, AGGGTC, GAGGTC,

AAGGTC, and so forth;

2. 5'-purine-purine-[X]-[Y]-pyrimidine-pyrimidine-3' IMSs containing GC

dinucleotide cores: GGGCTT, AGGCTT, GAGCTT, AAGCTT, GGGCCT, AGGCCT, GAGCCT, AAGCCT, GGGCTC, AGGCTC, GAGCTC,

AAGCTC, and so forth;

3. Guanine and inosine substitutions for adenine and/or uridine substitutions for cytosine or thymine can be made as set forth based on the guidelines above.

[0185] In certain embodiments of the present invention, the core hexamer region of the IMS is flanked at either the 5' or 3' end, or at both the 5' and 3' ends, by a polyG region. A "polyG region" or "polyG motif as used herein means a nucleic acid region consisting of at least two (2) contiguous guanine bases, typically from 2 to 30 or from 2 to 20 contiguous guanines. In some embodiments, the polyG region has from 2 to 10, from 4 to 10, or from 4 to 8 contiguous guanine bases. In certain preferred embodiments, the flanking polyG region is adjacent to the core hexamer. In yet other embodiments, the polyG region is linked to the core hexamer by a non-polyG region (non-polyG linker); typically, the non-polyG linker region has no more than 6, more typically no more than 4 nucleotides, and most typically no more than 2 nucleotides.

[0186] The immune modulatory nucleic acids can contain IMSs alone or incorporated in cis or in trans with other nucleic acid regions such as, for example, into a recombinant self-vector (plasmid, cosmid, virus or retrovirus) which may in turn code for any self- protein(s), - polypeptide(s), or -peptide(s) deliverable by a recombinant expression vector. In certain embodiments, the IMSs are incorporated into a vector such as, for example, an expression vector, which may be accomplished, for example, using conventional techniques as known to one of ordinary skill in the art (see, e.g., Ausubel, Current Protocols in Molecular Biology, supra). [0187] For example, construction of recombinant expression vectors employs standard ligation techniques. For analysis to confirm correct sequences in vectors constructed, the ligation mixtures may be used to transform a host cell and successful transformants selected by antibiotic resistance where appropriate. Vectors from the transformants are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., Nucleic Acids Res., 9:309, 1981 , the method of Maxam, et al , Methods in Enzymology, 65:499, 1980, or other suitable methods which will be known to those skilled in the art. Size separation of cleaved fragments is performed using conventional gel electrophoresis as described, for example, by Maniatis, et al , Molecular Cloning, pp. 133-134, 1982.

[0188] Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0189] If a recombinant vector is utilized as a carrier for the IMS-ON of the invention, plasmids and cosmids are particularly preferred for their lack of pathogenicity. However, plasmids and cosmids are subject to degradation in vivo more quickly than viruses and therefore may not deliver an adequate dosage of IMS-ON to prevent or treat an inflammatory or autoimmune disease.

[0190] In a related aspect, a nucleic acid vector is provided in which a non-CpG dinucleotide is substituted for one or more CpG dinucleotides of the formula 5'-punne-pyrimidine-C-G- pyrimidine-pyrimidine-3' or 5'-purine-purine-C-G-pyrimidine-pyrimidine-3', thereby producing a vector in which US-associated immunostimulatory activity is reduced. Such vectors are useful, for example, in methods for administering immune modulatory nucleic acids and/or for administering a self vector encoding one or more self-protein(s), -polypeptides(s), or -peptide(s). For example, the cytosine of the CpG dinucleotide can be substituted with guanine, thereby yielding an IMS region having a GpG motif of the formula 5'-purine-pyrimidine-G-G- pyrimidine-pyrimidine-3' or 5'-purine-purine-G-G-pyrimidine-pyrimidine-3'. The cytosine can also be substituted with any other non-cytosine nucleotide. The substitution can be

accomplished, for example, using site-directed mutagenesis. Typically, the substituted CpG motifs are those CpGs that are not located in important control regions of the vector {e.g., promoter regions). In addition, where the CpG is located within a coding region of an expression vector, the non-cytosine substitution is typically selected to yield a silent mutation or a codon corresponding to a conservative substitution of the encoded amino acid.

[0191] For example, in certain embodiments, a modified pVAXl vector is provided in which one or more CpG dinucleotides of the formula 5'-purine-pyrimidine-C-G-pyrimidine-pyrimidine- 3' is mutated by substituting the cytosine of the CpG dinucleotide with a non-cytosine nucleotide. The pVAXl vector is known in the art and is commercially available from Invitrogen (Carlsbad, CA). In one exemplary embodiment, the modified pVAXl vector has the following cytosine to non-cytosine substitutions within a CpG motif: cytosine to guanine at nucleotides 784, 1 161 , 121 8, and 1966; cytosine to adenine at nucleotides 1264, 1337, 1829, 1874, 1940, and 1997; and cytosine to thymine at nucleotides 1 158, and 1987; with additional cytosine to guanine mutations at nucleotides 1831 , 1876, 1942, and 1999. (The nucleotide number designations as set forth above are according to the numbering system for pVAXl provided by Invitrogen.) The vector thus constructed was named pBHTl .

[0192] Nucleotide sequences selected for use in the self-vector can be derived from known sources, for example, by isolating the nucleic acid from cells containing a desired gene or nucleotide sequence using standard techniques. Similarly, the nucleotide sequences can be generated synthetically using standard modes of polynucleotide synthesis that are well known in the art. See, e.g. , Edge et al , Nature 292:756, 1981 ; Nambair et al , Science 223: 1299, 1984; Jay et al, J. Biol Chem. 259:631 1 , 1984. Generally, synthetic oligonucleotides can be prepared by either the phosphotriester method as described by Edge et al. {supra) and Duckworth et al.

Nucleic Acids Res. 9: 1691 , 1981 ; or the phosphoramidite method as described by Beaucage et al. Tet. Letts. 22: 1859, 1981 and Matteucci et al, J. Am. Chem. Soc. 103:3185, 1981. Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. The nucleotide sequences can thus be designed with appropriate codons for a particular amino acid sequence. In general, one will select preferred codons for expression in the intended host. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge et al. {supra); Nambair et al. {supra) and Jay et al. {supra). [0193] Another method for obtaining nucleic acid sequences for use herein is by recombinant means. Thus, a desired nucleotide sequence can be excised from a plasmid carrying the nucleic acid using standard restriction enzymes and procedures. Site specific DNA cleavage is performed by treating with the suitable restriction enzymes and procedures. Site specific DNA cleavage is performed under conditions which are generally understood in the art, and the particulars of which are specified by manufacturers of commercially available restriction enzymes. If desired, size separation of the cleaved fragments may be performed by

polyacrylamide gel or agarose gel electrophoreses using standard techniques.

[0194] Yet another convenient method for isolating specific nucleic acid molecules is by the polymerase chain reaction (PCR) (Mullins et al, Methods Enzymol. 155:335-350, 1987) or reverse transcription PCR (RT-PCR). Specific nucleic acid sequences can be isolated from RNA by RT-PCR. RNA is isolated from, for example, cells, tissues, or whole organisms by techniques known to one skilled in the art. Complementary DNA (cDNA) is then generated using poly-dT or random hexamer primers, deoxynucleotides, and a suitable reverse transcriptase enzyme. The desired polynucleotide can then be amplified from the generated cDNA by PCR. Alternatively, the polynucleotide of interest can be directly amplified from an appropriate cDNA library. Primers that hybridize with both the 5' and 3' ends of the polynucleotide sequence of interest are synthesized and used for the PCR. The primers may also contain specific restriction enzyme sites at the 5' end for easy digestion and ligation of amplified sequence into a similarly restriction digested plasmid vector.

[0195] The expression cassette of the modified self-vector will employ a promoter that is functional in host cells. In general, vectors containing promoters and control sequences that are derived from species compatible with the host cell are used with the particular host cell.

Promoters suitable for use with prokaryotic hosts illustratively include the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as tac promoter. However, other functional bacterial promoters are suitable. In addition to prokaryotes, eukaryotic microbes such as yeast cultures may also be used.

Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, simian virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus and preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. β-actin promoter. The early and late promoters of the SV 40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll restriction fragment. Of course, promoters from the host cell or related species also are useful herein.

[0196] In one embodiment, DNA encoding two or more self-protein(s), -polypeptide(s), or - peptide(s) are encoded sequentially in a single self-vector utilizing internal ribosomal re-entry sequences (IRES) or other elements for expression of multiple proteins from a single DNA molecule.

[0197] The vectors used herein may contain a selection gene, also termed a selectable marker. A selection gene encodes a protein, necessary for the survival or growth of a host cell transformed with the vector. Examples of suitable selectable markers for mammalian cells include the dihydrofolate reductase gene (DHFR), the ornithine decarboxylase gene, the multidrug resistance gene (mdr), the adenosine deaminase gene, and the glutamine synthase gene. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. The second category is referred to as dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin (Southern and Berg (1982) J. Molec. Appl. Genet. 1, 327), mycophenolic acid (Mulligan and Berg ( 1980) Science 209, 1422), or hygromycin (Sugden et al. (1985) Mol. Cell. Bio. 5, 410-413). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug neomycin (G418 or genticin), xgpt (mycophenolic acid) or hygromycin, respectively.

[0198] Alternatively the vectors used herein are propagated in a host cell using antibiotic-free selection based on repressor titration (Cranenburgh et al., 2001 ). The vectors are modified to contain the lac operon either as part of the lac promoter or with the lacOi and lacOs operators with the optimal spacing found in the pUC series of plasmid vectors. Alternatively the lacOj operator or palindromic versions of the lacO can be used in isolation as single or multiple copies (Cranenburgh et al., 2004). The lac operon sequence may be incorporated at single or multiple sites anywhere within the vector so as not to interfere with other functional components of the vector. In preferred embodiments a synthetic Escherichia coli lac operon dimer operator (Genbank Acc. Num. K02913) is used. The lac operon may be added to a vector that lacks a suitable selective marker to provide selection, be added in addition to another selectable marker, or used to replace a selectable marker, especially an antibiotic resistance marker, to make the vector more suitable for therapeutic applications. Vectors containing the lac operon can be selected in genetically modified E. coli with an essential gene, including dapD, under the control of the lac promoter (lacOP) thus allowing the modified host cell to survive by titrating the lac repression from the lacOP and allowing expression of dapD. Suitable E. coli stains include OmiacdapD and OH\ lacP2dapD (Cranenburgh et al., 2001 )

[0199] The self-vector specific agents of this invention can be formulated as polynucleotide salts for use as pharmaceuticals. Polynucleotide salts can be prepared with non-toxic inorganic or organic bases. Inorganic base salts include sodium, potassium, zinc, calcium, aluminum, magnesium, etc. Organic non-toxic bases include salts of primary, secondary and tertiary amines, etc. Such self-DNA polynucleotide salts can be formulated in lyophilized form for reconstitution prior to delivery, such as sterile water or a salt solution. Alternatively, self-DNA polynucleotide salts can be formulated in solutions, suspensions, or emulsions involving water- or oil-based vehicles for delivery. In one preferred embodiment, the DNA is lyophilized in phosphate buffered saline with physiologic levels of calcium (0.9 mM) and then reconstituted with sterile water prior to administration. Alternatively the DNA is formulated in solutions containing higher quantities of Ca⁺⁺, between 1 mM and 2M. The DNA can also be formulated in the absence of specific ion species.

Administration of DNA antigen specific therapeutic agents

[0200] A wide variety of methods exist to deliver polynucleotide to subjects, as defined herein. For example, the polynucleotide encoding a self-polypeptide can be formulated with cationic polymers including cationic liposomes. Other liposomes also represent effective means to formulate and deliver self-polynucleotide. Alternatively, the self DNA can be incorporated into a viral vector, viral particle, or bacterium for pharmacologic delivery. Viral vectors can be infection competent, attenuated (with mutations that reduce capacity to induce disease), or replication-deficient. Methods utilizing self-DNA to prevent the deposition, accumulation, or activity of pathogenic self proteins may be enhanced by use of viral vectors or other delivery systems that increase humoral responses against the encoded self-protein. In other embodiments, the DNA can be conjugated to solid supports including gold particles, polysaccharide-based supports, or other particles or beads that can be injected, inhaled, or delivered by particle bombardment (ballistic delivery). Methods for delivering nucleic acid preparations are known in the art. See, e.g. ; U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. A number of viral based systems have been developed for transfer into mammalian cells. For example, retroviral systems have been described (U.S. Patent No. 5,219,740; Miller et al, Biotechniques 7:980-990, 1989; Miller, Human Gene Therapy 1 :5-14, 1990; Scarpa e/ a/., Virology 180:849-852, 1991 ; Burns et al, Proc. Natl. Acad. Sci. USA 90:8033-8037, 1993; and, Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. 3: 102-109, 1993). A number of adenovirus vectors have also been described, see e.g., (Haj-Ahmad et al. , J. Virol. 57:267-274, 1986; Bett er al. , J. Virol. 67:591 1 - 5921 , 1993; Mittereder et al, Human Gene Therapy 5:717-729, 1994; Seth et al, J. Virol.

68:933-940, 1994; Barr et al, Gene Therapy 1 :51 -58, 1994; Berkner, BioTechniques 6:616-629, 1988; and, Rich et al , Human Gene Therapy 4:461 -476, 1993). Adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5, 173,414 and 5, 139,941 ; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al, Molec. Cell. Biol. 8:3988-3996, 1988; Vincent et al, Vaccines 90 (Cold Spring Harbor

Laboratory Press) 1990; Carter, Current Opinion in Biotechnology 3:533-539, 1992; Muzyczka, Current Topics in Microbiol. And Immunol. 158:97-129, 1992; Kotin, Human Gene Therapy 5:793-801 , 1994; Shelling et al, Gene Therapy 1 : 165- 169, 1994; and, Zhou et al. , J. Exp. Med. 179: 1867-1875, 1994).

[0201] The polynucleotide of this invention can also be delivered without a viral vector. For example, the molecule can be packaged in liposomes prior to delivery to the subject. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, e.g., Hug et al, Biochim. Biophys. Acta. 1097: 1 -17, 1991 ; Straubinger et al, in Methods of Enzymology, Vol. 101, pp. 512-527, 1983.

[0202] Therapeutically effective amounts of self-vector are in the range of about 0.001 mg to about 1 g. A preferred therapeutic amount of self-vector is in the range of about 10 ng to about 10 mg. A most preferred therapeutic amount of self-vector is in the range of about 0.025 mg to 6 mg. In certain embodiments, the self- vector is administered monthly for 6-12 months, and then every 3-12 months as a maintenance dose. Alternative treatment regimens may be developed and may range from daily, to weekly, to every other month, to yearly, to a one-time

administration depending upon the severity of the disease, the age of the patient, the self- polypeptide or -polypeptides being administered, and such other factors as would be considered by the ordinary treating physician.

[0203] In one embodiment, the polynucleotide is delivered by intramuscular injection. In other variations, the polynucleotide is delivered intranasally, orally, subcutaneously, intradermal ly, intravenously, mucosally, impressed through the skin, or attached to gold particles delivered to or through the dermis (see, e.g. , WO 97/46253). Alternatively, nucleic acid can be delivered into skin cells by topical application with or without liposomes or charged lipids (see e.g. U.S. Patent No. 6,087,341 ). Yet another alternative is to deliver the nucleic acid as an inhaled agent. The polynucleotide is formulated in phosphate buffered saline with physiologic levels of calcium (0.9 raM). Alternatively, the polynucleotide is formulated in solutions containing higher quantities of Ca⁺⁺, between 1 mM and 2M. The polynucleotide may be formulated with other cations such as zinc, aluminum, and others. Alternatively, or in addition, the polynucleotide may be formulated either with a cationic polymer, cationic liposome-forming compounds, or in non-cationic liposomes. Examples of cationic liposomes for DNA delivery include liposomes generated using l ,2-bis(oleoyloxy)-3-(trimethylammionio) propane (DOTAP) and other such molecules.

