WO2010129959A1 - Compositions and methods for the prevention and treatment of lupus nephritis using anti-dsdna germline antibodies - Google Patents

Abstract

The invention provides IgM germline anti-dsDNA antibodies and their use in the prevention, amelioration, and treatment of lupus nephritis. As demonstrated herein, germline IgM antibodies that bind specifically to dsDNA were demonstrated to have a protective effect in preventing or ameliorating the development of lupus nephritis in the MRL/lpr mouse model of lupus. The effect was demonstrated to be specific to germline IgM antibodies rather than hypermutated antibodies, and further to antibodies with dsDNA antigen specificity.

Description

COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF LUPUS NEPHRITIS USING ANTI- dsDNA GERMLINE ANTIBODIES

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported in part by the National Institutes of Health Intramural Research Program, NIEHS, 1ZIAES101603-07. The government has certain rights in the invention.

CROSS REFERENCE TO RELATEDAPPLICATIONS

This application claims priority to US Provisional Patent Application Serial No. 61/176,615, filed on May 8, 2009. The application is incorporated by reference herein in its entirety.

BACKGROUND

MRL-Faslpr/lpr (MRL/lpr) mice develop a systemic autoimmune syndrome that shares many characteristics of human systemic lupus erythematosus (SLE) and is an accepted experimental model of the disease. Like the human disease, the MRL/lpr syndrome is characterized by polygenic inheritance, the presence of circulating autoantibodies, particularly to nuclear components, and lupus nephritis development through glomerular disease, mononuclear cell infiltration, and immune complex deposition. MRL/lpr mice also develop splenomegaly and lymphadenopathy, with mononuclear cell infiltration in lungs, liver, and other tissues. Multiple factors have been implicated in the development of this disease such as breakdown in lymphocyte tolerance, complement defects and defective apoptosis. Unlike human SLE with low monozygotic twin concordance, all MRL/lpr mice eventually develop the syndrome. Multiple loci contribute to autoimmunity in MRL/lpr mice, suggesting the involvement of various systems. Implicated are defects in B and T cell tolerance, complement activation, cytokine regulation, endothelial cell function, and apoptotic clearance.

Early work delineated the characteristics of autoantibodies in MRL/lpr mice but it was not clear whether autoantibodies were correlated with the syndrome in these mice or were actively contributing to disease development. Several pieces of evidence conclusively demonstrated an active role for autoantibodies and B cells in the lupus-like syndrome. These included the development of transgenic mice rendered autoimmune by the expression of autoreactive B -cell specificities, the identification of defects associated specifically with B-cell tolerance, and studies demonstrating that B cells play multiple (key) roles in the development of the lupus syndrome associated with MRL/lpr mice, both as secretors of autoantibodies and as cells that can stimulate autoreactive T lymphoytes. Additional studies strongly implicated defects in several B-cell tolerance checkpoints such as in early B-cell development and in peripheral tissues. That immunoglobulin G (IgG) autoantibodies are required for kidney damage is suggested by the reduction in glomerular injury in mice that are deficient in FcRc and FccRIII. Furthermore, it has been demonstrated that the kidney pathology associated with MRL/lpr mice is critically dependent on the presence of the activation-induced deaminase (AID) protein that triggers the generation of somatically mutated, high-affinity, isotype switched autoantibodies.

There is compelling evidence for a role of B cells in the MRL/ lpr syndrome, particularly affecting glomerulonephritis. B cell-deficient- MRL/lpr mice failed to develop glomerulonephritis. Also important in the development of lupus nephritis is a diverse lymphocyte repertoire, because MRL/lpr mice lacking terminal deoxynucleotidyl transferase, an enzyme that adds nucleotides to the V(D)J segments during recombination, have decreased glomerular disease. However, how B cells contribute to lupus nephritis might be more complicated than previously appreciated. In addition to secreting autoantibodies, B cells might contribute to lupus nephritis as antigen presenting cells (APCs) to autoreactive T cells and by promoting an inflammatory environment. MRL/lpr mice lacking secreted antibodies but with B cells bearing IgM receptors still develop a milder form of kidney disease and experience higher mortality rates than mice completely lacking B cells. A hallmark feature of MRL/lpr mice lacking B cells is a dramatic increase in the proportion of naive CD4⁺ T cells with a concomitant decrease in memory or activated T cells that was reconstituted to levels similar to those of conventional MRL/lpr mice in mice with B cells but without secreted antibodies.

These results suggest an additional, autoantibody-independent B cell role in the development of lupus nephritis in MRL/lpr mice, likely through the activation of autoreactive T cells. An aspect of B cell biology that impacts autoimmunity is the memory B cell response. B cells jointly activated by antigen and CD4⁺ T cells seed germinal centers (GCs) in secondary lymphoid tissues wherein their affinity to foreign antigen is enhanced by immunoglobulin (Ig) somatic hypermutation (SHM) and cellular selection. Isotype class switch recombination (CSR) also occurs in the GC environment, although not exclusively. In SHM, base pair substitutions are introduced into the DNA encoding the V regions of Ig receptors. Follicular dendritic cells provide foreign antigen to B cells in the GCs, selecting B cells with affinity-enhancing mutations to antigen in their receptors. Multiple rounds of division, mutation, and selection generate highly specific memory B cells. Interestingly, a majority of autoantibodies in patients with SLE and in MRL/lpr mice are hypermutated and isotype switched. In MRL/lpr mice, antibodies with mutations in the H chain Ig V region correlate with anti-dsDNA specificity, particularly those introducing arginines into the CDRs. One could envision that because SHM is random in relation to affinity, occasionally new mutations increase affinity to self-antigens or, catastrophically, that self-antigens drive the affinity maturation reaction. Evidence of the latter scenario is found in diseases such as rheumatoid arthritis, myasthenia gravis, and Sjogren's syndrome with ectopic GC formation resulting in high affinity autoantibodies against local self- antigens. The discovery of activation-induced deaminase (AID), a molecule critical to SHM and CSR, provides a direct approach at examining the contribution of mutated, class-switched antibodies to the MRL/lpr mice syndrome. Because AID is required for SHM and CSR, AID deficiency blocks the formation of high affinity, isotype-switched antibodies in activated B cells without impacting B or T cell development. These resulted in mice whose entire B cell repertoire was composed of unmutated IgM-bearing B cells,.

SUMMARY OF THE INVENTION

The invention provides anti-dsDNA germline IgM antibodies and methods of use of the antibodies, for example for the prevention and treatment of lupus erythematosus and its associated conditions, especially lupus nephritis.

The invention provides the use of an IgM germline antibody that binds specifically to dsDNA for preparation of a medicament for use in methods of prevention, amelioration, or treatment of lupus nephritis by providing a therapeutically effective amount of IgM germline antibody that specifically binds double stranded DNA and lupus nephritis is prevented, ameliorated, or treated. In an embodiment the effective dose of an antibody is an isolated population of antibodies enriched for an IgM germline antibody, for example a monoclonal germline antibody. Such antibodies include, but are not limited to monoclonal IgM germline antibodies produced by a hybridoma cell lines such as Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, and 16CJ-Hom5G91G12 defined by an Accession Numbers deposited at an appropriate cell repository. Antibodies for use in the methods of the invention also include IgM monoclonal antibodies including the variable heavy chain (V_H) or the variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line from any of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12. In an embodiment the V_H and V_L from a single antibody are paired (e.g., Horn 12H5 V_H with Horn 12H5 V_L). Antibodies for use in the methods of the invention also include IgM monoclonal antibodies including the CDRs of the variable heavy chain (V_H) or the variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line from any of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ- Hom5G91G12. In an embodiment the CDRs from the V_H and V_L chains from a single antibody are paired (e.g., Horn 12H5 V_H with Horn 12H5 V_L). Antibodies for use in the methods of the invention can further include IgM monoclonal antibodies having a V_H sequence at least 80% , 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a V_H of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, or 16CJ-Hom5G91G12, preferably paired with a corresponding V_L of at least 80% identical to a V_L of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, or 16CJ- Hom5G91G12. For example, the antibody can include a V_n at least 80% identical to Homl2H5_VH (SEQ ID NO: 2) and a V_L sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4); or a V_H at least 80% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a V_L at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); or a V_H sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10). In an embodiment, the invention provides for the use of antibodies in which the CDRs of any one of the antibodies can be placed in the context of a human IgM antibody framework to produce a humanized antibody. The changes from the sequences provided can be in the CDRs or in the framework regions or a combination thereof.

The invention provides methods for administration of a purified anti-dsDNA germline IgM antibody or an enriched population of anti-dsDNA germline IgM antibodies for the prevention, amelioration, or treatment of lupus nephritis. In an alternative embodiment, the invention provides methods for administration of a cell expressing an anti-dsDNA germline IgM for the prevention, amelioration, or treatment of lupus nephritis. In yet another embodiment, the invention provides for the administration of an agent to stimulate production of an anti-dsDNA germline IgM antibody by the subject for the prevention, amelioration, or treatment of lupus nephritis.

The methods of the invention can further include selecting a subject susceptible to or suffering from lupus nephritis and/ or monitoring the subject for prevention, amelioration, or treatment of lupus nephritis. In an embodiment, methods for prevention, amelioration, or treatment of a subject result in at least one of: significantly reducing a level of circulating immune complexes or a level of at least one inflammatory cytokine in serum of the subject; significantly increasing a level of at least one anti-inflammatory cytokine in a subject; and significantly decreasing proteinurea in a subject as compared to administration of an equivalent dose of a mature IgM that has undergone hypermutation, or as compared to a subject not administed an antibody of the invention. Inflammatory cytokines include, for example, TNF-alpha, and an anti-inflammatory cytokines include, for example, IL -4, IL-6, and IL-10. In an embodiment, the methods of the invention significantly reduces the level of macrophage activation in the subject as compared to administration of an equivalent dose of a mature IgM that has undergone hypermutation. The methods of the invention further include monitoring a subject for changes in one of the signs or symptoms of lupus nephritis, and/ or for a change in the level of one or more inflammatory cytokines.

The invention further provides compositions to practice the methods of the invention, and the use of such compositions for the preparation of a medicament, for example for the prevention, amelioration, or treatment of lupus nephritis.

In an embodiment, the invention provides composition including an isolated population of antibodies enriched for an IgM germline antibody that specifically binds double stranded DNA. The population of antibodies may include a population of antibodies having a single amino acid sequence (i.e., a single antibody) or a mixture of different antibodies (i.e., having different amino acid sequences) that are all germline anti-dsDNA IgM antibodies. Such antibodies include, for example, the monoclonal IgM germline antibody comprises an antibody produced by a hybridoma cell line such as Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, or 16CJ-Hom5G91G12 defined by an Accession

Numbers deposited at an appropriate cell repository. Such antibodies further include antibodies IgM monoclonal antibodies having a V_H and V_L, prefereably a paired V_H and V_L sequence of an antibody produced by a hybridoma cell line such as Horn Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ- HomlE21H3, Horn, or 16CJ-Hom5G91G12. Antibodies provided by the invention also include IgM monoclonal antibodies including the CDRs of the paired the variable heavy chain (V_H) and variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line from any of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12. Antibodies provided by the invention can also include IgM monoclonal antibodies having a V_H sequence at least 80% , 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, Horn, or 16CJ-Hom5G91G12, paired with a corresponding V_L of at least 80% identical to a V_L of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, or 16CJ-

Hom5G91G12. For example, the antibody can include a V_H at least 80% identical to Homl2H5_VH (SEQ ID NO: 2) and a V_L sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4); or a V_H at least 80% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a V_L at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence

Homl3D2_VL (SEQ ID NO: 8); or a V_H sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10). In an embodiment, the invention provides for the use of antibodies in which the CDRs of any one of the antibodies can be placed in the context of a human IgM antibody framework to produce a humanized antibody. The invention further provides a cell line expressing any one of the antibody compositions of the invention.

The invention further provides an antibody of the instant invention in pharmaceutically acceptable carrier.

The invention further provides kits having at least one antibody of the instant invention in appropriate packaging, optionally further including instructions for use.

The invention further provides method for providing a prognosis for a subject suffering from or suspected of suffering from lupus nephritis by obtaining a serum sample from the subject and detecting a germline anti-dsDNA IgM germline antibody in the subject serum sample, wherein detection of at least one germline anti-dsDNA IgM germline antibody in the subject sample is indicative of a positive prognosis.

DEFINITIONS

As used herein, "activation of specific B cells" is understood as administration of an agent or combination of agents to stimulate the production of antibodies by a specific B cell or population of B cells. For example, the administration of an agent or combination of agents to stimulate the production of germline IgM antibodies that specifically bind dsDNA.

An "agent" is understood herein to include a therapeutically active compound or a potentially therapeutic active compound. An agent can be a previously known or unknown compound. As used herein, an agent is typically a non-cell based compound, however, an agent can include a biological therapeutic agent, e.g., peptide or nucleic acid therapeutic, cytokine, antibody, etc.

An "agonist" is understood herein as a chemical substance capable of initiating the same reaction or activity typically produced by the binding of an endogenous substance or ligand to its receptor. An "antagonist" is understood herein as a chemical substance capable of inhibiting the reaction or activity typically produced by the binding of an endogenous substance (e.g., an endogenous agonist) to its receptor to prevent signaling through a receptor or to prevent downstream signaling that is the normal result of activation of the receptor. The antagonist can bind directly to the receptor or can act through other proteins or factors required for signaling, antigenonists and antagonists can modulate some or all of the activities of the endogenous substance or ligand that binds to the receptor. Antagonists are typically characterized by determining the amount of the antagonist is required to inhibit the activity of the endogenous agonist. For example, an inhibitor at 0.01-, 0.1-, 1-, 5-, 10-, 50-, 100-, 200-, 500-, or 1000-fold molar concentration relative to the agonist can inhibit the activity of the agonist by at least 10%, 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, or more.

As used herein "amelioration" or "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition. For example, amelioration or treatment of lupus nephritis can include prevention of progression of at least one sign or symptom from a diagnostic class of lupus nephritis to the next higher pathological designation, or decrease in signs or symptoms of inflammation associated with lupus nephritis as determined by the presence or absence of cytokines, inflammatory complexes, or activated macrophages, either in serum or kidney. In an embodiment, amelioration or treatment includes delay or prevention of the progression from one diagnostic class to the next diagnostic class. Amelioration and treatment can be viewed as a continuum and need not be understood as distinct activities.

As used herein, "antibody" is understood as a globular plasma protein having a molecular weight of about 15OkDa, also known as an immunoglobulin. The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can also be dimeric with two Ig units as with IgA, or pentameric with five Ig units, like mammalian IgM. The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains called Ig domains. These domains contain about 70-110 amino acids and are classified into different categories (for example, variable or IgV, and constant or IgC) according to their size and function. They have a characteristic immunoglobulin fold in which two beta sheets create a binding site, held together by interactions between conserved cysteines and other charged amino acids. As used herein, "antibody" also includes fragments, e.g., proteolytic fragments, of antibodies including Fab fragments, Fc fragments, and F(ab)₂ fragments.

As used herein, "antibody" can also include any of a number of single or multi chain containing a paired V_H/V_L domain that specifically binds an antigen. A single -chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V_H with the C-terminus of the V_L, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFvs can be made by transplanting V_H and V_L regions, from naturally occurring or synthetic (e.g., humanized), into the desired scFv sequence, or transplanting of CDRs from desired antibodies into an antibody framework present in an scFv sequence. Antigen binding portions (i.e., V_H/V_L pairs) can be optimized for the desired binding characteristics using methods such as antibody phage display (see, e.g., Antibody Phage Display: Methods and Protocols, Edited by P. M. O'Brien and R. Aitken, Humana Press, c. 2002, incorporated herein by reference). It is understood that scFvs can be modified to include sequences to facilitate multimerization of the single chains, either by expressing tandem scFvs from a single promoter, or by including sequences to allow for cross-linking, to include antigens to allow for multimerization by binding to a divalent antibody. Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Diabodies have been shown to have dissociation constants up to 40-fold lower than corresponding scFvs, corresponding to a much higher affinity to their target. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies and tetrabodies have also been produced (see, e.g., Adams, et al., (1998). British journal of cancer 77: 1405-12; Le Gall (1999). FEBS Letters 453: 164-168; and Mathew, (2004) Stroke; a journal of cerebral circulation 35: 2335-9, each incorporated herein by reference).

As used herein, the term "antigen" refers to a molecule that is bound by an antibody paired V_H/V_L domain. Typically, antigens are capable of raising an antibody response in vivo. An antigen can be a peptide, protein, nucleic acid, lipid, carbohydrate, hapten, or other molecule. Antigens can be non-self, e.g., from a pathogen, or in the case of various autoimmune diseases or disorders, antigens can include self-antigens.

As used herein, an "anti-double stranded DNA" or "anti-dsDNA" antibody or an antibody that "specifically binds dsDNA" is understood as an antibody that specifically binds double stranded DNA in the absence of proteins. The antibody binds dsDNA with at least a 10-fold, 20-fold, 50-fold, 100- fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 500-fold, or more preference over other nucleic acid antigens or nucleic acid containing antigens including single stranded DNA, single stranded or double stranded RNA, non-dsDNA nuclear antigens, or Smith antigens.

As used herein, "autoimmune" response is understood as the production of antibodies against self-antigens including, but not limited to, polypeptides, nucleic acids, and combinations thereof. B- cells capable of producing autoantibodies are typically cleared during development in a clonal selection process. An "autoimmune" response producing "autoantibodies" typically results in the development of a disease or disorder, including but not limited to the various forms of lupus including lupus nephritis. Autoantigens associated with lupus include, but are not limited to, dsDNA, Smith autoantigens, nuclear antigens, phospholipids, N-methyl-D-aspartic acid (NMDA), and ribonuclear protein particles (RNP).

As used herein, "class switching" or "isotype switching" refers to a biological process occurring after activation of the B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG). The different classes of antibody, and thus effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naϊve B cells express only cell- surface IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function, therefore, after activation, an antibody with a IgG, IgA, or IgE effector function might be required to effectively eliminate an antigen. Class switching allows different daughter cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy chain changes during class switching; the variable regions, and therefore antigen specificity, remain unchanged. Thus the progeny of a single B cell can produce antibodies, all specific for the same antigen, but with the ability to produce the effector function appropriate for each antigenic challenge. Class switching is triggered by cytokines; the isotype generated depends on which cytokines are present in the B cell environment. Class switching occurs in the heavy chain gene locus by a mechanism called class switch recombination (CSR). This process results in an immunoglobulin gene that encodes an antibody of a different isotype.

As used herein, "changed as compared to a control" sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator to be detected at a level that is statistically different than a sample from a normal, untreated, non-transgenic or other control geneotype, or control sample. Control samples include, for example, cells in culture, one or more laboratory test animals, or one or more human subjects. Methods to select and test control samples is within the ability of those in the art. An analyte can be a naturally occurring substance that is characteristically expressed or produced by the cell or organism (e.g., IgM or IgG antibodies, antibodies with a defined specificity) or a substance produced by a reporter construct (e.g, β-galactosidase or lucif erase). Change as compared to a control can be a change in the presence or severity of at least one sign or symptom of lupus nephritis as set forth in the classification table herein. Depending on the method used for detection the amount and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art.

As used herein, a "complementarity determining region" (CDR) is a short amino acid sequence found in the variable domains of antigen binding pocket (e.g. immunoglobulin and T cell receptor) proteins that complements an antigen and therefore provides a binding pocket with its specificity for that particular antigen. Each polypeptide chain of an antigen receptor contains three CDRs (CDRl, CDR2 and CDR3). Since the antigen binding pockets are typically composed of two polypeptide chains, there are six CDRs for each antigen receptor that can come into contact with the antigen (each heavy and light chain contains three CDRs), twelve CDRs on a single antibody molecule and sixty CDRs on a pentameric IgM molecule. Since most sequence variation associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable domains. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of the VJ (VDJ in the case of heavy chain) regions.

A "competition assay" as used herein is any type of test in which the binding of two agents, typically two antibodies, to the same agent is tested simultaneously in a single reaction mixture. For example, in a competition assay, a human antibody and a mouse antibody that bind to the same antigen are combined at various ratios (e.g., 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, 1:100) and contacted with the antigen to which both antibodies bind under conditions where the antibody to be detected is present in excess of the amount of antigen present. Specific binding of one of the antibodies is detected. If the binding of the detected antibody is decreased with increasing amounts of the not directly detected antibody, the antibodies are said to compete for binding. The antibodies can compete equally for binding to the antigen, e.g., when the antibodies are mixed at a 1:1 ratio, the amount of the detected antibody detected decreases by about half. As used herein, a first antibody competes for binding with a second antibody to a specific antigen when the presence of the first antibody decreases the binding of the second antibody by at least about 10%, or the second antibody decreases the binding of the first antibody by at least about 10%, when the antibodies are present in a mixture at about a 1 : 1 ratio. Similarly, a competition assay can be performed using two antigens to compete for binding to one antibody.

"Contacting a cell" is understood herein as providing an agent to a test cell or cell to be treated in culture or in an animal, such that the agent or isolated cell can interact with the surface of the test cell or cell to be treated, potentially be taken up by the test cell or cell to be treated, and have an effect on the test cell or cell to be treated. The agent or isolated cell can be delivered to the cell directly (e.g., by addition of the agent to culture medium or by injection into the cell or tissue of interest), or by delivery to the organism by an enteral or parenteral route of administration for delivery to the cell by circulation, lymphatic, or other means.

As used herein, "detecting", "detection" and the like are understood that an assay performed for identification of a specific analyte in a sample or a product from a reporter construct in a sample or one or more specific signs or symptoms of lupus nephritis. The amount of analyte detected in the sample or the change in one or more signs or symptoms of a disease or condition can be none or below the level of detection of the assay or method.

As used herein, a "diagnostic marker" is understood as one or more signs or symptoms of a disease or condition that can be assessed, preferably quantitatively to monitor the progress or efficacy of a disease treatment or prophylactic treatment or method. A diagnostic marker can be one or more of the diagnostic classification criteria set forth in the classification table herein.

As used herein, the terms "effective" and "effectiveness" includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side -effects) resulting from administration of the treatment. On the other hand, the term "ineffective" indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population. (Such a treatment may be ineffective in a subgroup that can be identified by the expression profile or profiles.) "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of a drug, a drug which is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease signs or symptoms, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

As used herein, "enriched" is understood increasing the relative portions of some desirable quality, attribute, or agent to a mixture, e.g., enriching a population of antibodies for the presence of IgM antibodies for example relative to the amount of IgG antibodies in the sample so that the IgM antibodies are present at a substantially higher proportion than IgG antibodies in the original mixture, e.g., naturally occurring mixture of antibodies present in a sample not enriched for a specific antibody type, the relative amount of germline antibodies as compared to hypermutated antibodies (e.g., at least about 10-fold higher, at least about 20-fold higher, at least about 50-fold higher, at least about 100- fold higher, at least about 250-fold higher, at least about 500-fold higher, at least about 1000-fold higher w/w than would typically be present in an unenriched sample). A sample may be enriched for a particular agent by adding the isolated agent to the mixture, e.g., addition of a monoclonal IgM antibody to a pharmaceutically acceptable carrier, or by selecting the source of the sample, e.g., a subject deficient in somatic hypermutation and antibody class switching to provide an enriched population of IgM antibodies or B cells for generation of hybridoma cells to express IgM antibodies.

As used herein, the term "epitope" refers to a unit of structure conventionally bound by an immunoglobulin V_H/V_L pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody.

A "germline antibody" is an antibody that arises exclusively from V(D)J recombination of CDR regions present in the germline of the individual producing the antibodies without somatic hypermutation or class switching. They are polyreactive, tend to use VH families proximal to the JH region and in spite of binding auto antigens, they constitute a small fraction of the normal repertoire. B cells secreting JH region -proximal germline autoantibodies are predominantly expressed in the neonatal stage of development but represent a small portion of the B cell population (2-15% of which only a small fraction are specifically against ds-DNA) present in an adult unless elicited by a specific antigen. Nuclear autoantigens like dsDNA, are not typically available to the immune system of normal subjects; therefore the presence of antibodies to such antigens, and B-cells expressing antibodies to such antigens would be uncommon in a normal subject. Moreover, cells expressing germline IgM antibodies would be expected to undergo class switching upon stimulation to produce IgG antibodies rather than IgM antibodies upon activation. Therefore, autoreactive IgM antibodies in germline configuration antibodies are unusual even in patients chronically displaying autoantigens due to cell death and inflammation.

As used herein, "heterologous" as "heterologous protein" is understood as a protein not natively expressed in the cell in which it is expressed.

"Humanized antibodies" or "chimeric antibodies" are a type of monoclonal antibody that have been synthesized using recombinant DNA technology to circumvent the clinical problem of immune response to foreign antigens. The standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences, and the human immune system recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects. Humanized antibodies are produced by operably linking the DNA that encodes the binding portion of a monoclonal mouse antibody with human antibody-producing framework and constant regions. Humanized antibodies are typically expressed in culture, however, the method of making the antibodies is not a limitation of the composition. Methods for generation of constructs for the expression of humanized antibodies, and methods of expression of humanized antibodies are well known in the art. (e.g., Hay et al.,

"Bacteriophage cloning and Escherichia coli expression of a human IgM Fab," Hum. Antibod. Hybridomas, 3:81-85 (1992); and Pascalis et al., Grafting of "abbreviated" complementarity- determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody. Journal of Immunology, 2002. 169: 3076-3084, both incorporated herein by reference.)

"Hypermutation" is understood as the process of random mutation in rapidly proliferating, stimulated B -cells to produce a population of cells expressing antibodies with differences in antigen binding specificities. The process depends on the enzyme Activation-Induced (Cytidine) Deaminase, or AID which causes the deamination of cytidine to uracil in the DNA. The uracil in DNA can either be replicated over to yield C to T or G to A transition mutations, or can be removed and the patch resynthesized by an error prone DNA repair mechanism. The end result of this mutational process is to generate variation in the DNA sequence, and subsequently in the antibody polypeptide sequence.

As used herein, "identity" is understood as the percent of matching of nucleic acid or amino acid sequence over at least a portion of a nucleic acid or amino acid sequence. Sequence identity can be determined by those of skill in the art, for example, by computer programs that compare sequences such as BLAST or ClustalW. Identity is typically expressed as a percent, for example 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. Identity can be determined for the length of a domain (e.g., for a CDR, for an antibody variable domain) or over the length of an entire peptide (e.g., for a light chain or heavy chain). Determining identity is well within the ability of those of skill in the art. As used herein, "isolated" or "purified" when used in reference to a polypeptide means that a natural polypeptide or protein has been removed from its normal physiological environment (e.g., protein isolated from plasma or tissue) or is synthesized in a non-natural environment (e.g., artificially synthesized in a heterologous system), and optionally further removed from the artificial synthetic environment. Thus, an "isolated" or "purified" polypeptide can be in a cell-free solution or placed in a different cellular environment (e.g., expressed in a heterologous cell type or heterologous organism). The term "purified" does not imply that the polypeptide is the only polypeptide present, but that it is essentially free (about 90-95%, up to 99-100% pure) of cellular or organismal material naturally associated with it, and thus is distinguished from naturally occurring polypeptide. Similarly, an isolated nucleic acid is removed from its normal physiological environment. "Isolated" when used in reference to a cell means the cell is in culture (i.e., not in an animal), either cell culture or organ culture, of a primary cell or cell line. Cells can be isolated from a normal animal, a transgenic animal, an animal having spontaneously occurring genetic changes, and/or an animal having a genetic and/or induced disease or condition.

As used herein, "kits" are understood to contain at least one non-standard laboratory reagent for use in the methods of the invention in appropriate packaging and with instructions for use, or a composition of the invention in appropriate packaging. A therapeutic kit can include one or more anti-dsDNA IgM germline antibodies and a device for delivery of the antibody such as a syringe, or a solution for reconstitution of the antibody when the antibody is provided as a dry powder. A diagnostic kit can include reagents for detection of IgM antibodies, and for differentiation of germline antibodies from antibodies that have undergone somatic hypermutation. The kit can further include any other components required to practice the method of the invention, as dry powders, concentrated solutions, or ready to use solutions. In some embodiments, the kit comprises one or more containers that contain reagents for use in the methods of the invention; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.

The term "label" or "detectable label" as used herein refers to any atom or molecule which can be used to provide a readily detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for include, but are not limited to, the following: radioisotopes (e.g., ³H), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. In others, the label is part of the fusion protein, e.g. Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP).

"Lupus nephritis" is acute or chronic renal impairment that may develop in conjunction with SLE, leading to acute or end-stage renal failure. Classification of the stages of lupus nephritis into classes was performed by the International Society of Nephrology/Renal Pathology Society (ISN/RPS) in 2003. The classification method was published in /. Am. Soc. Nephrol. 15:241-250 in 2004, which is incorporated herein by reference. A summary table reproduced from the reference is provided below.