[0204] Prior to delivery of the polynucleotide, the delivery site can be preconditioned by treatment with bupivicane, cardiotoxin or another agent that may enhance the subsequent delivery of the polynucleotide. Such preconditioning regimens are generally delivered 12 to 96 hours prior to delivery of therapeutic polynucleotide; more frequently 24 to 48 hours prior to delivery of the therapeutic polynucleotide. Alternatively, no preconditioning treatment is given prior to polynucleotide therapy.

[0205] "Antigen," as used herein, refers to any molecule that can be recognized by the immune system that is by B cells or T cells, or both.

[0206] "Autoantigen," as used herein, refers to an endogenous molecule, typically a protein or fragment thereof, that elicits a pathogenic immune response. When referring to the autoantigen or epitope thereof as "associated with an autoimmune disease," it is understood to mean that the autoantigen or epitope is involved in the pathophysiology of the disease either by inducing the pathophysiology (i.e., associated with the etiology of the disease), mediating or facilitating a pathophysiologic process; and/or by being the target of a pathophysiologic process. For example, in autoimmune disease, the immune system aberrantly targets autoantigens, causing damage and dysfunction of cells and tissues in which the autoantigen is expressed and/or present. Under normal physiological conditions, autoantigens are ignored by the host immune system through the elimination, inactivation, or lack of activation of immune cells that have the capacity to recognize the autoantigen through a process designated "immune tolerance."

[0207] As used herein the term "epitope" is understood to mean a portion of a polypeptide having a particular shape or structure that is recognized by either B-cells or T-cells of the animal's immune system. "Autoantigenic epitope" or "pathogenic epitope" refers to an epitope of an autoantigen that elicits a pathogenic immune response.

[0208] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0209] "Self-protein," "self-polypeptide," or self-peptide" are used herein interchangeably and refer to any protein, polypeptide, or peptide, or fragment or derivative thereof that: is encoded within the genome of the animal; is produced or generated in the animal; may be modified post- translationally at some time during the life of the animal; and, is present in the animal non- physiologically. The term "non-physiological" or "non-physiologically" when used to describe the self-protein(s), -polypeptide(s), or -peptide(s) of this invention means a departure or deviation from the normal role or process in the animal for that self-protein, -polypeptide, or - peptide. When referring to the self-protein, -polypeptide or -peptide as "associated with a disease" or "involved in a disease" it is understood to mean that the self-protein, -polypeptide, or -peptide may be modified in form or structure and thus be unable to perform its physiological role or process or may be involved in the pathophysiology of the condition or disease either by inducing the pathophysiology; mediating or facilitating a pathophysiologic process; and/or by being the target of a pathophysiologic process. For example, in autoimmune disease, the immune system aberrantly attacks self-proteins causing damage and dysfunction of cells and tissues in which the self-protein is expressed and/or present. Alternatively, the self-protein, - polypeptide or -peptide can itself be expressed at non-physiological levels and/or function non- physiologically. For example in neurodegenerative diseases self-proteins are aberrantly expressed, and aggregate in lesions in the brain thereby causing neural dysfunction. In other cases, the self-protein aggravates an undesired condition or process. For example in

osteoarthritis, self-proteins including collagenases and matrix metalloproteinases aberrantly degrade cartilage covering the articular surface of joints. Examples of posttranslational modifications of self-protein(s), -polypeptide(s) or -peptide(s) are glycosylation, addition of lipid groups, reversible phosphorylation, addition of dimethylarginine residues, citrullination, and proteolysis, and more specifically citrullination of fillagrin and fibrin by peptidyl arginine deiminase (PAD), alpha β-crystallin phosphorylation, citrullination of MBP, and SLE

autoantigen proteolysis by caspases and granzymes. Immunologically, self-protein, -polypeptide or -peptide would all be considered host self-antigens and under normal physiological conditions are ignored by the host immune system through the elimination, inactivation, or lack of activation of immune cells that have the capacity to recognize self-antigens through a process designated "immune tolerance." A self-protein, -polypeptide, or -peptide does not include immune proteins, polypeptides, or peptides which are molecules expressed physiologically exclusively by cells of the immune system for the purpose of regulating immune function. The immune system is the defense mechanism that provides the means to make rapid, highly specific, and protective responses against the myriad of potentially pathogenic microorganisms inhabiting the animal's world. Examples of immune protein(s), polypeptide(s) or peptide(s) are proteins comprising the T-cell receptor, immunoglobulins, cytokines including the type I interleukins, and the type II cytokines, including the interferons and IL- 10, TNF, lymphotoxin, and the

chemokines such as macrophage inflammatory protein -1 alpha and beta, monocyte-chemotactic protein and RANTES, and other molecules directly involved in immune function such as Fas- ligand. There are certain immune protein(s), polypeptide(s) or peptide(s) that are included in the self-protein, -polypeptide or -peptide of the invention and they are: class I MHC membrane glycoproteins, class II MHC glycoproteins and osteopontin. Self-protein, -polypeptide or - peptide does not include proteins, polypeptides, and peptides that are absent from the subject, either entirely or substantially, due to a genetic or acquired deficiency causing a metabolic or functional disorder, and are replaced either by administration of said protein, polypeptide, or peptide or by administration of a polynucleotide encoding said protein, polypeptide or peptide (gene therapy). Examples of such disorders include Duchenne' muscular dystrophy, Becker's muscular dystrophy, cystic fibrosis, phenylketonuria, galactosemia, maple syrup urine disease, and homocystinuria. Self-protein, -polypeptide or -peptide does not include proteins, polypeptides, and peptides expressed specifically and exclusively by cells which have characteristics that distinguish them from their normal counterparts, including: (1 ) clonality, representing proliferation of a single cell with a genetic alteration to form a clone of malignant cells, (2) autonomy, indicating that growth is not properly regulated, and (3) anaplasia, or the lack of normal coordinated cell differentiation. Cells have one or more of the foregoing three criteria are referred to either as neoplastic, cancer or malignant cells.

[0210] "Modulation of," "modulating", or "altering an immune response" as used herein refers to any alteration of an existing or potential immune responses against self-molecules, including, e.g. , nucleic acids, lipids, phospholipids, carbohydrates, self-polypeptides, protein complexes, or ribonucleoprotein complexes, that occurs as a result of administration of a polynucleotide encoding a self-polypeptide. Such modulation includes any alteration in presence, capacity, or function of any immune cell involved in or capable of being involved in an immune response. Immune cells include B cells, T cells, NK cells, NK T cells, professional antigen-presenting cells, non-professional antigen-presenting cells, inflammatory cells, or any other cell capable of being involved in or influencing an immune response. "Modulation" includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration in the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response.

[0211] "Modulation of an immune response" includes, for example, the following:

elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells (APCs), or inflamatory cells; induction of an unresponsive state in immune cells (i.e. , anergy); increasing, decreasing, or changing the activity or function of immune cells or the capacity to do so, including but not limited to altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors; or any combination of these modulatory events. [0212] "Preventing," "prophylaxis" or "prevention" of a disease or disorder as used in the context of this invention refers to the administration of a polynucleotide encoding a self- protein^), -polypeptide(s), or -peptide(s) either alone or in combination with another compound as described herein, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.

[0213] The terms "treating," "treatment" and "therapy" and the like as used within the context of the present invention, are meant to include therapeutic as well as prophylactic, or suppressive measures for a disease or disorder leading to any clinically desirable or beneficial effect, including but not limited to alleviation of one or more symptoms, regression, slowing or cessation of progression of the disease or disorder. Thus, for example, the term treatment includes the administration of one or both agent(s) of the instant invention prior to or following the onset of a symptom of an autoimmune disease or disorder thereby preventing or removing all signs of the autoimmune disease or disorder. As another example, the term includes the administration of one or both agent(s) after clinical manifestation of the autoimmune disease to combat the symptoms of the disease. Further, administration of one or both agent(s) of the instant invention after onset and after clinical symptoms have developed where administration affects clinical parameters of the autoimmune disease or disorder, such as the degree of tissue injury, whether or not the treatment leads to amelioration of the autoimmune disease, comprises "treatment" or "therapy" within the context of the invention.