Class I Minimal mesangial lupus nephritis

Normal glomeruli by light microscopy, but mesangial immune deposits by immunofluorescence

Class II Mesangial proliferative lupus nephritis

Purely mesangial hypercellularity of any degree or mesangial matrix expansion by light microscopy, with mesangial immune deposits

May be a few isolated subepithelial or subendothelial deposits visible by immunofluorescence or electron microscopy, but not by light microscopy

Class III Focal lupus nephritis³

Active or inactive focal, segmental or global endo- or extracapillary glomerulonephritis involving < 50% of all glomeruli, typically with focal subendothelial immune deposits, with or without mesangial alterations

Class III(A) Active lesions: focal proliferative lupus nephritis Class III(A/C) Active and chronic lesions: focal proliferative and sclerosing lupus nephritis Class III(C) Chronic inactive lesions with glomerular scars: focal sclerosing lupus nephritis

Class IV Class IV Diffuse lupus nephritis

Active or inactive diffuse, segmental or global endo- or extracapillary glomerulonephritis involving >50% of all glomeruli, typically with diffuse subendothelial immune deposits, with or without mesangial alterations. This class is divided into diffuse segmental(IV-S) lupus nephritis when > 50% of the involved glomeruli have segmental lesions, and diffuse global (IV-G) lupus nephritis when >50% of the involved glomeruli have global lesions. Segmental is defined as a glomerular lesion that involves less than half of the glomerular tuft. This class includes cases with diffuse wire loop deposits but with little or no glomerular proliferation

Class IV-S (A) Active lesions: diffuse segmental proliferative lupus nephritis Class IV-G (A) Active lesions: diffuse global proliferative lupus nephritis

Class IV- Active and chronic lesions: diffuse global proliferative and sclerosing lupus S(A/C) nephritis Class IV-S (C) Chronic inactive lesions with scars: diffuse segmental sclerosing lupus nephritis

Class IV-G (C) Chronic inactive lesions with scars: diffuse global sclerosing lupus nephritis Class V Membranous lupus nephritis

Global or segmental subepithelial immune deposits or their morphologic sequelae by light microscopy and by immunofluorescence or electron microscopy, with or without mesangial alterations

Class V lupus nephritis may occur in combination with class III or IV in which case both will be diagnosed

Class V lupus nephritis show advanced sclerosis Class VI Advanced sclerosis lupus nephritis

> 90% of glomeruli globally sclerosed without residual activity a Indicate the proportion of glomeruli with active and with sclerotic lesions. b Indicate the proportion of glomeruli with fibrinoid necrosis and/or cellular crescents.

Indicate and grade (mild, moderate, severe) tubular atrophy, interstitial inflammation and fibrosis, severity of arteriosclerosis or other vascular lesions.

Other more simplified classification systems are provided in the Examples.

"Monoclonal antibody" or "mAb" as used herein is a monospecific antibody produced by immortalized immune cells that are all clones of a single parent cell. Monoclonal antibody expressing cells are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. The coding sequences of mouse monoclonal antibodies can be modified to include the CDR antigen binding portions from mouse in the context of a human antibody. Such antibodies are particularly useful for therapeutic applications in humans.

"Obtaining" is understood herein as manufacturing, purchasing, or otherwise coming into possession of.

"Operably linked" as used herein is understood as joining in a manner such that each component in the linkage performs the desired activity. For example, coding sequences for mouse CDRs can be operably linked to the coding sequences for human constant chains and frameworks by fusing the sequences in frame, and the chimeric sequence can further be operably linked to a promoter such that the expression of the chimeric protein is controlled by the promoter sequence, which can be a constitutive or inducible promoter.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. For example, pharmaceutically acceptable carriers for administration of cells typically is a carrier acceptable for delivery by injection, and do not include agents such as detergents or other compounds that could damage the cells to be delivered. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intraperotineal, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.

As used herein, "prevention" is understood as delaying the onset of at least one sign or symptom of a disease in a subject prone to the disease or condition. Prevention does not require that the disease or condition never develop in the subject. Prevention can include administration of multiple doses of an agent, for example, multiple doses of anti-dsIgM antibody, can be administered to a subject having a family history of lupus or having lupus with no detectable renal involvement, to delay the onset of at least one sign or symptom of lupus nephritis.

As used herein, "prone to" as in a subject prone to a specific disease or condition, such as SLE or lupus nephritis, refers to a subject more likely than the general population to develop the disease or condition. For example, a subject prone to SLE or lupus nephritis is a woman of non- European descent who is between the ages of about 15 to about 50. A woman having close relatives having autoimmune diseases or conditions, particularly lupus, may be further prone to the disease. A subject having previously been diagnosed with lupus erythematosus or other localized or systemic form of lupus is prone to lupus nephritis. One of skill in the art, such as a physician, can identify a subject prone to SLE or lupus nephritis.

As used herein, "providing" is understood as to supply or make available.

"Reporter construct" as used herein is understood to be an exogenously inserted gene, often present on a plasmid, with a detectable gene product, under the control of a promoter sequence. Preferably, the gene product is easily detectable using a quantitative method. Common reporter genes include lucif erase and beta-galactosidase. The reporter construct can be transiently inserted into the cell by transfection or infection methods. Alternatively, stable cell lines can be made using methods well known to those skilled in the art, or cells can be obtained from transgenic animals expressing a reporter construct. The specific reporter gene or method of detection is not a limitation of the invention.

A "sample" as used herein refers to a biological material that is isolated from its environment

(e.g., blood, cells, or tissue from an animal, or conditioned media or cells from tissue culture) and is suspected of containing, or known to contain an analyte or a characteristic physiological, histological, or morphological characteristic diagnostic for or indicative of a disease or condition (e.g., a kidney biopsy to be assayed for particular histological characteristics of lupus nephritis such as those provided herein). A sample can also be a partially purified fraction of a tissue or bodily fluid (e.g., serum, or fractionated serum). A reference sample can be a "normal" sample, from a donor not having the disease or condition fluid, or from a normal tissue in a subject having the disease or condition (e.g., normal tissue vs. tumor tissue). A reference sample can also be from an untreated donor or cell culture not treated with an active agent (e.g., no treatment or administration of vehicle only) and/or stimulus. A reference sample can also be taken at a "zero time point" prior to contacting the cell or subject with the agent or cell to be tested.

"Small molecule" as used herein is understood as a compound, typically an organic compound, having a molecular weight of no more than about 1500 Da, 1000 Da, 750 Da, or 500 Da. In an embodiment, a small molecule does not include a polypeptide or nucleic acid. As used herein, "specifically binds" is understood as binding the specific target antigen with a higher relative affinity (e.g., as determined by a competition assay) than to a non-specific antigen, e.g., at least 10-fold higher, at least 10²-fold higher, at least 10³-fold higher, at least 10⁴-fold higher, or at least 10⁵-fold higher.

A "subject" as used herein refers to living organisms. In certain embodiments, the living organism is an animal. In certain preferred embodiments, the subject is a mammal such as primate including a non-human primate. In certain embodiments, the subject is a domesticated mammal. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A human subject may also be referred to as a patient. Subjects can also include non-mammals. A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Methods for identification of subjects suffering from or suspected of suffering from conditions such as lupus and/ or lupus nephritis is within the ability of those in the art for example using the classification criteria set forth herein. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

"Systemic lupus erythematosus" (SLE or lupus) is a chronic autoimmune connective tissue disease that can affect any part of the body. As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage.

Autoantibodies present in subjects suffering from SLE include antibodies that specifically bind to dsDNA, ssDNA, RNA, ribonucleoprotein complexes such as RNPs that form the Smith (Sm) antigen, anti-nuclear antigens (ANA), and phospholipids. SLE most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system. The course of the disease is unpredictable, with periods of illness (called flares) alternating with remissions. The disease occurs nine times more often in women than in men, especially between the ages of 15 and 50, and is more common in those of non-European descent. SLE is treatable through addressing its symptoms, mainly with corticosteroids and immunosuppressants; there is currently no cure. SLE can be fatal, although with recent medical advances, fatalities are becoming increasingly rare. Survival for people with SLE in the United States, Canada, and Europe is approximately 95% at five years, 90% at 10 years, and 78% at 20 years.

"Therapeutically effective amount," as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder beyond that expected in the absence of such treatment.

An agent can be administered to a subject, either alone or in combination with one or more therapeutic agents, as a pharmaceutical composition in mixture with conventional excipient, e.g., pharmaceutically acceptable carrier, or therapeutic treatments such as radiation.

The pharmaceutical agents may be conveniently administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in Remington's Pharmaceutical Sciences (Lippincott Williams & Wilkins; Twenty first Edition, 2005). Formulations for parenteral administration may contain as common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.

It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to e.g., the specific compound being utilized, the particular composition formulated, the mode of administration and characteristics of the subject, e.g., the species, sex, weight, general health and age of the subject. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests conducted with regard to the foregoing guidelines. Doses, for example, would typically fall within the range of about 500μg/kg/week to 10 mg/kg/week.

The term "transfection" as used herein refers to the introduction of a transgene into a cell. The term "transgene" as used herein refers to any nucleic acid sequence which is introduced into the genome of a cell by experimental manipulations. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of a transgene into the genome of the transfected cell. The term "stable transfectant" refers to a cell which has stably integrated one or more transgenes into the genomic DNA.

The term "transient transfection" or "transiently transfected" refers to the introduction of one or more transgenes into a transfected cell in the absence of integration of the transgene into the host cell's genome. The term "transient transfectant" refers to a cell which has transiently integrated one or more transgenes.

As used herein, "V(D)J recombination" refers to the process of somatic recombination of immunoglobulins, also known as V(D)J recombination, involves the generation of a unique immunoglobulin variable region. The variable region of each immunoglobulin heavy or light chain is encoded in several gene segments. These segments are called variable (V), diversity (D), and joining (J) segments. V, D and J segments are found in Ig heavy chains, but only V and J segments are found in Ig light chains. Multiple copies of the V, D, and J gene segments exist, and are tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell assembles an immunoglobulin variable region by randomly selecting and combining one V, one D, and one J gene segment (or one V and one J segment in the light chain). As there are multiple copies of each type of gene segment, and different combinations of gene segments can be used to generate each immunoglobulin variable region, this process generates a huge number of antibodies, each with different paratopes, and thus different antigen specificities.

The term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild- type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product which displays modifications (e.g. deletions, substitutions, etc.) in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, the term "or " is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean, antibodyout can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1. AID deficiency-associated reduction in glomerulonephritis and mononuclear cell infiltrates in MRL/lpr mice. A., Average severity scores for amount of mesangial matrix, mononuclear cell infiltrates, and overall glomerulonephritis in kidneys of AID^+/+ MRl/lpr (n = 19), AID⁷⁺ MRl/lpr (n = 18), and AID^7" MRl/lpr (n = 19) F5 mice. The increase in the mesangial matrix and glomerular change severity (due to increased cellularity and matrix) was graded as follows: 0, no increase (matrix occupied up to 10% of the glomerulus; 10% increased cellularity and mesangial matrix); 1, minimal (up to 25%); 2, mild (up to 50%); 3, moderate (up to 75%); and 4, marked (>75%). B. The average number of glomerular cells in similar-size glomeruli is depicted. Nonautoimmune AID-deficient mice (n = 6), C57BL/6 mice (n = 4), and BALB/c mice (n = 3) were used as controls. Mesangial matrix, glomerulonephritis, and mononuclear cell scores in control mice were set at 0 for comparison.

FIGURE 2. A. Accumulation of protein in the urine of F5 AID-deficient and wild-type MRL/lpr mice. Sample sizes were 22 AID^+/+MRL/lpr and 19 AID^7" MRL/lpr. In addition, five nonautoimmune AID^7" and six C57BL/6 mice were used. Error bars represent SD values. The differences between genotypes at various time points remained identical when gender was considered. B. The levels of blood urea nitrogen and creatinine in the serum are reduced in 52-wk-old AID- deficient MRL/lpr mice. Each circle depicts data from an individual mouse.

FIGURE 3. Increased lifespan in AID-deficient MRL/lpr mice. AID^+/+MRL/ lpr (n = 34), AID^+Λ MRL/lpr (n = 58), AID^7" MRL/lpr (n = 42), and nonbackcrossed MRl/lpr mice (n = 39) were set aside for lifespan study. Mice were euthanized when they reached moribund stage as determined by two veterinarians. At 52 wk (not shown) the results were nearly identical with survival at 50 wk.

FIGURE 4. High levels of autoreactive IgG were observed in AID wild-type MRL/lpr mice, whereas the sera from AID-deficient MRL/lpr mice contained high levels of autoreactive IgM antibodies. A. AID-deficient MRL/lpr mice have increased levels of IgM in the serum and, as expected, lack any IgG or IgA antibodies. Each circle depicts data from an individual mouse of the F5 generation (16-18 wk of age). B. Anti-dsDNA IgG scores for AID^+/+ MRL/lpr (n = 34), AID⁷⁺ MRL/lpr (n = 35), and AID^7" MRL/lpr (n=27). C. Anti-dsDNA IgM scores in the same mice. D. Anti- dsDNA IgM in nonautoimmune AID^7" and C57BL/6 mice. Each individual symbol in A-D is a single mouse score and the line across each lane depicts the average for that genotype. B-D depict data combined for F5 and F6 mice.

FIGURE 5A-D. Sequences of the (A) heavy and (B) light chains of the dsDNA specific IgM germline monoclonal antibodies Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ- Hom5G91G12 that were obtained from the hybridomas from the AID-deficient MRL/lprm mice. These sequences all belong to murine Vh5 or VH6 families and are in germline; and the (C) heavy and (D) light chains of the dsDNA specific IgM monoclonal antibodies raise in AID competent mice (i.e., non-germline). Sequences were aligned using ClustalW2. CDRs are indicated by underlining. X indicates any amino acid wherein each X is selected independently. Consensus sequences of CDRs can readily be determined using the alignment provided.

FIGURE 6. Transfer of serum from AID-deficient MRL/lpr mice into asymptomatic MRL/lpr mice conferred protection against kidney damage. MRL/lpr mice of 8-10 weeks of age were treated with pooled sera collected from either old MRL/lpr mice (WT), gender-matched, age-matched AID ^ΛMRL/lpr mice (AID⁷ ), and PBS by i.p. injection, 200μl/mouse, twice a week for 8 weeks. A. Decreased levels of urine protein in mice receiving the AID-deficient derived serum (N = 6) but not the AID-wild type serum (N = 6) or PBS control (N = 6). B. Decreased immune complex deposition in the glomeruli of kidneys from mice receiving the AID-deficient derived serum. Fluorescent antibodies against C3 complement factor were used to visualize immune complexes. Urine protein was scored in all experiments as follows: 1, trace; 2, 30 mg/dl; 3, 100 mg/dl; 4, 300 mg/dl; and 5, 2000 mg/dl or more. For C3 staining, scoring was done for all experiments as follows: +, weak staining with limited localization in the glomerulus(<50%); ++, moderate stain intensity where localization in the glomerulus was more diffuse (50% -75%); and +++, intense stain with diffused and homogeneous stain covering most of the glomerulus (>75%) as depicted on top of the figure. Mann- Whitney Rank Sum Test was used to test for significance. Error bars depict standard error.

FIGURE 7. Transfer with multiple anti-dsDNA IgM clones into young asymptomatic MRL/lpr mice conferred protection against kidney damage. At least 3 different anti-dsDNA IgM antibodies were used either derived from AID-deficient (AID^"Λ) or wild type (WT) MRl/lpr mice (numbers in parenthesis depict clone number) and compared against a non-autoreactive IgM (NA) and PBS control. lOOug of antibody were injected twice a week for 8 to 15 weeks depending on the experiment. Decreased levels of urine protein (A and C) and immune complex deposition (B and D) in mice receiving the anti-dsDNA IgM (For A and B, all groups were N = 8; and for C and D: N = 11). Mann-Whitney Rank Sum Test was used to test for significance. Error bars depict standard error.

FIGURE 8. Not all autoreactive IgM protected against lupus nephritis. A. Decreased levels of urine protein in mice receiving the AID-deficient derived anti-dsDNA IgM (AID-/-) compared to mice receiving an anti-phospholid IgM (AP), anti-Smith antigen IgM (AS), a non- autoreactive IgM (NA) and PBS. The anti-dsDNA IgM derived from wild type MRL/lpr mice hybridomas did not protect as well as the ones derived from AID-deficient mice (this IgM had the highest affinity to dsDNA) B. Similar results were observed in terms of immune complex deposition as measured by C3 staining in the glomeruli. There were at least 8 mice per group and Mann-Whitney Rank Sum Test was used to test for significance. Error bars depict standard error.

FIGURE 9. Histopathology analysis revealed less mononuclear cell infiltration and glomerulonephritis in mice receiving the AID-deficient-derived anti-dsDNA IgM (AID⁷ ). A. Mononuclear cell infiltration in the kidneys as visualized with hematoxylin and eosin stain. B. Glomerulonephritis scores for each group were based from analysis of 20 randomly selected glomeruli from each group visualized with hematoxylin and eosin stain and Periodic Acid Schiff stain. Scoring in this figure was done blindly by pathologists. Next to the dot plots are representative images from the named groups. Abbreviations are the same as for Figure 8. Mann- Whitney Rank Sum Test was used to test for significance. FIGURE 10. Therapeutic and dose-related effects of anti-dsDNA IgM in MRl/lpr mice. A.

Increasing dose correlates with better protection up to a certain point (lOOμg). MRL/lpr mice, at 10-11 weeks of age, were divided into five groups, 9-10 mice/group. The mice were i.p. injected with anti- dsDNA IgM rnAb (clone 12H5) derived from AID^7~MRL/lpr mice or PBS only, twice a week for 8 weeks, each time, at the indicated dose. B and C. Therapeutic effect of anti-dsDNA IgM in mice with significant proteinuira at start of treatment. B. Decreased levels of urine protein in mice receiving the anti-dsDNA IgM (N = 12 for all groups). C. Decreased immune complex deposition in the glomeruli of kidneys from symptomatic mice receiving the anti-dsDNA IgM compared to PBS group, as measured by C3 staining. Mann-Whitney Rank Sum Test was used to test for significance. Error bars depict standard error. FIGURE 11. Electron microscopy of kidney sections from all mice except those receiving anti-dsDNA IgM reveals the presence of inflammatory cells and apoptosis. At least 6 mice were examined from each of the following groups: mice receiving anti-dsDNA IgM from AID^7" mice, those receiving anti-dsDNA IgM from wild type mice, mice receiving a non-autoreactive IgM and mice receiving PBS control. Images are representative of general findings in mice receiving the non- autoreactive IgM or PBS. A. Image showing apoptotic cells (letter A) and B. Image showing inflammatory cells such as macrophages (MA), and lymphocytes (L).

FIGURE 12. Immunohistochemistry of the kidneys from mice reveal decreased infiltration by macrophages and apoptosis in mice receiving anti-dsDNA IgM. A. Staining of kidneys with anti- F4/80 antibody which recognizes a glycoprotein expressed by macrophages. B. Staining of kidneys with anti-cleaved-caspase 3 antibody which binds to the large fragment of activated caspase-3 following cleavage during apoptosis. The arrows indicate positive staining within a glomerulus. The images are representative of corresponding groups. Mann-Whitney Rank Sum Test was used to test for significance. Error bars depict standard error.

FIGURE 13. Increased secretion of TNF-OC and decreased levels of circulating IgG-containing immune complexes in mice receiving anti-dsDNA IgM. A. Serum TNF-OC levels in all groups (N = 6 per group: mice receiving IgM from wild type (WT), from AID knockout (AID -/-), mice receiving a non-autoreactive IgM (NA), and PBS group. B. Splenic macrophages were cultured with LPS and supernatants measured for TNF- α production. Macrophages from mice receiving anti-dsDNA IgM derived from AID-deficient hybridomas secreted lower levels of TNF-OC compared to PBS group. The sample sizes were: N= 9 for WT, 8 for AID, and N = 6 for PBS). C. Circulating IgG-containing immune complexes (IgG-IC ) were reduced in mice receiving the anti-dsDNA IgM from AID- deficient hybridomas (N = 11 mice/group). The amount of IgG-IC in each well was calculated according to the standard curve of the reference sera which is defined as a value of 1. Significant P values were obtained with the Mann-Whitney Rank Sum Test. Error bars depict standard error.

FIGURE 14. Similar levels of proteinuria in AID-deficient MRL/lpr mice and μS ^ΛAID ^Λ MRL/lpr mice with no secreted IgG. By crossing μS^"Λ MRL/lpr mice to AID-deficient MRL/lpr) mice, we generated mice unable to secrete antibodies of any kind. The resulting μS^"/"AID^"/" MRL/lpr mice (N = 22) were followed for at least 52 weeks and levels of proteinuria compared to those of AID-deficient MRL/lpr mice (N = 26), secreting only IgM. There was no difference between the two groups suggesting IgM protects from lupus nephritis through an IgG-mediated mechanism. Error bars depict standard error.

FIGURE 15. AID-deficient MRL/lpr mice experienced increased survival levels compared to μS ' AID^7" MRL/lpr. Even though both μS^"ΛAID^7" MRL/lpr and AID-deficient MRL/lpr mice had similar levels of proteinuria, AID-deficient mice experienced significantly higher survival levels (p < 0.05). This suggests an additional protective role for IgM, besides lupus nephritis. The two survival curves were compared by using Log-rank (Mantel-Cox) Test.

FIGURE 16. Repertoire of anti-dsDNA IgM secreting hybridomas from AID-deficient MRL/lpr mice is different from the repertoire derived from AID wild type counterparts. A. Sequencing of hybridomas AID^"ΛMRL/lpr hybridomas (N = 11) revealed that at least half the clones used Vh7183 while AID wild type hybridomas (N = 10) used mostly J558. This difference was significant (P < 0.05) using the Likelihood Ratio test. B The repertoire differences in the hybridomas cannot be explained by a repertoire shift with AID deficiency. Splenic B cells were taken from 3 mice from each group and over 70 clones/ group from PCR-amplified regions were sequenced.

TABLE 1. Table of SEQ ID NOs corresponding to heavy and light chain antibody amino acid sequences.

TABLE 2. Summary of results of electron microscopy analysis.

DETAILED DESCRIPTION Lupus nephritis is a common complication of systemic lupus. At present, there are no specific treatments approved for lupus nephritis. Methods and agents for limiting kidney damage include methods and agents used to limit damage systemically by limiting inflammation, including administration of steroids and immunosuppressants which can cause long term complications.

The invention provides anti-dsDNA IgM antibodies that are in the germ line configuration

(e.g., without somatic mutations) and derived from B cells bearing receptors of a subset of the murine VH5 and VH6 subgroups (7183 and J606) that normally populate the neonatal repertoire that are useful for the prevention, amelioration, and treatment of lupus nephritis. In lupus nephritis, patients develop autoantibodies that form pathogenic immune complexes which are deposited in kidneys and elicit an inflammatory response leading to kidney failure. The subset of IgM antibodies were identified using a new mouse strain that is incapable of generating hypermutated switched antibodies that was backcrossed over 8 generations into an autoimmune background.

A "germline antibody" is an antibody that arises exclusively from V(D)J recombination of CDR regions present in the germline of the individual producing the antibodies without somatic hypermutation or class switching. They are polyreactive, tend to use V_H families proximal to the J_H region and in spite of binding auto antigens, they constitute a small fraction of the adult repertoire. Instead, the V_H families used by B cells secreting germline autoantibodies are predominantly expressed in the neonatal stage of development but represent a small portion of the B cell population (2-15% of which only a small fraction are specifically against ds-DNA) present in an adult unless elicited by a specific antigen.

Autoantigens, e.g., dsDNA, are not typically accessible to the immune system of normal subjects; therefore the presence of antibodies to such antigens, and B-cells expressing antibodies to such antigens would be uncommon in a normal subject. Moreover, cells expressing germline IgM antibodies in individuals capable of undergoing class switching (most individuals) would be expected to produce specific IgG antibodies upon stimulation rather than specific IgM antibodies. Therefore, autoreactive IgM antibodies in germline configuration derived from neonate-like V_H families are very unusual even in patients chronically displaying autoantigens due to cell death and inflammation.

We found that these mice experienced a dramatic increase in survival that exceeded that expected from the absence of pathogenic (switched) antibodies, suggesting that they carry a protective factor. To confirm this, we performed serum transfer from these mice into asymptomatic lupus-prone mice and this resulted in a decrease in the severity of nephritis. Passive transfer experiments wherein IgM antibodies derived from hybridomas from AID-deficient and AID-wild type MRL/lpr mice were given to young asymptomatic MRL/lpr mice revealed that mice receiving the anti-dsDNA IgM experienced a dramatic improvement in all measures of lupus nephritis, such as proteinuria, immune complex deposition, infiltration of inflammatory cells into the kidney and glomerulonephritis. These results suggest that treatment with anti-dsDNA IgM may provide a novel therapy in the treatment of kidney disease in SLE. Indeed, treatment with anti-dsDNA IgM antibodies of MRL/lpr mice having significant proteinuria resulted in a significant reduction in the accumulation of urine protein over time when compared to PBS controls. There was also a positive correlation between increasing dosage and a reduction in porteinuria .

To further examine what specific antibodies may be responsible for this "protective" effect, we generated hybridomas and examined the impact of passive transfers using a variety of monoclonal antibodies. This led us to the identification of autoreactive IgM, specifically to ds-DNA as the protective factor, since mice receiving this IgM, experienced reduced immune complex deposition in the kidneys and displayed low levels of proteinuria. These antibodies were named Horn 12H5,

Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, and 16CJ-Hom5G91G12. The antibodies have the heavy and light chain sequences provided in Figure 5. All tested thus far have been demonstrated to be useful for the prevention, amelioration, and treatment of lupus nephritis. The monoclonal antibody producing hybridomas are deposited under the Budapest Treaty and are available under Accession Nos. .

Not all autoreactive IgM were equally effective: other antinuclear antibodies not binding to dsDNA did not protect and while all anti-dsDNA IgM show some protection, only the germline anti- dsDNA IgM dramatically reduced the levels of inflammatory cytokines such as TNF-alpha, both in the serum and upon activation of macrophages; and increased the serum levels of anti-inflammatory cytokines including IL-4 and IL-10. Inflammation is a major culprit in the development of the immune cascade that leads to lupus nephritis.

The human equivalent of the mouse antibodies can be readily identified or prepared. Given the high relatedness between mouse and man, the corresponding antibodies in humans are expected to provide similar relief from this disease in humans. The equivalent families in humans (VH3) are also normally associated with the early repertoire and have been found to occasionally encode for autoantibodies without any mutations.

Using a cell line system that secretes germline antibodies (a transfectoma) with V_H segments derived from human VH families, autoantibodies will be tested for protection in mouse models of lupus nephritis (MRL/lpr). Since the antigen is dsDNA, the human antibodies should be able to cross react with mouse dsDNA and in fact in antinuclear antibodies tests (ANA) using human Hep-2 cells, we have shown that mouse anti-dsDNA can be detected. This is not surprising considering how exquisitely conserved dsDNA is across all organisms. In addition, human antibodies have been shown to properly elicit an immune response in mice. Therefore, we will be able to screen transfectoma generated germline human antibodies in MRL/lpr mice. This type of human germline anti-dsDNA IgM can be used for inhibiting or reducing the severity of nephritis in patients with lupus. The differential contribution of autoreactive IgM versus IgG to autoimmunity remains controversial. IgG autoreactive antibodies are thought to be pathogenic in autoimmune disease because of both isotype and affinity to self-antigen. Somatic hypermutation and class switch recombination tend to occur together in the germinal center reaction which means that much of the IgG in the serum has been fine-tuned to a specific antigen through the process of affinity maturation. In SLE, this could mean enhanced affinity to self-antigen. Indeed, AID -heterozygous MRL/lpr mice experienced a delay in the onset of lupus nephritis that correlated with a paucity in the generation of high affinity anti-dsDNA IgG antibodies. The IgG isotype contributes to the pathogenicity of the antibodies as certain IgG subclasses are associated more frequently with autoimmune disease than others, and FcR and FcRIII deficient mice experienced a reduction in kidney damage. So it is apparent that both, switching to IgG and affinity maturation against self-antigen through somatic hypermutation contribute to the generation of pathogenic IgG antibodies.