[0214] "Therapeutically effective amounts" of the self-vector comprising polynucleotide encoding one or more self-protein(s), -polypeptide(s) or -peptide(s) is administered in accord with the teaching of this invention and will be sufficient to treat or prevent the disease as for example by ameliorating or eliminating symptoms and/or the cause of the disease. For example, therapeutically effective amounts fall within broad range(s) and are determined through clinical trials and for a particular patient is determined based upon factors known to the ordinarily skilled clinician including the severity of the disease, weight of the patient, age and other factors.

Therapeutically effective amounts of self-vector are in the range of about 0.001 micrograms to about 1 gram. A preferred therapeutic amount of self-vector is in the range of about

10 micrograms to about 5 milligrams. A most preferred therapeutic amount of self-vector is in the range of about 0.025 mg to 5 mg. Polynucleotide therapy is delivered monthly for 6- 12 months, and then every 3-12 months as a maintenance dose. Alternative treatment regimens may be developed and may range from daily, to weekly, to every other month, to yearly, to a one-time administration depending upon the severity of the disease, the age of the patient, the self- protein(s), -polypeptide(s) or -peptide(s) being administered and such other factors as would be considered by the ordinary treating physician.

[0215] For example, the method of treating an autoimmune disease by administering a combination of specific agents comprising a B cell specific agent in combination with a DNA antigen specific therapeutic agent comprising a polynucleotide encoding a self-polypeptide can modulate an immune response by eliminating, sequestering, or inactivating immune cells mediating or capable of mediating an undesired immune response; inducing, generating, or turning on immune cells that mediate or are capable of mediating a protective immune response; changing the physical or functional properties of immune cells; or a combination of these effects. Examples of measurements of the modulation of an immune response include, but are not limited to, examination of the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measurement of the functional capacity of immune cells including ability or resistance to proliferate or divide in response to a signal (such as using T cell proliferation assays and pepscan analysis based on ³H-thymidine incorporation following stimulation with anti-CD3 antibody, anti-T cell receptor antibody, anti-CD28 antibody, calcium ionophores, PMA, antigen presenting cells loaded with a peptide or protein antigen; B cell proliferation assays); measurement of the ability to kill or lyse other cells (such as cytotoxic T cell assays); measurements of the cytokines, chemokines, cell surface molecules, antibodies and other products of the cells (e.g., by flow cytometry, enzyme-linked immunosorbent assays, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of immune cells or signaling pathways within immune cells (e.g., Western blot and

immunoprecipitation analysis of tyrosine, serine or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array analysis; DNA transcriptional, profiling using DNA arrays or subtractive hybridization); measurements of cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assays, gel electrophoresis to measure DNA laddering, histology; fluorogenic caspase assays, Western blot analysis of caspase substrates); measurement of the genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarrays, protein microarrays, 2-dimentional gel electrophoresis, Western blot analysis, enzyme linked immunosorbent assays, flow cytometry); and measurement of clinical symptoms or outcomes such as improvement of autoimmune, neurodegenerative, and other diseases involving self proteins or self polypeptides (clinical scores, requirements for use of additional therapies, functional status, imaging studies) for example, by measuring relapse rate or disease severity (using clinical scores known to the ordinarily skilled artisan) in the case of multiple sclerosis, measuring blood glucose in the case of type I diabetes, or joint inflammation in the case of rheumatoid arthritis.

EXAMPLE 1

B Cell Specific Agent Treatment of T1D in the NOD Mouse

[0216] To perform these studies, 20 prediabetic 8-10 weeks old female Non-Obese diabetic- mice (NOD-mice) and recent onset type- 1 -diabetic (T1 D) female NOD-mice per group were used. Blood glucose was measured 2-times a week using ONE TOUCH ULTRA® glucose monitors (Lifespan Inc). Mice were considered diabetic when two consecutive readings exceed >250mg/dL of blood glucose were obtained. To determine the half-life of a murine monoclonal antibody that binds murine CD20 designated 5D2 (Genentech), 200 g of the antibody was administered intravenously (i.v.) into prediabetic NOD mice. 50 uL of blood were taken daily and the amount of anti-CD20 in serum was quantified by ELISA using a synthetic CD20-peptide containing its extracellular sequence from amino acids 140 to 184, with biotin fusion at amino acid 140, as described previously (P. J. Adamson, H. Zola, I. C. Nicholson, G. Pilkington, A. Hohmann, Leuk Res 25, 1047 (Dec, 2001 )). Mice were given varying doses (5, 10, 25, 50, or 100 μg) of the B cell specific agent, i.e., anti-CD20 mAb, only on day 1 (lx) or on days 1 , 4, 7 and 10 (4x) intravenously. Alternatively, mice not treated with an antibody or those mice treated with an isotype matched control antibody were used as controls. To determine the protection offered by anti-CD20 mAb treatment, blood glucose was monitored in treated and untreated mice twice weekly for 10 weeks. Mice were considered protected when the blood glucose values were <200ug/dL. Expression of insulin in the pancreas, 5 weeks post treatment, was determined by immunohistochemistry (IHC) staining of 6 μΜ frozen pancreatic sections with guinea-pig anti- swine insulin (1 :300) antibody, as described (D. Bresson et al , J Clin Invest 116, 1371 (May, 2006), U. Christen et al Clin Invest 114, 1290 (Nov, 2004)) To measure insulitis, frozen pancreatic sections were stained with anti-CD4 RM4.5 and anti-CD8a IHC (BD Biosciences) [dilution 1 :50] (D. Bresson et al, J Clin Invest 116, 1371 (May, 2006), U. Christen et al, J Clin Invest 114, 1290 (Nov, 2004)). To determine the mechanism of protection observed, frequencies and total numbers of various cell types in peripheral blood were determined on days 1 , 3, 8 and 15 post treatment by staining with anti-CD4, -CD8, and -IgM/-lgD (BD pharmingen) and analyzed by FACS. Further, splenocytes or PLN cells were surface-stained with anti-CD4, -CD8, -CD25, -CD 127, -CTLA4 ( 1 :20 dilution, BD Biosciences), followed by intracellular staining with anti-Foxp3 (ebioscience) or anti-GITR (R&D systems) (D. Bresson et al, J Clin Invest 116, 1371 (May, 2006)). Frequencies of nTreg cells were determined by analyzing the expression of Foxp3, CTLA4 or GITR in CD4⁺ CD127^low CD25⁺ or CD4⁺ CD25- cells, and as a control in CD8⁺ cells. Further, production of cytokines was assessed by ELISA, ELISPOT or ICS.

Splenocytes or PLN cells were stimulated for 3-days in vitro using anti-CD3/CD28 or isotype matched control antibodies (l ug/mL each). Culture supernatants were harvested and cytokines were quantified using IFN-γ, TNF-a, IL-10, IL-4 (BD pharmingen), TGF-βΙ (Quantikine kit, R&D systems) ELISA kits or using multiplexed cytokine assay (Beadlyte Mouse Multi-Cytokine Detection System; Upstate USA Inc), as per manufacturer's instructions. Intracellular cytokine staining and analysis were performed as described (D. Bresson et al., J Clin Invest 116, 1371 (May, 2006)). To enumerate adaptive insulin specific Tregs, cytokine assays (as described above) were performed following stimulation with insulin 9-23 and proinsulin peptides (IA^g7 restricted). Additionally, to determine the effect on autoaggressive CD8⁺ T cells, frequencies of antigen specific T cells were determined in the peripheral blood by FACS analysis, or their presence were detected in frozen pancreatic sections by IHC, using NRP-V7 tetramers (obtained from Pere Santamaria, University of Calgary, Canada) (D. Bresson et al., J Clin Invest 116, 1371 (May, 2006)). Functional status of these antigen specific T-cells were determined by stimulating them in vitro with anti-CD3/anti-CD28 (l ug/mL each) and measuring the production of IFN-γ and TNF-a by ELISA and ICS. And finally, to determine the minimal effective dose of the anti- CD20 antibody that induces partial remission of recent onset TI D, long-term protection in recent onset TI D mice after various treatments (l d/4d treatment with 10, 50 & 100 ug antibody i.v.) were determined. Blood glucose values were determined as earlier, and mice with <200 ug/dL of glucose in blood were considered cured. The dose of the antibody that induces partial remission (10-20% decrease in diabetes in recent onset TI D NOD-mice) was calculated from these analyses.