The story for IgM is much less clear. Since much of the IgM antibody in the serum is secreted by cells participating in primary B cell responses, many are in germline configuration, i.e. unmutated, and therefore unlikely to have undergone affinity maturation. In addition, most of the serum IgM is in pentameric form and many have questioned whether in complex with antigen, pentameric IgM may be too big to generate the characteristic immune complex deposits seen in the kidneys of lupus nephritis, although we see IgM complex depositions in glomeruli of MRL/lpr mice. The dramatic increase in survival experienced by AID-deficient MRL/lpr mice demonstrated herein, which secrete nothing but IgM, much of it, autoreactive, casts serious doubts on the pathogenecity of autoreactive IgM in lupus nephritis. In fact, the data presented in this study strongly suggest that anti-dsDNA IgM is actually protective. MRL/lpr mice that only secrete switched antibodies because of a defect in the IgM secretory exon, experienced an acceleration of the autoimmune syndrome. The combined data strongly points to anti-dsDNA IgM as a protective antibody in lupus nephritis.

This does not mean that all autoreactive IgM is protective, i.e. anti-phospholipid and anti-

Smith IgM did not protect animals from lupus nephritis as demonstrated herein. Similarly, some autoreactive IgM has been associated with pathogenesis in autoimmune disease. Natural IgM antibodies that tend to be in germline configuration and polyreactive, have been associated with exacerbation of ischemia/reperfusion injury (Zhang M, et al.. J Immunol. 2006; 177:4727-4734.). There is an increased incidence of immune cytopenias and other autoimmune disorders with AID deficiency in humans (Quartier P, et al. Clin Immunol. 2004; 110:22-29.), suggesting autoreactive IgM may play a role in these diseases. However, this is observed not just with AID deficiency but with most immunodeficiencies suggesting that the immune dysregulation that is usually found in immunodeficiency results in increased activation of autoreactive lymphocytes or activation of poorly discriminating inflammatory cells to handle persistent infections (Haymore BR, et al., Autoimmun Rev. 2008;7:309-312.). Given that B cells from AID-deficient MRL/lpr mice can only secrete germline IgM, much of it autoreactive, this novel strain represents a new tool to better define the role of germline autoreactive IgM and natural antibodies in autoimmunity.

The mechanism by which anti-dsDNA IgM antibodies protect against lupus nephritis is unknown. We identified a general trend for macrophages cultured from mice receiving the protective antibodies to secrete less pro-inflammatory cytokines such as TNF-alpha and interpheron γ which is consistent with the decrease in the number of inflammatory cells in the kidneys of these mice. A correlation between anti-dsDNA IgG antibodies and activation of macrophages to secrete proinflammatory cytokines has been made previously (Jang EJ, et al. Immunol Lett. 2009; 124:70-76.); perhaps IgM has the opposite effect on macrophages. If true, it suggests that protective IgM helps create an environment less prone to inflammation-induced tissue injury. One possibility is that anti- dsDNA IgM efficiently clears apoptotic debris and immune complexes from the kidneys preventing the inflammatory response brought on by autoreactive IgG-bearing immune complexes. Indeed, mice receiving the protective IgM, had lower levels of circulating IgG immune complexes. Also there was decreased levels of cleaved-caspase 3 antibody staining in the kidneys of these mice. Interestingly, MRL/lpr mice with the defect in secreted IgM and rendered AID deficient (therefore lacking any secreted antibodies) revealed very low levels of proteinuria that were similar to AID-deficient MRL/lpr mice with secreted IgM, suggesting that anti-dsDNA IgM protects against lupus nephritis through an IgG-mediated process. Despite of this, mice secreting only IgM still experienced significantly higher survival levels than the non-secreting mice, suggesting an additional protective role for IgM in MRL/lpr mice, besides lupus nephritis.

Our data suggests that the IgM isotype and an anti-dsDNA specificity are important to provide protection from lupus nephritis. However, in contrast to pathogenic IgG, high affinity to dsDNA did not enhance protection by anti-dsDNA IgM. This suggests that either dsDNA specificity of the protective IgM is a correlate to other self-antigen important for protection, or that high affinity correlates with reduced polyreactivity and it is the polyreactivity that is important. Intriguingly, other autoreactive IgM, such as those against phospholipids and against the Smith antigen (a ribonuclear protein) did not confer any protection against lupus nephritis, proving that not all autoreactive IgM is protective and suggesting that it is only anti-dsDNA IgM.

There was a trend for anti-dsDNA IgM antibodies derived from AID-deficient MRL/lpr mice to protect better than those derived from AID-wild type MRL/lpr mice. To examine if this trend reflected a difference in the repertoire of B cells from AID-deficient-derived hybridomas, we sequenced at least 20 anti-dsDNA IgM-secreting hybridomas from each group. There was a significant difference in Vh usage wherein over half of the hybridomas derived from AID-deficient mice used Vh7183 and one used J606, Vh genes most commonly seen in the neonatal repertoire (Kearney JF, et al. Int Rev Immunol 1992;8:247-257.). None of the hybridomas derived from AID- wild type mice used Vh7183 or J606; most used J558. This difference was specific to the anti-dsDNA hybridomas and not the result of a change in the overall B cell repertoire from AID deficiency. Interestingly, Vh7183 and J606 are commonly seen in neonatal repertoire usage, which tends to secrete germline natural antibodies that are autoreactive or that have evolved recognition to particular antigens (Kearney JF, et al. Int Rev Immunol. 1992;8:247-257). The Vh usage in AID-deficient

MRL/lpr-derived anti-dsDNA IgM hybridomas suggests the interesting possibility that there may be a population of B cells that have evolved for the specific function of secreting protective antibodies. Such a population could be specifically targeted to secrete protective IgM in SLE.

The studies herein provide a mouse-based assay for testing potential mouse and human germline anti- dsDNA IgM for a protective role against nephritis. Potential candidates for human antibodies are determined from germline human Variable region homologs to mouse germline Variable regions demonstrated to play a protective role using various similarity search tools such as IgBlast, as well as chromosomal location and expression in neonatal repertoire. Anti-dsDNA binding specificity is determined by ELISA. The confirmed V regions are be cloned into transfectoma cell lines that generate chimeric human-mouse with human variable regions and mouse constant domains. This is a well established technology and since dsDNA is highly conserved across species, it will translate well.

Two systems are established to demonstrate the efficacy of the method, one with IgM constant domains to check for protection, and one with IgG to ensure the antibodies cannot be pathogenic in their switched form if cells expressing the antibodies are to be administered rather than administration of isolated antibodies. For delivery of isolated antibodies, secreted antibodies are collected from the supernatant of the transfectoma cells and optionally further purified or concentrated. The antibodies are then used for passive transfers into young asymptomatic MRL/lpr mice and into AID-deficient young asymptomatic MRL/lpr mice. Passive transfer inlcudes injecting antibodies 2x per week for at least 8 weeks. The end-points include protein in the urine, survival, serum cytokine levels (particularly tumor necrosis factor (TNF)-alpha), C3 deposition in the kidneys, and circulating immune complexes; and increase anti-inflammatory cytokines including IL-4, IL-6, and IL-10 in response to each the IgM and IgG antibodies.

Having identified human variable domains useful for the treatment of lupus nephritis in mice, IgM antibodies can be produced having the same specificity (e.g., by grafting the CDRs, V_H CDRs, V_L CDRS, or both, on to a human framework) for passive transfer of germline natural protective anti- dsDNA IgM from the human VH3 family for the treatment of lupus.

In an alternative embodiment, the paired variable domains, or the CDRs from the paired variable domains, in the Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, or 16CJ-Hom5G91G12 antibodies can be expressed in the context of a human IgM antibody. As the antigen, DNA, is conserved between mice and humans it is expected that the antibodies would be effective for the treatment of lupus nephritis in humans.

In an alternative embodiment, the human germline antibodies for use in the treatment methods will compete for binding to dsDNA with Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, or 16CJ- Hom5G91G12 antibodies. In a preferred embodiment, the antibodies have about the same avidity for dsDNA, i.e., when mixed at a 1:1 ratio for use in a competition assay, the human antibody decreases the binding of the mouse antibody to the dsDNA by about 40% to about 60%. This demonstrates that the antibodies have about the same avidity for dsDNA.

In an embodiment, the invention provides a method for reducing systemic inflammation in a subject suffering from or suspected of suffering from SLE or lupus nephritis by administering to the subject an isolated population of antibodies enriched for an IgM germline antibody wherein the antibody specifically binds double stranded DNA. Systemic inflammation can be determined, for example, by determining serum levels of TNF-alpha, 11-4, IL-6, or IL-10; or by determining activation of macrophages. Administration of an isolated population of antibodies enriched for an IgM germline antibody is more effective at reducing systemic inflammation than a population of anti-dsDNA IgM antibodies enriched for hypermutated antibodies, such as would be found in serum. In an embodiment, the decrease in systemic inflammation as determined by the concentration of TNF-alpha is at least 10%, 20%, 30%, 40%, 50% or more with the population of germline antibodies as compared to control than the hypermutated antibodies as compared to control. It is noted that B cells used to make hybridoma cells are most likely antibodies that have undergone somatic hypermutation and produce non-germline antibodies.

Because the germline anti-dsDNA antibodies for use in the methods of the invention originate from a subset of B cells that populate the neonatal repertoire and secrete natural antibodies, it may be possible to induce the expression and secretion of these protective antibodies in lupus patients by stimulation of those cells or other means. Specific stimulation of antibody production from such cells can be used for the treatment of lupus nephritis. Such stimulating agents would also preferably reduce or eliminate class switching to IgG class antibodies.

The invention allows those of skill in the art to distinguish protective antibodies from non- protective, and pathogenic antibodies. For example, the disclosure provided herein demonstrates that a certain type of autoreactive IgM, specifically a germline IgM, is protective, and that it generates from a small fraction of the immunoglobulin locus. The ratio of protective antibodies versus non- protective antibodies is expected to be predictive of disease. The invention includes analysis of the antibodies expressed by a subject having or suspected of having lupus nephritis, for a diagnostic or prognostic tool, or to monitor the progression of the disease. The relatively high expression of IgG anti-dsDNA antibodies is indicative of a less positive or poor prognosis, wherein the relatively high expression of germline IgM anti-dsDNA antibodies is indicative of a less poor or more positive prognosis. Example 1 — Materials and Methods

Generation of AID-deficient MRL/lpr mice. AID-deficient MRL/lpr mice were prepared essentially as described in Jiang et al., 2007 (Abrogation of lupus nephritis in activation-induced deaminase-deficient MRL/lpr mice. /. Immunol. 178:7422-7431, incorporated herein by reference). AID-deficient C57BL/6 mice were provided by T. Honjo (Kyoto University, Kyoto, Japan) and D. Schatz (Yale University School of Medicine, New Haven, CT). MRL/MpJ-Faslpr/J (MRL-lpr), C57BL/6J, and BALB/c strains were purchased from The Jackson Laboratory. AID^7" mice were backcrossed to MRL/lpr mice and, at the fifth and sixth generations AID⁷⁺ MRL/lpr mice (with >96% MRL/lpr background) were intercrossed to generate AID^+/+, AID^+Λ, and AID^7' MRL/lpr mice (n = 34, 33, 34, respectively). The mice were housed in specific pathogen-free facilities, maintained in microisolator cages on hardwood bedding, and provided with autoclaved food and reverse osmosis, deionized water. AID alleles were analyzed by PCR.

Lifespan analysis In addition to the mice described above, 134 MRL/lpr mice from the F5 generation, AID^+/+ MRL/lpr (n = 34) AID^+Λ MRL/lpr (n = 58), and AID^7" MRL/lpr, (n = 42), were used to examine survival. The non-backcrossed MRL/lpr mice (n = 39) were used as controls. Similar numbers of males and females were used in each group. The mice were closely monitored for at least 12 mo and euthanized when moribund.

For survival studies, mouse strain B6; 129S4-Igh-6^tmlch7J was purchased from The Jackson Laboratory. This strain was generated by targeted mutagenesis resulting in deletion of the μ_s exon and its three downstream polyadenylation sites (μS^~). This resulted in mice deficient in secreted IgM but able to express membrane -bound IgM (42). They are also able to secrete downstream isotypes like IgG. This strain was bred with AID-/-MRL/lpr mice. At the 5th generation of backcrossing, siblings of heterozygous mutant (μS^+Λ) AID^7"MRL/lpr mice were bred to obtain μS^+/+AID^7"MRL/lpr mice and μS^7"AID^7"MRL/lpr mice. Because of the AID -deficiency combined with a defect in secreted IgM, μS^{" Λ}AID⁷ MRL/lpr mice are unable to secrete any antibodies. μS^7"AID ^Λ MRL/lpr mice (N = 22) and AID-deficient MRL/lpr mice (N = 26) were followed for at least 56 weeks to examine mortality with or without secreted IgM.

All mice were housed in specific pathogen-free facilities at NIEHS/NIH.

Histology Formalin-fixed tissues were embedded in paraffin, cut into-5 μm sections, and stained with H&E. The severity of any abnormalities observed was graded as follows: 1, minimal; 2, mild; 3, moderate; and 4, marked. Additional sections of kidney were stained with periodic acid- Schiff stain. Glomerular change severity was graded based upon an increase in the size of affected glomeruli due to increased cellularity and the mesangial matrix. The severity of mononuclear cell infiltrate was graded based upon the total amount of infiltrate present.

The number of cells in each of 20 glomeruli per mouse was scored for the kidneys of each mouse. C57BL/6 and BALB/c mice of similar age were used as controls; the amount of mesangial matrix present in the glomeruli of controls, (-10% of glomerulus), was considered the amount normally present. Lungs, lymph nodes, spleen, liver, and bone marrow from each animal were examined for mononuclear cell infiltration. Electron microscopy Kidneys from 16- to 18 wk-old mice were collected in 3% paraformaldehyde at necropsy. A section from the renal cortex was taken, minced into lmm x lmm cubes then placed into EM fixative. The tissues were then processed routinely for Transmission Electron Microscopy (TEM). Semi-thin (or thick sections, 800 nm sections stained with Toluene blue) were examined and areas were chosen for thin sectioning (90nm). Those areas chosen for thin sectioning were sectioned, placed on formvar coated copper grids and digital photomicrographs were taken of randomly selected glomeruli and proximal convoluted tubules. At least 10 different glomeruli and 4 different tubules were examined for most mice, and embedded in Spurr's resin. Approximately 80-nm sections from epoxy blocks were cut, mounted on 200-mesh copper grids, stained with methanolic uranyl acetate and Reynolds lead citrate, and examined on a Zeiss® 900 transmission electron microscope. A total of 40 photomicrographs from two representative mice of each genotype were evaluated.

Detection of urine protein level Urine protein levels, collected monthly by expressing urine from the urethra directly, were tested with Multistix 10 SG (Bayer), and scored as follows: 0, negative; 1, trace; 2, 30 mg/dl; 3, 100 mg/dl; 4, 300 mg/dl; and 5, 2000 mg/dl or more. The average urine protein score from asymptomatic young MRL/lpr mice was below 1.2, while the score at 2.5 or higher was considered as proteinuria.

Blood urea nitrogen and creatinine levels in the serum. Blood urea nitrogen and creatinine levels were determined by urease with the glutamate dehydrogenase reaction and alkaline picrate (Jaffe Reaction), respectively. Both reagents were purchased from Olympus® America and the determinations were run using an Olympus® AU400e clinical analyzer (Olympus America).

Immunofluorescence and immunohistochemistry. To examine complement component 3 (C3) staining in glomeruli, kidneys from 16- to 18-wk-old mice were frozen in Tissue-Tek® OCT (Sakura®) and sectioned on a Leica® CM 3050 S cryostat (6 μm). Sections were fixed in acetone, washed in Ix automation buffer (Biomedia®), and blocked with Dako® serum-free protein block (DakoCytomation®). The slides were incubated in a 1/200 dilution of fluorescein-conjugated anti- mouse C3 antibodies (ICN Biomedicals®) for 1 h, mounted with Vectashield® mounting medium (Vector Laboratories®), and viewed with a fluorescent microscope. For negative controls, FITC- conjugated goat serum (Caltag Laboratories®) was used. IgG deposition analysis was performed similarly except for the use of a FITC-conjugated goat anti-mouse antibody (Sigma-Aldrich®) at a 1/100 dilution.

To examine GC morphology, biotin-labeled peanut agglutinin (PNA) (Vector Laboratories®) was used following standard avidin-biotin-peroxidase protocols. Briefly, frozen spleens from 16- to 18 wk-old F5 mice were sectioned on a cryostat (6 μm), affixed to slides, and placed into Rapid Fix solution (Shandon-Lipshaw®) and Ix automation buffer solution. The sections were placed in 3%

H₂O₂ and rinsed in Ix automation buffer. Protein blocking was done with an avidin-biotin blocking kit (Vector Laboratories®). Incubation with PNA was done at a 1/1000 dilution (1 mM CaCl₂, MgCl₂, and MnCl₂) and labeling with Biogenex® streptavidin label. The stain was developed with diaminobenzidine chromogen (DakoCytomation®) and the slides were counterstained with hematoxylin, dehydrated with graded ethanol, and visualized with a fluorescence microscope.

Detection of antibodies by ELISA Beginning at 2 mo of age, mice were bled monthly by retro-orbital puncture. Serum, culture supernatant, and ascites fluid IgM, IgG, and IgA levels were determined with commercial ELISA kits (Bethyl Laboratories) following the manufacturer's instructions Cell culture supernatants were diluted with sample diluent (blocking buffer plus 0.05% Tween 20) at 1 :2 to 1 : 128 serial dilutions. Ascites fluid was diluted at 1 :2000 to 1 :256000. Mouse sera were used at 1:1000 to 1:5000 depending on mouse age. Mouse anti-dsDNA IgM and IgG antibodies were measured as previously reported with modification (Radic et al., IgH and L chain contributions to autoimmune specificities. /. Immunol. 146:176-182, incorporated herein by reference). Briefly, the diluted samples were added to plates at 100 μl/well. Goat anti-mouse IgM- HRP conjugator (Bethyl Laboratories or SouthernBiotech) was appropriately diluted and added at 100 μl/well. Following incubation and washing, tetramethylbenzidine (TMB) enzyme substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added at 100 μl/well and incubated for 20-30 mins at room temperature. The reaction was stopped by adding 50μl of 2M H₂SO₄. The absorbance at 450 nm was measured in a Multiskan Ascent microplate reader (Thermo Electron, Waltham, MA).

To screen anti-dsDNA antibody-producing hybridomas, similar protocol as above was used except that hybridoma culture supernatants were used at 1:5 dilution and goat anti-mouse kappa chain-HRP conjugator (SouthernBiotech, Birmingham, AL) was used. Reading at 3-folds of background reading was set as cut-off value for selecting positive hybridomas. Generation of hybridoma cells from mice: Anti-dsDNA IgM -producing hybridomas were generated following standard protocols (43). Briefly, spleens were collected from uninmunized AID^+/+, AID^+/ , and AID^"'^" MRL/lpr mice (10-12 weeks of age, 3-6 mice/group). Splenocytes with the same genotype were pooled and fused with murine myeloma NSl at a ratio of 5:1 in 50% polyethylene glycol (1500 PEG, Roche, Basel, Switzerland) according to the manufacturer's directions. The fused cells were resuspended in the selection medium (DMEM plus 10% fetal bovine serum and HAT medium supplement, all from Invitrogen, Carlsbad, CA) and seeded into 96-well polyvinyl plates.

After two weeks, supernatants from the 96-well plates were screened for anti-dsDNA antibody-producing hybridomas by ELISA (see below). The positive hybridomas were cloned and recloned by limiting dilution at 1 to 10 cells per well of the 96-well plates. The cell clones stably secreting anti-dsDNA IgM were expanded, injected (2x10⁶ cells) into the peritoneal cavity of Rag- 1 ko mouse (The Jackson Laboratory) that had been pretreated with 0.2ml Pristane (Sigma, St. Louis, MO) 10 days earlier to induce ascites fluid. The ascites fluid was collected 2-3 weeks after injection and filtered through a Syringe driven filter unit (Millex, Bedford, MA). IgM concentrations were titrated by ELISA kit (Bethyl Laboratories, Montgomery, TX) and its specificity to dsDNA was confirmed by ELISA (see below). A non-autoreactive IgM-producing hybridoma clone was also generated from the same AID^~'^~MRL/lpr mice and confirmed to be non autoreactive by anti-nuclear antibody assay. A mouse lymphocyte cell line secreting anti-phospholipid IgM antibodies was a gift from Laurent Verkoczy at Duke University. An anti-Sm IgM-producing hybridoma clone was provided by Barbara Vilen at the University of North Carolina in Chapel Hill. Both of these lines were used to generate their respective IgM by injecting into the peritoneal cavity of RAG-I deficient mice pretreated with pristine to generate ascites. All monoclonal antibodies (mAb) were generated using the same strategy.

Generation of monoclonal antibodies from AID MRL/lpr mice: 50 RAG-I mice were intraperitoneally injected with 0.2 ml pristane per mouse (proportionate to mouse weight) 10-14 days prior to inoculation with cells. Then, mice were injected i.p. with 0.5 ml hybridoma cells (2.5 x 10⁶ cells/ml) from AID MRL/lpr mice. When ascites forms (1 to 2 weeks), and before abdominal distention is great enough to cause discomfort, ascites was harvested by inserting an 18-G needle into the abdominal cavity and allowing the ascites to drip into a sterile tube. Antibodies generated were used for passive transfers (see below).

Passive transfers: 13 weeks old MRL/lpr mice divided into 4 groups (8 mice/group), were injected I.P. with lOOμg/ml, twice a week i.p. of monoclonal antibody (mAb) IgM or PBS, for 9 weeks. Monoclonal Ab included IgMs derived from MRL/lpr mice (dsDNA-specific), from AID^"'^" .MRL/lpr (dsDNA-specific), and from MRL/lpr mice (IgM positive but not anti-dsDNA).

Adoptive transfer of serum or monoclonal antibodies In serum transfer experiments, MRL/lpr mice of 8-10 weeks of age (just before the onset of proteinuria) were treated with pooled sera which had been collected from relatively old MRL/lpr mice (WT), sera from age-matched AID-/-MRL/lpr mice (AID-/-), and PBS by i.p. injection, 200ul/mouse, twice a week for 8 weeks. Wild type sera contained both IgM and IgG autoantibodies, while AID-deficient sera contained high levels of IgM autoantibodies but no IgG. Urine samples were collected every one or two weeks for testing urine protein. After 8 weeks of treatment, mice were euthanized. Similarly, in monoclonal antibody transfer experiments, MRL/lpr mice were treated with anti-dsDNA IgM mAbs at lOOug, twice a week for 8 to 15 weeks depending on the experiment. For therapeutic experiments, MRL/lpr mice were not given mAbs until the mice had moderate proteinuria (over 50 mg/dl) . The mice were treated with anti-dsDNA IgM mAbs for 8 weeks as in these experiments. At the end of treatment with sera/mAb, all mice were euthanized and tissues and sera collected for various analysis.

Measurement of circulating IgG complexes To determine circulating IgG-containing immune complexes (IgG-IC), Costar High Binding 96-well EIA/RIA Plate was coated with goat IgG fraction to mouse complement C3 (MP Biomedicals, Solon, Ohio) in carbonate-bicarbonate buffer, 0.05M, pH9.6 (Sigma) at lOug/ml, lOOul/well at 4°C overnight. The plate was washed with washing buffer (PBS, pH7.4, 0.05% Tween 20, Sigma), treated with blocking buffer (PBS, pH7.4, with 1% BSA, Sigma) at 200ul/well at RT for Ih. Reference sera which had been collected and pooled from 12 MRL/lpr mice at 5-7 months of age were prepared by serial two-fold dilutions starting at 1 : 1000 to 1:256000 (9 points) with sample diluent (Blocking buffer plus 0.05% Tween 20) and added to the plates at 100ul/well. Serum samples were diluted at 1:8000 with sample diluent and were added to the plate at 100ul/well. After incubation at room temperature for lhour, the plate was washed, HRP- conjugated goat anti-mouse IgG added (SouthernBiotech, Birmingham, Alabama) at 1:15000 dilution at lOOμl/well, and incubated for lhour. After washing, lOOul TMB substrate was added to each well, incubated for 15 - 20 min in dim light. The reaction was stopped by adding 50μl 2M H₂SO₄ to each well. The absorbance at 450nm was measured in a Multiskan Ascent microplate reader. The amount of IgG-IC in each well was calculated according to the standard curve of the reference sera which is defined as a value of 1.

Sequencing of V_H regions from hybridomas and splenic B cells Anti-dsDNA monoclonal antibody-producing hybridoma cells were lysed with TRIzol Reagent (Invitrogen, Carlsbad, CA) for total RNA preparation. Two micrograms of total RNA was used as template to synthesize the first strand cDNA by Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen) following manufacturer's instructions. Five microliters of cDNA reactions was used to amplify V_H regions of immunoglobulins by AccuPrimeTM Pfx DNA polymerase (Invitrogen). Universal primers were designed according to Liang and colleagues ( Liang Z, et al.. J Exp Med. 2004;199(3):381-398, incorporated herein by reference) as follows: universal primer MV_H-F (5'- AGGTSMARCTGCAGSAGTCWGG-S' (SEQ ID NO: 33) -(M = A+C; R = A+G; S = C +G; W = A+T ) paired with MCμ-R (5'-CAGGGGGCTCTCGCAGGAGACGAGG-S') (SEQ ID NO: 34) for Vμ amplification, while MV_n-F paired with MCγ-R ( 5'-GGACAGGGATCCAGAGTTCC-S') (SEQ ID NO: 35) for Vγ amplification. The PCR cycle conditions included: denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 1 min, and extension at 68°C for 1 min; 68°C for 6 min. The amplified V_H DNA fragments were purified with QIAquick® Gel Extraction Kit (Qiagen, Valencia, CA) following the kit instructions. The purified V_H fragments were sequenced using BigDye Terminator vl.l Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) plus MCμ-R primer or MCγ-R primer depending on the V_H fragments. Following cycle conditions were used: 96°C 1.5min; 25 cycles of 96°C lOsec, 50⁰C 5sec, and 60⁰C 4min. The sequencing reactions were purified with DyeEx™ 2.0 Spin Kit (Qiagen), vacuum dried, and run through 3130x1 Genetic Analyzer (Applied Biosystems) for sequencing analysis. Sequence data were analyzed online by the blast programs at NCBI and IgBLAST (http://www.ncbi.nlm.nih.gov/projects/igblast/).

For general repertoire analysis, spleens were collected from AID-wt and -deficient MRL/lpr mice (3 mice per group, 10-14 weeks of age). Single cell suspensions were made and pooled for each group. CD19⁺ B cells were purified with anti-CD19 MACS MicroBeads and magnet columns (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and lysed with TRIzol for total RNA preparation. The first strand cDNA was synthesized and Vμ fragments were amplified exactly as mentioned above. The amplified Vμ fragments were gel-purified and cloned into ZeroBlunt®TOPO PCR Cloning Kit for Sequencing (Invitrogen) following manufacturer's instructions. MAX Efficiency® DH5α™ competent cells (Invitrogen) were transformed with the TOPO cloning reactions, spread on LB agar plates (with Amp at 50ug/ml), and incubated at 37°C overnight. Colonies were inoculated into LB medium (with Amp at 75ug/ml) to grow overnight. Plasmid DNA was minipreped using QIAprep® Spin Miniprep Kit (Qiagen, Valencia, CA). The cloned V_H fragments were sequenced using BigDye Terminator vl.l Cycle Sequencing Kit plus MCμ-R primers as mentioned above.

Immunofluorescence and histoimmunochemical staining Frozen kidney samples were cut and stained for C3 as reported previously (Jiang C, et al. J Immunol. 2007; 178:7422-7431). For each animal 20 randomly selected glomeruli were evaluated to obtain an average score. Scoring was done as follows: +, weak staining with limited localization in the glomerulus(<50%); ++, moderate stain intensity where localization in the glomerulus was more diffuse (50% -75%); and +++, intense stain with diffused and homogeneous stain covering most of the glomerulus (>75%).

For F4/80 staining, paraffin-fixed kidney slides were first deparaffinized, hydrated, and then treated with 3% hydrogen peroxide to quench endogenous peroxidase. Slides were placed in a Decloaking Chamber (Biocare Medical, Concord, CA) at 120⁰C for 5 min. Slides were blocked with 5% normal rabbit serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 20 minutes, and avidin block from the Avidin /Biotin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA). Rat anti-F4/80 monoclonal antibody (BM8) (Santa Cruz Biotechnology Santa Cruz, CA) was applied at a 1:25 dilution and incubated for 1 hour. Biotinylated Rabbit Anti-Rat IgG (H+L) (Vector Laboratories, Inc. Burlingame, CA) was used at a 1:200 dilution and incubated for 30 minutes. Label Complex from Vectastain Standard Elite ABC Kit (Vector Laboratories, Inc.