Results

[0217] Administration of anti-CD20 once was only protective when higher concentrations of the antibody were used (Fig. 1 A); administration of 5 or 10 μg anti-CD20 was not protective while 50 and 250 μg of anti-CD20 reduced the incidence of diabetes by 40% and 50% respectively. Further increasing the dose of anti-CD20 had no protection. Next, anti-CD20 was administered four times, on day 1 , 2, 3 and 4. At week 20, when at least 70% of the control mice have turned diabetic, most of the anti-CD20 treated mice stayed diabetes free (Fig. I B). At each dose tested, 5, 10, 50 and 100 μg anti-CD20, diabetes incidence is reduced by 75%. Thus, 4x anti-CD20 antibody treatment regimen offered the best protection observed using only anti- CD20.

EXAMPLE 2

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Treat T1D in the NOD Mouse

[0218] To perform these studies, groups of 20-40 recent-onset type- 1 -diabetic (T1 D) female NOD-mice or prediabetic (20 mice per group) 8-10 weeks old female NOD-mice were used. Blood glucose was measured 2-times a week using ONE TOUCH ULTRA® glucose monitors (Lifespan Inc). Mice were considered diabetic when two consecutive readings measure

>250mg/dL of glucose in blood. Mice were administered a single 10, 50 and 100 μg dose of B cell specific agent, i.e., murine anti-CD20 antibody 5D2. DNA plasmid vector encoding proinsulin (SEQ ID NO: 20) was prepared, (see WO 2007/044394) and used at >95% purity. Subsequent to the administration of the anti-CD20, mice were administered DNA plasmid vector encoding proinsulin on days 1 , 8, 15 and 22. Recent onset T1 D NOD-mice treated with anti- CD20 only, insulin only or untreated mice were included as controls. After combination therapy incidence of diabetes in these mice was monitored by measuring blood-glucose levels and the mice were considered protected when blood glucose values are <200ug/dL. The increase in efficacy following combination therapy over anti-CD20 or insulin peptide administration was determined as ([% protection with combination therapy - %protection with peptide only] / [% protection with peptide only])* 100 and data was evaluated in respect to the age of the mouse when it became diabetic as well as the blood glucose level at entry into the mouse trial (it has been observed that mice with BG>400mg/dl hardly ever revert from recent-onset diabetes, because the remaining beta cell mass is too low). In mice with increased efficacy of protection, pancreatic insulin expression, insulitis, and the effect on antigen specific CD8⁺ T cells was determined. To identify the cells that are responsible for tolerance induction, splenocytes or peripheral lymph node (PLN) cells or CD4⁺ or CD8⁺ or B220⁺ cells (purified with the respective auto-MACS purification kits from Miltenyi Biotec, USA) were used as described above.

Enumeration of various cell types by FACS and cytokine secretion profiles of these cells by ELISA or ICS was performed as described above. In co-culture studies, CFSE-labeled BDC2.5 CD8⁺ T cells were used as responders while DCs pulsed for six hours with DNA plasmid or controls will be used as antigen presenting cells (APC) to stimulate the responders. Responder CD8⁺ T cells (l xl O⁵) were mixed with an equal number of or serial dilutions of purified CD4 or CD8⁺ or B220⁺ cells. Following co-culture for 2-days, production of regulatory cytokines in the supernatants were quantified by ELISA and, inhibition of the proliferation of antigen specific T cells was determined by measuring CFSE dilution by FACS. nTregs and adaptive Tregs were enumerated and tested for cytokine secretion as described above. To determine if these regulatory cell population can transfer dominant tolerance to the recipients, either whole splenocytes, or PLN cells or purified CD4⁺ or CD8⁺ or B220⁺ cells was expanded in vitro for 3- days using polyclonal stimuli (such as l ug/mL anti-CD3/CD28 for T cells, or 1 μg/mL anti- B220/CD40 for B cells) or the DNA plasmid (1 μg/mL). Such expanded cells (5x10⁶) cells will be adoptively transferred into recent onset T1 D or prediabetic NOD mice and blood glucose levels will be monitored. Concurrently, such expanded cells themselves were labeled with CFSE and adoptively transferred into recipient mice. Two days after the transfer, pooled cells from spleen or PLN were stained with anti-CD4, anti-CD8 or anti-B220 (BD pharmingen) and expansion of the transferred cells in these locations were determined by measuring CFSE dilution. To determine the transfer of tolerance in a more stringent model of recent onset T1D, regulatory cell populations (5xl 0⁶ cells), expanded in vitro, were adoptively transferred into RIP- LCMV-NP mice, 5-days after the initiation of diabetes by LCMV-armstrong infection, as described. Protection from diabetes offered by the transferred cells were determined by measuring blood-glucose levels, by determining insulitis and by determining the extent of Ag- specific CD8⁺ T cell infiltration of the pancreas, by IHC, using NP 1 1 8 tetramers, as described (D. Bresson et al., J Clin Invest 116, 1371 (May, 2006)).

Results

[0219] To improve the efficacy of anti-CD20 mediated T l D treatment, we combined the administration of anti-CD20 with that of a DNA plasmid encoding proinsulin. l (day 1 ) intravenous administration of anti-CD20 was given in combination with either l x (dl ) or 4x (d l , 8, 15, 21 ) intra-muscular injections of DNA plasmid encoding proinsulin. The lowest dose of anti-CD20 used in these studies, 10 μg, did not offer any protection either alone or in

combination with proinsulin plasmid (Fig. 2A). Higher doses of anti-CD20, 50 (Fig. 2B) and 100 μg (Fig. 2C), both offered some protection from T1 D by themselves. Co-administering DNA self-vector encoding proinsulin with anti-CD20 increased the efficacy of protection. While 50^g anti-CD20 alone reduced the incidence of T1 D by 40%, co-administering DNA plasmid encoding proinsulin 4x further reduced the incidence by 75%. A one-time administration of anti- CD20 with one time administration of proinsulin peptide did not achieve better responses than anti-CD20 alone. Interestingly, when 100 μg anti-CD20 was used in combination with DNA plasmid encoding proinsulin plasmid, both lx administration of DNA plasmid encoding proinsulin and 4x administration of the DNA self-vector were able to reduce the incidence of diabetes by 75% exhibiting synergy compared to single-agent administration. Thus the combination therapy of anti-CD20 and DNA self-vector encoding proinsulin plasmid, is more efficacious than using either of these reagents alone.

[0220] Further experiments were performed to improve the efficacy of anti-CD20 mediated T1D prevention. For this set of experiments, the concentration of proinsulin plasmid was always the same, 50 μg per/animal per injection. The frequency of dosing was either lx (day 1 ) or 4x (dl , 2, 3 & 4) for i.v. anti-CD20 without (Fig. 3A) or with (Fig. 3B) continuous administration of proinsulin plasmid. When prediabetic NOD mice received only anti-CD20, the best protection was seen at the highest dose studied, such that 25% of the mice were protected upon receiving 100 μg anti-CD20. Surprisingly, administration of anti-CD20 only once or four times achieved similar protection in these mice (Fig. 3A, right panel). The lower doses studied did not offer significant protection from T1 D onset. Note that in our NOD colony, T1 D incidence reaches 90% or greater by 30 wks of age.