Burlingame, CA) added and incubated for 30 minutes. Slides were incubated with DAB chromagen for 6 minutes and counterstained with Harris Hematoxylin for 20 seconds. F4/80 staining in glomeruli was scored on a scale of 0-3 in 20 randomly selected high-power microscopic fields (x400) per animal as follows: 0, No F4/80 positive cell; +, 1-9 F4/80 positive cells; ++, 10-20 F4/80 positive cells; +++, > 20 F4/80 positive cells.

Cleaved caspase 3 staining was performed similarly as F4/80 staining except rabbit anti- cleaved Caspase-3 Antibody (Promega Corporation, Madison, WI) was used at 1:500 dilution and the biotinylated goat anti-rabbit IgG (H+L) (Vector Laboratories, Inc. Burlingame, CA) was used at 1:1000 dilution. Caspase3 positive cells were counted in 10 randomly selected views from High- Power Microscopic Field (x200) per animal. The presence of apoptotic cells was graded according to apoptotic index which equals Caspase3 positive cell number/per HPMF: 0, No Caspase3 positive cell;+, 1-4 Caspase3 positive cells; ++, 5-10 Caspase3 positive cells; +++, > 10 Caspase3 positive cells.

Flow cytometric analysis Mouse spleens were collected at the end of antibody treatment experiments. Single cell suspensions were made and red blood cells were lysed as reported previously 1. One million of cells were treated with FcB lock at lug/100ul staining buffer on ice for 10 min, then stained with appropriate conjugated antibodies on ice for 30 min. After washing, the cells were run through LSR II (BD Biosciences, San Jose, CA) and the acquired data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). The conjugated antibodies used included: anti-mouse CD4 PE, anti-Mouse CD62L FITC, CD44 APC or PE-Cy5, anti-mouse CD25 PE-Cy7,anti-mouse CD154 APC, anti-mouse CD19 FITC or PE-Cy7, anti-mouse CD86 FITC, anti-mouse CD69 PE or PE-Cy5, anti-mouse I- Ak biotin/streptavidin-APC, anti-mouse IgG2a+b PE, and their corresponding isotype controls. All antibodies were purchased from BD Biosciences.

Detection of cytokines The levels of serum cytokines were determined with Bio-Plex Mouse Cytokine Assays (Bio-Rad Laboratories, Inc., Hercules, CA) and Mouse TH1/TH2 9-PlexUltra- Sensitive Kit (Meso Scale Discovery, Gaithersburg, Maryland). To test cytokine production in cultured macrophages, splenic macrophages were purified with CDl Ib Micro beads and MACS Seperation Columns (Miltenyi Biotec Inc., Auburn, CA), plated to 96-well plate at 2 x 10⁵cells/well, incubated with LPS (lug/ml) for 6Oh. Culture supernatants were used to measure cytokine production by Mouse TH1/TH2 9-PlexUltra-Sensitive Kit (Meso Scale Discovery). Antinuclear antibody test To ensure a non-autoreactive IgM control was used in the passive transfers experiments, we examined several hybridoma candidates secreting IgM but that were negative against dsDNA. Supernatants were tested for autoreactivity using the indirect fluorescent antibody assay (ANA HEp-2 Ag substrate slide; Bion Enterprises) following the manufacturer's instructions and as described previously (Jiang C, et al. J Immunol. 2007; 178:7422-7431.).

Statistical analysis Pairwise associations between the outcomes (lymphoid hyperplasia, glomerulonephritis, and mononuclear cell infiltrate) were examined with the Kruskal-Wallis ANOVA and Spearman's correlation coefficient. When significant differences were detected, Mann-Whitney tests were used to compare them to the mild severity group. To assess which combination of measures best predicted outcome severity, stepwise linear regression analysis was used. Kruskal-Wallis ANOVA was used to test for differences among genotypes followed by Mann-Whitney tests to identify the differing pair of genotypes. For urine data, paired measurements on wk 12-14 and wk 17- 19 were compared using the Wilcoxon signed-ranks test. Differences were considered statistically significant at the 0.05 level using the Bonferroni correction for multiple testing where appropriate. For survival analysis, Log-rank (Mantel-Cox) Test was used to compare survival curves. For repertoire analysis, the Likelihood Ratio test was used. All p values of less than 0.05 were deemed significant.

Example 2— AID deficiency in the MRL/lpr background alleviated glomerulonephritis and mononuclear cell infiltration in the kidneys

Multifocal mononuclear cell infiltration and glomerulonephritis were prominent findings in the kidney of MRL/lpr mice. The average severity of glomerulonephritis and mononuclear cell infiltrates among the F5 mice was significantly higher in the AID-wild type (^+/+) and AID- heterozygous ^(+Λ) MRL/lpr mice than in the AID-deficient ^{( Λ)} MRL/lpr mouse littermates (Fig. 1; Kruskal-Wallis ANOVA, p < 0.0001). Glomerulonephritis was characterized by varying increases in the mesangial matrix due to a homogeneous eosinophilic material filling the mesangial spaces between glomerular capillary loops (Fig. 1C). In glomeruli with severe glomerulonephritis, mesangial matrix increase was associated with increasing numbers of glomerular cells. Both the mesangial matrix average score and the number of glomerular cells were dramatically reduced with AID deficiency (Fig. IA and B) wherein glomerular cell numbers in AID-deficient MRL/lpr mice were similar to those observed in C57BL/6 and BALB/c mice (Fig. IB). The glomerular cell increase was due primarily to inflammatory cells, particularly mesangial macrophages.

Mononuclear cell infiltrates consisted of mixed mononuclear inflammatory cells, primarily lymphocytes and macrophages in the kidney interstitium. AID-deficient MRL/lpr mice mononuclear cell infiltrate scores were reduced compared with those of AID wildtype and heterozygous MRL/lpr littermates (Fig. IA; Kruskal-Wallis ANOVA, p < 0.0001). These cells accumulated adjacent to the renal pelvis, and in AID-deficient MRL/lpr mice they were seen only in that location. As the amount of infiltrate increased, the cells formed large cuffs around arcuate arteries in the cortex, and in AID wild- type MRL/lpr mice the cells were also scattered in the inters titium between clusters of tubules. Mononuclear cell infiltration in AID-deficient MRL/lpr mice, although reduced over that in AID wild-type littermates, was above background compared with AID/C57BL/6, C57BL/6, and BALB/c mice. The kidney weights of F5 and F6 AID^7" MRl/lpr mice were significantly reduced compared with those of AID wild-type littermates.

Consistent with the reduced kidney pathology observed in the histology, urine protein levels in F5 AID-deficient MRL/lpr mice older than 10 wk of age were significantly lower than those in AID wild-type MRL/lpr mice (Fig. 2A; Kruskal-Wallis ANOVA, p = 0.0002) and undistinguishable from those in either nonautoimmune AID-deficient mice in the B16 background or conventional C57BL/6 mice. Urine protein scores from AID wild-type MRL/lpr mice of the F6 backcrossed generation were also significantly higher than those from AID-deficient MRL/lpr siblings at 17-19 wk (n = 24, mean of 2.8 + 1.32 in wild type compared with 1.39 + 0.41 for AID-deficient mice; Wilcoxon signed-ranks test, p < 0.01). Similarly, F7 and F9 combined data from mice with >99.22% MRL/lpr background that were between 20 and 30 wk old displayed the same trend (n = 17, mean = 3.11 + 1.19 for AID- competent mice vs 1.5 ±0.40 for AID-deficient MRL/lpr mice; Wilcoxon signed-ranks test, p < 0.01). Furthermore, this difference in correlates of kidney pathology persisted over time, as significantly higher levels of blood urea nitrogen and creatinine in the serum of AID wild-type and heterozygous MRL/lpr mice were detected when compared with AID-deficient MRL/lpr siblings of the F5 generation at 52 wk of age (Fig. 2B; p < 0.05 for creatinine analysis and p < 0.01 for blood urea nitrogen analysis; Wilcoxon ranks test).

AID-deficient MRL/lpr mice kidneys revealed lower C3 levels in their glomeruli than AID wild-type MRL/lpr littermates (Kruskal-Wallis ANOVA, p < 0.02), suggesting that the abrogation of glomerulonephritis is associated with a reduction in immune complex deposition. Also, IgG deposition in the glomeruli of AID wild-type MRl/lpr mice was detected at 16- to 18-wk of age but, as expected, was absent in glomeruli from AID-deficient MRL/lpr mice. IgM deposition was detected in AID-wild type, heterozygous, and deficient MRL/lpr mice, but no differences among the groups were seen. To determine whether any evidence of the early stages of glomeruli damage could be observed in AID-deficient MRL/lpr mice, electron micrographs of glomeruli from representative mice were taken. Although the glomeruli from AID-wild type MRL/lpr mice had severe lesions consisting of fusion of the glomerular podocytes' foot processes and infiltration by intravascular macrophages, the glomeruli from AID-deficient MRL/lpr mice were intact and undistinguishable from those of nonautoimmune C57BL/6 mice. Glomerulonephritis scores were similar between males and females (Kruskal-Wallis ANOVA, p > 0.15), but females tended to have more severe mononuclear cell infiltrate scores (Kruskal-Wallis ANOVA, p = 0.007). Gender differences in mononuclear cell infiltrates cannot account for the differences among genotypes because similar gender ratios were used and, when analyzed separately for gender, the differences between the various MRL/lpr littermates remained intact.

Pathological manifestations in other tissues were similar among all MRL/lpr mice and different from those of normal mice. Tissues analyzed included lymphoid tissues, spleen, liver, lung, and bone marrow. Example 3— Improved survival with AID deficiency in MRL/lpr mice

To examine the impact of AID deficiency on lifespan, a group of F5 mice were allowed to live until multiple signs of impending death were evident as determined by at least two veterinarians (i.e., decreased activity, lowered body temperature, respiratory distress, weigh loss, etc.). After 50 wk, -75% of the AID-wild type MRL/ lpr mice, 65% of AID -heterozygous MRL/lpr mice, and 75% of the nonbackcrossed MRL/lpr controls had perished whereas only 22% of the AID-deficient MRL/lpr had died, indicating a dramatic increase in lifespan with AID deficiency in MRL/lpr mice (Fig. 3; Wilcoxon test; p < 0.0001).

Example 4— B and T cell subsets in AID-deficient MRL/lpr mice

The total numbers of CD19⁺B220⁺ B cells, the percentage of naϊve and activated B cells (based on the expression of CD40, 1-A^k, PNA, or CD44) from spleen and lymph nodes were similar among F5 and F6 MRL/lpr mice regardless of AID status. Marginal zone B cells (based on CD21/CD23 expression) in all MRL/lpr mice were increased over those of BALB/c and C57BL/6 mice (-26% in MRL/lpr vs -10% in C57BL/6 mice) with a concomitant reduction in follicular zone B cells. The germinal centers of AID-deficient MRL/lpr mice were similar in morphology and number to AID wild-type MRL/lpr littermates as revealed by PNA staining of GC B cells.

Within T cells, the fractions of CD4⁺ or CD8⁺ T cells were similar among MRL/lpr mice regardless of AID status. The intriguing CD4^" CD8^"B220⁺ T cell population characteristic of MRL/lpr mice was similar among all MRL/lpr mice, (-50% of CD3⁺ cells in the spleen and lymph nodes). B cell-deficient MRL/lpr mice had been previously reported to have a large increase in the percentage of naive CD4⁺ T cells with a concomitant decrease in activated or memory T cells, suggesting B cell mediated activation of autoreactive T cells. Because in mice with B cells but lacking secreted antibodies the alteration in the proportions of naive, activated, and memory T cells were restored to those seen in MRL/lpr mice, (naive T cell population was reduced by >90%), this effect on splenic T cells was directly attributed to an antibody independent role by B cells. F5 and F6 AID-deficient MRL/ lpr mice consistently displayed a slight increase in the splenic naϊve CD4⁺T cell population that was significant in the F6 mice, but this increase was only 2-fold (3% in AID^+/+ vs 6% in AID^7" mice; Kruskal-Wallis ANOVA test, p < 0.05). No consistent pattern emerged in the mean values for a concomitant decrease in the memory or activated CD4⁺ T cell population in neither F5 (10.4% activated, 69.4% memory in AID^+/+ vs 10.8% activated and 68.6% memory in AID^7") nor F6 mice (15% activated and 77.1% memory in AID^+/+ mice vs 17.8% activated and 71.8% memory in AID^7" mice). Further analysis of the lymph nodes from these mice revealed no difference in the levels of naive, activated, or memory CD4⁺ T cells in AID-deficient vs AID wild-type, MRL/lpr mice.

Example 5- Autoreactive IgG and IgM levels in AID^7", AID⁷⁺, and AID^+/+ MRL/lpr mice Serum anti-dsDNA IgG antibodies are characteristic of MRL/lpr mice and humans with SLE.

These autoantibodies contribute to glomerulonephritis via their deposition in glomeruli as part of immune complexes. We first examined total serum Ig antibodies and as expected, AID-deficient MRL/lpr mice had increased levels of IgM but lacked total IgG and IgA because they lack the ability to undergo CSR (Fig. 4A). Anti-dsDNA IgG antibodies levels in the sera of F5 and F6 AID heterozygous- and AID-wild type MRL/lpr mice increased over time while, as expected, AID- deficient MRL/lpr mice had undetectable levels of anti-dsDNA IgG (Fig. 4B). Intriguingly, AID heterozygous MRL/lpr mice had significantly lower levels of anti-dsDNA IgG than their AID wild- type MRL/lpr littermates (Fig. 4B; Kruskal-Wallis ANOVA, p = 0.0001). In fact, there was a small, statistically insignificant but consistent trend for AID heterozygous mice to display lower severity scores in the histology measures of kidney pathology (Fig. 1) that disappeared with age. These combined data suggest an AID dosage effect in MRL/lpr mice.

We also looked at levels of anti-dsDNA IgM. Strikingly, AIDdeficient MRL/lpr mice displayed a 5-fold increase in the levels of IgM autoantibodies (Fig. 4C; Kruskal-Wallis ANOVA, p = 0.0001). To address whether this is strictly due to the AID defect, we examined anti-dsDNA IgM levels in AID-deficient C57BL/6 mice. There was a modest increase in the levels of anti-dsDNA IgM (< 2-fold) in the AID-deficient mice compared with those in C57BL/6 mice (Fig. 5D), but these levels were 60-fold lower than those in AID-deficient MRL/lpr mice, suggesting that it is the combination of AID deficiency in the MRL/lpr background that contributes to exaggerated anti-dsDNA IgM levels.