[0221] Interestingly, co-administering proinsulin plasmid with anti-CD20 increased the efficacy of protection. At lower doses studied, 10 and 50 μg anti-CD20, proinsulin coadministration was able to help prevent the onset of Tl D in more mice than anti-CD20 alone (Fig. 3B, left and middle panels). When 50 μg anti-CD20 was administered alone, there was no protection observed (Fig. 3A, middle panel). However, co-administration of proinsulin plamid protected -50% of the mice from disease onset (Fig. 3B, middle panel). Increasing the dose of anti-CD20 further did not result in any increased protection (Fig. 3B, right panel).

[0222] Frequent dosing of anti-CD20 does not favor combination therapy. In order to find the optimal doses of anti-CD20 and Proinsulin to achieve the highest synergy, in this set of experiments we varied the frequency of administration of both anti-CD20 and Proinsulin plasmid. Anti-CD20 administration was performed only once (day 1 , Fig. 4A, C) or four times (Fig. 4B). Similarly, Proinsulin plasmid was administered either only once (Fig. 4A, B) or four times (Fig. 4C). Frequent dosing of anti-CD20, in combination with a single dose of Proinsulin was not beneficial as this dosing frequency did not show any protection from T1 D onset (Fig. 4B). Suprisingly, a one time administration of anti-CD20 in combination with either a one time (Fig. 4A) or four times (Fig. 4C) administration of Proinsulin plasmid exhibited synergy in protecting the NOD mice from T1 D onset. Further, a four-time administration of Proinsulin plasmid exhibited more synergy than a one time administration of Proinsulin plasmid.

[0223] The observation that a single dose of 100 μg anti-CD20 combined with one

administration of proinsulin plasmid was able to exhibit synergy is a significant finding. Thus, the combination therapy of anti-CD20 and proinsulin plasmid is more efficacious than using either of these reagents alone.

EXAMPLE 3

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Treat Pemphigus Vulgaris (PV)

[0224] Pemphigus vulgaris (PV) is a skin blistering disease caused by a targeted autoantibody immune response against the self protein, desmoglein III (DMGIII) (Perez and Patton. Drugs Aging.. 26:833-46, 2009). Current first line therapies include high dose systemic corticosteroids (e.g., prednisone l mg/kg/day) . Steroid-sparing immunosuppressives such as azathioprine, cyclosphosphamide, mycophenolate mofetil are also utilized extensively. Rituximab (anti-CD20) has been used to treat pemphigus vulgaris that is refractory to conventional therapy (Fernando and Broadfoot. G. Ital. Dermatol. Venereol. 144:363-77, 2009.) . Intravenous immunoglobulin (IVIG) may be useful as monotherapy in patients who do not respond to steroids or who have contraindications for Rituximab.

[0225] Human desmogleinlll (DMGIII) is a protein of 107.53 molecular weight and its nucleotide sequence is set forth in SEQ ID NO: 3. An animal model of PV used to screen potential therapeutics for treating PV is developed using anti-DMGIII antibodies obtained from immunized DMGIII knockout mouse (Koch et al. J Cell Biol. 137: 1091 -1 102, 1997; Amagai et al. J Clin. Invest.. 105:625-631 , 2000). Anti- DMGIII antibodies are adoptively transferred to wild type mice that subsequently develop skin lesions similar in pathophysiology to those in patients with PV.

[0226] For experiments demonstrating efficacy of sequential or simultaneous therapy of an anti-B cell specific agent, such as anti-CD20, combined with a DNA antigen specific therapeutic agent, a pDNA self-vector is constructed encoding the self-antigen polypeptide, desmoglein III (SEQ ID NO: 1 ), comprising the appropriate control and regulatory elements (See for exampleWO 2007/044394). A second pDNA self-vector is constructed encoding DMGIII non- secreted (SEQ ID NO: 2) and is also tested as follows. Treatment commences in DMGIII knockout animals after antibodies to DMGIII are induced and detectable by ELISA. A dose range is tested from 5 to 250ug/mouse of anti-CD20 alone defining the kinetics of response and extent of disease modification of anti-CD20 in this disease model. A sub-optimal and optimal dose level of anti-CD20 is tested in combination with pDNA self vector encoding DMGIII. A sub-optimal dose of anti-CD20 mAb is evaluated to identify a lower dose of anti-CD20 mAb for administration in combination with the DNA antigen specific therapeutic agent encoding DMGIII to improve the anti-CD20 mAb's safety profile while maintaining efficacy. Anti- CD20 mAb (both sub-optimal and optimal dose levels) and pDNA self vector encoding DMGIII is administered to DMGIII knock out mice randomized to 7 treatment groups based on antibody levels. The treatment regimens are shown in Table 3.

[0227] Group 1 is left untreated. Three groups are treated with sub-optimal anti-CD20 mAb, including two with the combination therapy of this invention, and three groups are treated with the optimal anti-CD20 mAb dose, including two with the combination therapy of this invention The effect of the anti-CD20 mAb alone is seen in Groups 2 and 5. The effect of combination therapy, by combining the pDNA self vector encoding autoantigen with the anti-CD20 mAb is seen in groups 3, 4, 6 and 7. The effect of the timing of the administration (simultaneous therapy versus sequential therapy) on the efficacy is demonstrated by comparing groups 3 with 4 and 6 with 7. pDNA self vector is administered weekly by IM injection (5C^g/mouse, 25μg/quad). For groups 4 and 7, the pDNA therapy is started at 2, 4 or 6 weeks after the anti-CD20 mAb is administered. Once started, the pDNA therapy is continued weekly until sera is collected for the adoptive transfer.

[0228] Effects on anti-DMGIII antibody levels are evaluated in sera collected from DMGIII knockout animals . Autoantibody levels to DMGIII is monitored by ELISA using DMGIII protein or peptide as the antigen. Adoptive transfer of serum collected from treated mice to wild type mice is performed. Efficacy is demonstrated in recipient mice by monitoring body weight and histopathologic scoring of skin lesions. (Takae et al. Exp. Dermat.. 18:252-60, 2009).

EXAMPLE 4

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Treat Multiple Sclerosis (MS)

[0229] The experimentally induced autoimmune encephalomyelitis (EAE) mouse model is used to evaluate compounds for treating multiple sclerosis. EAE is induced in animals of different background following immunization of mice with adjuvant in combination with myelin proteins or peptides. Disease ensues within 2 weeks of immunization. Scoring scales have been established which measure the extent of limb paralysis and histological evaluations have demonstrated lymphocytic infiltrate in the brains of mice with EAE. EAE mice have been used in preclinical studies to validate and develop certain MS drugs such as Copaxone (Webb et al. Immunochemistry.,13(4):333-7, \ 976.), and Tysabri (Yednock et al. Nature. ,356:63-6, 1992) confirming the usefulness of this model in drug discovery.

[0230] Antigen specific therapy has been previously demonstrated to decrease symptoms of EAE. One of the myelin proteins targeted during the autoimmune response in EAE is the protein, proteolipid protein or PLP. Using the pDNA self-vector approach, pDNA self-vector encoding PLP has demonstrated ability to lower relapse rates in preclinical EAE studies (Garren et al. Immunity., 15(1 ): 15-22, 2001 ) in mice, however, animals continue to have symptoms of disease and could benefit from combination therapy of a DNA antigen specific therapeutic agent combined with another immunomodulatory compound such as B cell specific agent.

[0231] Murine PLP is a polypeptide of 30.08 molecular weight. For experiments

demonstrating efficacy of combination therapy using a B cell specific agent, such as anti-B cell antibody, in combination with a pDNA antigen specific therapeutic agent, EAE is induced in the SJL mouse model using PLP peptide emulsified in CFA. A DNA self-vector is constructed encoding the self antigen polypeptide, proteolipid protein, PLP (SEQ ID NO: 5), comprising the appropriate control and regulatory elements (See WO 2007/044394). The anti-CD20 monoclonal antibody is tested across a dose range from 5 to 250ug/mouse of anti-CD20 alone defining the kinetics of response and extent of disease modification of anti-CD20 in this model. A sub-optimal and optimal dose level of anti-CD20 mAb is tested alone and in combination with pDNA self-vector encoding PLP. A sub-optimal dose of anti-CD20 mAb is evaluated to identify a lower dose of anti-CD20 mAb for administration in combination with the DNA antigen specific therapeutic agent encoding PLP to improve the anti-CD20 mAb's safety profile while maintaining efficacy. Anti-CD20 (both sub-optimal and optimal dose levels) and pDNA self- vector encoding PLP is administered to EAE mice randomized to seven treatment groups based on disease severity. The treatment regimen is shown in Table 3 above.

[0232] Group 1 is left untreated. Three groups are treated with sub-optimal anti-CD20 mAb, and two groups are treated with the optimal anti-CD20 mAb dose. The effect of the anti-CD20 mAb alone is seen in Groups 2 and 5. The effect of combination therapy by combining the pDNA self-vector encoding autoantigen with anti-CD20 mAb is seen in groups 3, 4, 6 and 7. The effect of the timing of the administration (simultaneous therapy versus sequential therapy) on the efficacy is demonstrated by comparing groups 3 with 4 and 6 with 7. Once started, the pDNA antigen specific therapy is continued weekly for the length of the study .The pDNA self- vector is administered weekly by IM injection at a 50ug/mouse dose (25ug/quad). For groups 4 and 7, the pDNA antigen specific therapy is started at 2, 4 or 6 weeks after the anti-CD20 is administered. Once started, the pDNA therapy is continued weekly for the length of the study.

[0233] Effectiveness of the combination approach is determined by comparing disease scores and brain histology from mice that received only the anti-CD20 antibody with animals that received combination therapy with anti-CD20 mAb and pDNA self-vector encoding PLP. EXAMPLE 5

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Treat Rheumatoid Arthritis (RA)

[0234] The experimentally induced mouse model of arthritis which specifically targets citrullinated fibrinogen (FIA) has recently been established (Ho et al. J. Immunol. 184:379-90, 2010.). Antibodies to citrullinated proteins are readily detectable in patients with rheumatoid arthritis and the development of this mouse model will aid in the development of antigen specific tolerization approaches for this debilitating disease. Mice that are induced to develop FIA possess fibrinogen-reactive T cells that produce proinflammatory cytokines, however the disease appears to be antibody mediated since the disease can be adoptively transferred to naive animals by plasma alone.

[0235] Fibrinogen is a polypeptide of 69.76 molecular weight. For experiments demonstrating efficacy of concurrent or sequential combination therapies of anti-CD20 and pDNA self-vector encoding autoantigen in FIA mice. A DNA self-vector is constructed encoding the self antigen polypeptide, fibrinogen (SEQ ID NO:6), comprising the appropriate control and regulatory elements (See WO 2007/044394). The anti-CD20 monoclonal antibody is tested across a dose range from 5 to 250ug/mouse of anti-CD20 alone defining the kinetics of response and extent of disease modification of anti-CD20 in this model. A sub-optimal and optimal dose level of anti- CD20 mAb is tested alone and in combination with pDNA self-vector encoding fibrinogen. A sub-optimal dose of anti-CD20 mAb is evaluated to identify a lower dose of anti-CD20 mAb for administration in combination with the DNA antigen specific therapeutic agent encoding fibrinogen to improve the anti-CD20 mAb's safety profile while maintaining efficacy. Anti- CD20 (both sub-optimal and optimal dose levels) and pDNA self vector encoding fibrinogen is administered to EAE mice randomized to seven treatment groups based on disease severity. The treatment regimen is shown in Table 3 above.

[0236] Group 1 is left untreated. Three groups are treated with sub-optimal anti-CD20 mAb, and two groups are treated with the optimal anti-CD20 mAb dose. The effect of the anti-CD20 mAb alone is seen in Groups 2 and 5. The effect of combination therapy by combining the pDNA self vector encoding autoantigen with anti-CD20 mAb is seen in groups 3, 4, 6 and 7. The effect of the timing of the administration (simultaneous therapy versus sequential therapy) on the efficacy is demonstrated by comparing groups 3 with 4 and 6 with 7. Once started, the pDNA antigen specific therapy is continued weekly for the length of the study. The pDNA self vector is administered weekly by IM injection at a 50ug/mouse dose (25ug/quad). For groups 4 and 7, the pDNA antigen specific therapy is started at 2, 4 or 6 weeks after the anti-CD20 is administered. Once started, the pDNA therapy is continued weekly for the length of the study.

[0237] Effectiveness of the combination approach is determined by comparing disease scores and joint histology from mice that received only the anti-CD20 antibody with animals that received combination therapy of anti-CD20 mAb and pDNA self vector encoding fibrinogen.

EXAMPLE 6

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Idiopathic Pulmonary Fibrosis (IPF)

[0238] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive autoimmune disease that is ultimately a fatal lung disease of unknown etiology. Its prognosis is poor and the outcome even worse than in many malignant diseases. The bleomycin mouse model of IPF (bIPF) has been used for decades as a model system for evaluating therapeutics for evaluation in clinical studies (Moeller et al., Int. J. Biochem Cell Biol. 2008. 40(3):362-382). When administered to the lungs, bleomycin causes inflammatory and fibrotic reactions within a short period of time. Proinflammatory cytokines are elevated followed by increased expression of pro-fibrotic markers with a peak at about 14 days. The switch between inflammation and fibrosis occurs about day 9. Part of the inflammation that is induced is thought to be the result of an autoimmune response to collagen V that is exposed during the remodeling of lung tissue after exposure to injury (Wilkes, DS., Arch. Immunol. Ther. Exp. 2003. 51 :227-30).

[0239] Collagen V is a polypeptide of 183.56 molecular weight. For experiments

demonstrating efficacy of concurrent or sequential combination therapies of anti-CD20 and pDNA self-vector encoding autoantigen in bleomycin induced IPF mice (bIPF) a DNA self- vector is constructed encoding the self-antigen polypeptide, collagen V (SEQ ID NO: 10), comprising the appropriate control and regulatory elements (See WO 2007/044394). A second pDNA self-vector is constructed encoding collagen V non-secreted and is also tested as follows. The anti-CD20 monoclonal antibody is tested across a dose range from 5 to 250ug/mouse of anti-CD20 alone defining the kinetics of response and extent of disease modification of anti- CD20 in this model. A sub-optimal and optimal dose level of anti-CD20 mAb is tested alone and in combination with pDNA self-vector encoding collagen V. A sub-optimal dose of anti- CD20 mAb is evaluated to identify a lower dose of anti-CD20 mAb for administration in combination with the DNA antigen specific therapeutic agent encoding collagen V to improve the anti-CD20 mAb's safety profile while maintaining efficacy. Anti-CD20 (both sub-optimal and optimal dose levels) and pDNA self vector encoding collagen is administered to EAE mice randomized to seven treatment groups based on disease severity. The treatment regimen is shown in Table 3 above.

[0240] Group 1 is left untreated. Three groups are treated with sub-optimal anti-CD20 mAb, and two groups are treated with the optimal anti-CD20 mAb dose. The effect of the anti-CD20 mAb alone is seen in Groups 2 and 5. The effect of combination therapy by combining the pDNA self vector encoding autoantigen with anti-CD20 mAb is seen in groups 3, 4, 6 and 7. The effect of the timing of the administration (simultaneous therapy versus sequential therapy) on the efficacy is demonstrated by comparing groups 3 with 4 and 6 with 7. Once started, the pDNA antigen specific therapy is continued weekly for the length of the study. The pDNA self vector is administered weekly by IM injection at a 50ug/mouse dose (25ug/quad). For groups 4 and 7, the pDNA antigen specific therapy is started at 2, 4 or 6 weeks after the anti-CD20 is administered. Once started, the pDNA therapy is continued weekly for the length of the study.

[0241] Effectiveness of the combination approach is determined by comparing disease scores and histopathological evaluation of lung tissues from mice that received only the anti-CD20 antibody with animals that received combination therapy of anti-CD20 mAb and pDNA self vector encoding collagen V.

EXAMPLE 7

B Cell Specific Agent Combined with a DNA Antigen Specific Therapeutic Agent to Acetylcholine Receptor (AChR)

[0242] Myasthenia gravis (MG) is a chronic progressive autoimmune disease characterized by an autoantibody response to the alpha chain of the nicotinic acetylcholine receptor (AChR) that leads to fluctuating muscle weakness and fatiguability (Grob et al. Muscle Nerve. 2008.

37: 141 -49).

[0243] The disease is mediated by circulating autoantibodies that block acetylcholine receptors at the post-synaptic neuromuscular junction inhibiting the stimulative effect of the

neurotransmitter acetylcholine. Myasthenia is treated medically with cholinesterase inhibitors or immunosuppressants, and, in selected cases, thymectomy. Recently, anti-CD20 has been used in some patients with MG {Chan et al. J. Neurol. 2007. 254: 1604-06). [0244] Development of therapeutics for myasthenia gravis has been aided by the establishment of an induced model of MG called experimentally induced autoimmune myasthenia gravis (EAMG) in rats. EAMG is induced by immunization of rats with purified acetylcholine receptor (usually purified from torpedo fish) emulsified in CFA. Animals demonstrate signs of muscle fatigability after exercise and a clinical disease scoring system has been established to monitor severity of disease.

[0245] AChR is a polypeptide of 51 .84 molecular weight. For experiments demonstrating efficacy of simultaneous or sequential combination therapies of anti-CD20 mAb and pDNA self- vector encoding autoantigen, a DNA self-vector is constructed encoding the self-antigen polypeptide acetylcholine receptor (SEQ ID NO: 14) comprising the appropriate control and regulatory elements (See WO 2007/044394). The anti-CD20 monoclonal antibody is tested across a dose range from 5 to 250ug/rat of anti-CD20 alone defining the kinetics of response and extent of disease modification of anti-CD20 mAb in the EAMG model. A sub-optimal and optimal dose level of anti-CD20 mAb is tested alone and in combination with pDNA self-vector encoding AChR. A sub-optimal dose of anti-CD20 mAb is evaluated to identify a lower dose of anti-CD20 mAb for administration in combination with the DNA antigen specific therapeutic agent encoding AChR to improve the anti-CD20 mAb's safety profile while maintaining efficacy. Anti-CD20 (both sub-optimal and optimal dose levels) and pDNA self vector encoding AChR is administered to EAMG rat randomized to seven treatment groups based on disease severity. The treatment regimen is shown in Table 3 above.

[0246] Group 1 is left untreated. Three groups are treated with sub-optimal anti-CD20 mAb, and two groups are treated with the optimal anti-CD20 mAb dose. The effect of the anti-CD20 mAb alone is seen in Groups 2 and 5. The effect of combination therapy by combining the pDNA self vector encoding autoantigen with anti-CD20 mAb is seen in groups 3, 4, 6 and 7. The effect of the timing of the administration (simultaneous therapy versus sequential therapy) on the efficacy is demonstrated by comparing groups 3 with 4 and 6 with 7. Once started, the pDNA antigen specific therapy is continued weekly for the length of the study. The pDNA self vector is administered weekly by IM injection at a 50ug/rat dose (25ug/quad). For groups 4 and 7, the pDNA antigen specific therapy is started at 2, 4 or 6 weeks after the anti-CD20 is administered. Once started, the pDNA therapy is continued weekly for the length of the study. [0247] Effectiveness of the combination approach is determined by comparing disease scores, muscle fatiguability and histopathological evaluation of muscle tissues from rats that received only the anti-CD20 antibody with animals that received combination therapy of anti-CD20 mAb and pDNA self vector encoding AChR.

EXAMPLE 8

Immunosuppressive Agent Combined with a DNA Antigen Specific Therapeutic Agent to Acetylcholine Receptor (AChR)

[0248] This example provides data showing that DNA vaccine (rBHT-3034) and corticosteroid (Solu-Medrol) combination therapy is more effective than either monotherapy at suppressing disease scores in a rat model of EAMG.

[0249] Previous studies have demonstrated that weekly dosing of a DNA vaccine encoding a non-secreted form of the rat acetylcholine receptor subunit alpha protein was effective at reducing disease scores in a rat experimental autoimmune myasthenia gravis (EAMG) model. Similarly, a published study has shown dose dependent benefits of corticosteroid therapy in rat EAMG. In this study, we evaluated the efficacy of DNA vaccine/corticosteroid combination therapy in a rat EAMG model. As shown in Figure 5, the rats receiving rBHT-3034/Solu-medrol combination therapy show a statistically significant reduction in disease scores relative to the PBS treated control group and the rBHT-3034 and Solu-Medrol monotherapy groups.

[0250] EAMG was induced in Lewis rats as previously described. Prior to the onset of disease, animals were randomized to treatment groups and dosing of Solu-Medrol began 7 days post immunization. Weekly IM injections of rBHT-3034 began at Day 28. Animals were scored weekly for disease and disease scores are plotted above ± SEM. P-values <0.05 were obtained from days 76-105 comparing the Solu-Medrol+rBHT-3034 to the PBS control group using a ruskal-Wallis test.

[0251] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1 . A method for the treatment of an autoimmune disease in a mammal, the method comprising administering to the mammal a therapeutically effective amount of an immunosuppressive or immunomodulatory agent in combination with a DNA antigen specific therapeutic agent encoding a self-protein.

2. The method according to claim 1 wherein the autoimmune disease is type 1 diabetes.

3. The method according to claim 1 wherein the autoimmune disease is multiple sclerosis.

4. The method according to claim 1 wherein the autoimmune disease is rheumatoid arthritis.

5. The method according to claim 1 wherein the autoimmune disease is pemphigus vulgaris.

6. The method according to claim 1 wherein the autoimmune disease is myasthenia gravis.

7. The method according to claim 1 wherein the administration of the immunosuppressive or immunomodulatory agent and the DNA antigen specific therapeutic agent is simultaneous.

8. The method according to claim 1 wherein the administration of the immunosuppressive or immunomodulatory agent and the DNA antigen specific therapeutic agent is sequential.

9. The method according to claim 1 wherein the self-protein is associated with the autoimmune disease.

10. The method according to claim 1 , wherein the immunomodulatory agent is a B cell specific agent.

1 1. The method according to claim 10 wherein the B cell specific agent is a CD20 antibody.

12. The method according to claim 1 1 wherein the CD20 antibody is selected from the group consisting of rituximab, ocrelizumab, ofatumumab, and veltuzumab.

13. The method according to claim 2 wherein the self-protein encoded by the DNA antigen specific therapeutic agent is selected from the group consisting of insulin, proinsulin, glutamic acid decarboxylase 65 or islet cell antigen.

14. The method according to claim 13 wherein the self-protein encoded by the DNA antigen specific therapeutic agent is proinsulin.

15. The method according to claim 3 wherein the self-protein encoded by the DNA antigen specific therapeutic agent is selected from the group consisting of myelin basic protein, proteolipid protein, myelin associated glycoprotein, cyclic nucleotide phosphodiesterase, myel in-associated glycoprotein, myelin-associated oligodendrocytic basic protein; alpha-B- crystalin or myelin oligodendrocyte glycoprotein

16. A method for the treatment of type I diabetes (T 1 D) in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a CD20 antibody in combination with a DNA plasmid encoding proinsulin.

17. The method of 16 wherein the CD20 antibody is rituximab and the DNA plasmid encoding proinsulin is proinsulin tolerizing plasmid (PTP).

1 8. The method of claim 16 wherein administration of the CD20 antibody and the DNA plasmid encoding proinsulin is simultaneous or sequential.

19. The method of claim 16 wherein the combined administration is synergistic compared to administration of either agent alone.

20. The method of claim 16 wherein the combined administration reduces the frequency of administration of the CD20 antibody as compared to administration of the CD20 antibody alone.

21 . The method of claim 16 wherein the mammal is a human.

22. A pharmaceutical composition comprising in an amount effective for the treatment of an autoimmune disease: (a) a CD20 specific agent; (b) a DNA antigen specific therapeutic agent and (c) a pharmaceutically acceptable carrier.

23. A kit comprising (a) a CD20 specific agent; (b) a DNA antigen specific therapeutic agent and (c) a pharmaceutically acceptable carrier.