To confirm serum autoantibody results, ANA tests using HEp-2 cells were done. Sera from AID wild-type MRL/lpr mice showed bright staining for IgG in the nucleus of cells, whereas the sera from AID-deficient MRL/lpr mice were negative. Confirming the high levels of autoreactive IgM antibodies, the ANA IgM stain was bright for serum from all AID^7"MRL/lpr mice and displayed two distinct patterns: strictly nuclear and nuclear with an additional cytoplasmic punctate distribution. There was weak IgM staining for AID wild-type MRL/lpr mice in most samples, with a few showing moderate staining. Example 6 — Passive transfer of anti-dsDNA germline IgM antibodies prevent kidney damage and inflammation in MRL/lpr mice

To examine the role of memory B cells we generated a new mouse strain that is incapable of generating hypermutated switched antibodies that was backcrossed over 8 generations into an autoimmune background. These mice, AID ^~'^~ MRL/lpr mice experienced a dramatic increase in survival levels that exceeded even those of mice without secreted IgG. This suggested that something else in addition to the lack of pathogenic IgG contributed to their survival.

We hypothesized that the dramatic increase in the survival of AID-deficient MRL/lpr mice, was not only due to the lack of IgG, but also to the presence of a protective factor in their serum. Given the high levels of autorective IgM, we speculated that the factor might be anti-dsDNA antibodies. To test this hypothesis we carried out passive transfer experiments using sera or monoclonal anti-dsDNA IgM antibodies from AID-deficient MRL/lpr mice. The results are discussed below.

To first determine whether a factor in the sera of AID deficient MRL/lpr mice contributed to their dramatic increase in survival, we transferred sera from AID deficient, AID wild type, and AID heterozygous MRL/lpr into young asymptomatic MRL/lpr mice for at least eight weeks. These mice were approximately 8-10 weeks of age and they did not exhibit evidence of proteinuria at the start of the experiment (negative to trace levels). There was a significant reduction in the levels of proteinuria in the mice receiving the sera from the AID-deficient MRL/lpr mice compared to the PBS and the wild type group (Fig. 6a). Accordingly, AID-deficient MRl/lpr mice revealed significantly decreased levels of immune complex deposition in the kidneys, as measured by the amount of complement factor 3 immunofluorescence staining in glomeruli (Fig. 6b). The group receiving serum from AID wild-type MRL/lpr mice exhibited a trend for increased kidney damage but it was not significantly different from PBS. Example 7- Anti-dsDNA treatment of MRL/lpr mice reduced the severity of kidney damage

To further delineate the protective factor in the sera of AID ^~'^~ MRL/lpr, we generated hybridomas from AID ^''^' MRL/lpr, AID ^+/~ MRL/lpr, and AID ^+/+ MRL/lpr, and cloned those secreting antidsDNA IgM antibodies. Passive transfers into young asymptomatic mice were done using antidsDNA IgM from AID deficient and AID wild type MRL/lpr mice and found that while both offered some level of protection, those derived from AID deficient mice resulted in the best improvement in lupus nephritis in several scores: urine protein and anti-inflammatory cytokines were increased. Sequencing of hybridomas derived from AID ^~'^~ MRL/lpr, AID ^+Λ MRL/lpr and AID ^+/+ MRL/lpr revealed that a different repertoire of variable regions was used in those derived from the AID deficient MRL/lpr mice in two ways: they were unmutated and unexpectedly appeared to be derived from a different set of variable region genes than those derived from wild type, which were mutated.

These data demonstrate the utility of germline (unmutated) anti-dsDNA antibodies as for IgM-mediated protection from lupus nephritis. These protective anti-dsDNA IgM antibodies from AID deficient mice not only lacked somatic mutations but tended to be derived from B cells bearing receptors of a subset of the murine VH5 and VH6 subgroups (7183 and J606) that normally populate the neonatal repertoire and are associated with natural antibodies.

The human equivalent of the mouse antibodies are identified and prepared using various methods. The equivalent families in humans are also normally associated with the neonatal repertoire and have also been found to predominantly be derived from the equivalent family to 7183: VH3. The genes within that family that are useful for protection are being determined by generating a cell line system that secretes germline antibodies (a transfectoma) with Vh segments derived from human VH3 families that are associated with natural autoantibodies in sufficient amounts to test for protection in mouse models of lupus nephritis (MRL/lpr). Since the antigen is dsDNA, the human antibodies should be able to cross react with mouse dsDNA and in fact in antinuclear antibodies tests (ANA) using human Hep-2 cells, we have shown that mouse anti-dsDNA can be detected. This is not surprising considering how exquisetely conserved dsDNA is across all organisms. In addition, human antibodies have been shown to properly elicit an immune response in mice. Therefore, we will be able to screen transfectoma generated germline human antibodies in MRL/lpr mice.

Mouse antibodies offering the best protection (germline anti-dsDNA) are humanized by generating human-mouse chimeric antibodies with human framework regions and constant domains for effector function using well known methods. Again, since the antigen is dsDNA, a highly conserved molecule between mouse and human, this type of approach wherein mouse complementarity regions from germline mouse anti-dsDNA antibodies are used within the context of human framework regions is a viable approach in inhibiting or reducing the severity of nephritis in patients with lupus.

Example 8- Anti-dsDNA IgM treatment of MRL/lpr mice reduced the severity of kidney damage. To determine whether the increased levels in autoreactive IgM contributed to the protection conferred by serum from AID-deficient MRL/lpr mice, anti-dsDNA IgM secreting hybridomas were generated from AID- deficient, and AID wild-type MRL/lpr mice. Screening only for kappa light- chain secreting hybridomas with anti-dsDNA specificity, there was a 6.5-fold increase in the number of anti-dsDNA clones in the hybridomas derived from the AID-deficient MRL/lpr mice. As expected, all the clones from the AID-deficient mice were IgM.

Clones secreting anti-dsDNA IgM from AID wild type and AID deficient MRL/lpr mice were taken to monoclonality and used in passive transfer experiments. At least 2 from each group were used. In addition, a non-autoreactive, IgM-secreting clone as determined by anti-nuclear antibody assay, was used as a control. Following treatment with anti-dsDNA IgM, asymptomatic MRL/lpr mice experienced a significant delay in the onset of lupus nephritis as measured by proteinuria and C3 deposition (Fig. 7a-d). This was seen with several clones regardless of whether the anti-dsDNA IgM originated from AID-deficient or AID wild type MRL/lpr mice (Fig. 7a-d). Accordingly, the kidneys of mice receiving anti-dsDNA IgM treatment were smaller than those from the other groups in three out of the four groups receiving the anti-dsDNA IgM. In most cases, mice receiving this treatment failed to develop significant kidney disease even after 10 weeks of treatment, at a point where most mice were at least 6 months of age and when all of the mice in the PBS or the non-autoreactive group had developed moderate to severe kidney damage. To ask if other autoreactive IgM antibodies could also confer protection against lupus nephritis, we repeated these experiments this time including an anti-phospholipid IgM and an anti- Smith antigen IgM. The anti-Smith antigen IgM has been previously described and it cross-reacts with single-stranded DNA but not with dsDNA. Mice receiving the anti-dsDNA IgM derived from AID- deficient MRL/lpr mice fared best compared to all groups including mice treated with anti-Smith and anti-phospholipid IgM which displayed little or no protection (Fig. 8a-b). The anti-dsDNA IgM derived from wild type MRL/lpr mice in this experiment tended to protect less than the anti-dsDNA IgM from AID-deficient MRL/lpr mice (Fig. 8) similarly to seen in one of the wild type groups when examining kidney weights in the previous experiment. Histopathology analysis of these groups confirmed that only the anti-dsDNA IgM -receiving groups experienced a reduction in kidney damage as revealed by a reduction in mononuclear cell infiltration (Fig. 9a) and glomerulonephritis (Fig. 9b). Again, mice receiving the anti-dsDNA IgM from AID-deficient MRl/lpr mice fared better than when derived from wild type mice (Fig. 9).

Similar experiments to those described above, but testing various doses of anti-dsDNA IgM confirmed a positive correlation for better protection with increasing dose up to lOOμg, twice a week, the dose used in our studies. There was little gain when using double that amount (Fig. 10a). Using the 100 μg, twice a week regimen, we then treated symptomatic mice (proteinuria of >60 mg/dl). Even in mice with significant proteinuria initially, there was a significant reduction in kidney damage with anti-dsDNA IgM treatment (Fig. lOb-c). Example 9- Anti-dsDNA IgM treatment was associated with a reduction in macrophage infiltration and apoptotic debris in the kidneys.

Electron micrographs of glomeruli and surrounding areas taken from representative mice from each group revealed two characteristics see in all groups except mice receiving anti-dsDNA IgM from AID-deficient hybridomas: extensive macrophage and lymphocytic infiltration and in some cases, the presence of what appeared to be apoptotic cells/debris in the mesangium (Fig. 11). To see if this was a general trend, kidney tissues were stained for immunohistochemistry with either f4/80 antibody for detection of macrophages or anti-cleaved caspase-3 antibody for detection of ongoing apoptosis. There was a significant reduction in the intensity of signal for both antibodies in mice receiving the anti-dsDNA IgM treatment compared to the PBS group, confirming the electron microscopy observations (Fig. 12a-b). Similarly, experiments including anti-phospholipid and anti- Smith IgM revealed significantly less f4/80 and cleaved-caspase 3 antibody staining in kidneys from mice receiving anti-dsDNA IgM, particularly if derived from AID-deficient MRL/lpr mice.

Interestingly, electron microscopy analysis suggested that when the anti-dsDNA IgM antibody is derived from AID-deficient MRL/lpr mice, the level of protection is slightly better than when derived from wild type MRL/lpr mice as evidenced by an increase in electron dense deposits in the glomerular filtration membrane and the glomerular tuft in mice receiving the anti-dsDNA IgM derived form AID wild type hybridomas (Table 1). This trend for better protection from AID-deficient MRL/lpr mice- derived anti-dsDNA antibodies was seen in all antibody transfer experiments described above.

Example 10- Anti-dsDNA IgM did not impact autoreactive T cells in the spleen, but splenic macrophages from anti-dsDNA IgM -treated mice secreted less inflammatory cytokines and the serum of these mice had lower levels of circulating IgG-immune complexes.

Previously, it was demonstrated that in addition to secreting pathogenic autoantibodies, B cells may also contribute to autoimmunity by activating autoreactive T cells (Chan et al., J Immunol. 1999;163:3592-3596). Indeed, MRL/lpr mice have a 10-fold increase in the proportion of activated/memory T cells but in the absence of B cells, these activated T cells are dramatically reduced with a concordant increase in the proportion of naϊve T cells. Introduction of an IgM transgene that rescued B cells but not secreted antibodies, reconstituted the activated/memory T cell population, indicating an antibody-independent role of B cells by activating T cells, possibly as antigen-presenting cells. To examine if anti-dsDNA IgM somehow prevents the activation of autoreactive T cells by B cells, we examined the proportion of naϊve, activated, and memory splenic T cells. The evidence does not support this as the mechanism for IgM protection, as the proportion of memory/activated T cells, and of naive T cells was unaltered with IgM treatment. It was also possible, that anti-dsDNA IgM imparts protection by somehow modulating the proportion of regulatory T cells. However, there was no alteration in the numbers of splenic regulatory T cells either.

We also examined whether anti-dsDNA IgM protects in part by preventing the secretion of inflammatory cytokines and/or promoting secretion of anti-inflammatory cytokines by B cells and/or macrophages. B cells and macrophages were taken from the spleen of mice from the various groups and examined for cytokine secretion following activation with LPS. Serum levels of various cytokines were also measured. There was a consistent trend for a reduction in serum TNF-alpha levels and for reduced secretion of TNF-alpha by cultured macrophages when derived from mice receiving the anti- dsDNA IgM, particularly when the IgM was derived from AID-deficient MRL/lpr mice (Fig. 13a-b). In addition, activated macrophages from mice receiving the protective IgM, secreted less interferon γ (Supplemental Fig. 11). There was a trend for mice receiving the anti-dsDNA IgM to have more of the anti-inflammatory cytokines IL4 and ILlO in the serum, but it was not consistent across experiments (data not shown). No differences were detected in the levels of cytokine IL-12, IL- Ib, IL-2, and IL-5 (data not shown). The levels of circulating IgG-containing immune complexes in the serum were lower in mice receiving the anti-dsDNA IgM but this decrease was only statistically significant when the IgM was derived from AID-deficient MRL/lpr mice (Fig. 13c).

Example 11- AID deficient MRL/lpr mice with secreted IgM, outlived MRL/lpr mice lacking secreted antibodies despite both groups having normal levels of proteinuria

Given the decreased levels of circulating IgG-containing immune complexes in mice receiving the anti-dsDNA IgM, we asked whether IgM-mediated protection was through an IgG- dependent process. μS7-MRL/lpr mice were crossed to AID-deficient MRL/lpr mice to generate MRL/lpr mice with no secreted antibodies, since μS7-MRL/lpr mice lack secreted IgM because of a targeted mutation to the IgM secretory exon, and without AID, they are unable to switch to IgG or other isotypes. This enabled the comparison of two nearly identical strains differing only on whether there is secretion of IgM. Urine protein was examined for at least 52 weeks and no detectable difference was observed between the two strains suggesting anti-dsDNA IgM protects against lupus nephritis through an IgG-mediated process (Fig. 14). We then examined mortality in both strains and were surprised to see that AID-deficient MRL/lpr mice still experienced significantly lower mortality rates than the mice without secreted IgM (Fig. 15), despite both groups having only minimal proteinuria. This suggested an additional mechanism of IgM-mediated protection.

Example 12- The repertoire of B cells secreting protective anti-dsDNA IgM derived from AID- deficient MRL/lpr mice is different from the repertoire derived from AID wild type MRL/lpr mice. Prompted by a trend for anti-dsDNA IgM antibodies derived from AID-deficient MRL/lpr mice to protect better than those derived from their AID wild type counterparts, we sequenced hybridomas from both groups. Over half of the hybridomas derived from AID-deficient MRL/lpr mice were of the Vh7183 family, while most derived from the wild type mice were from the VhJ558 family (Fig. 16a). The rest from AID-deficient hybridomas were either J558 or J606. These results cannot be explained by a bias in the B cell repertoire of AID-deficient MRL/lpr mice (Fig. 16b). IgM repertoire analysis of splenic B cells from AID-deficient and AID-wild type MRL/lpr mice using the same primers as done for hybridoma analysis did not reveal an increase in the usage of Vh7183 (Figure 16b). Therefore, the repertoire difference in the hybridomas could not be explained by an overall B cell repertoire alteration in AID-deficient MRL/lpr mice. Another possible explanation for the better protection by anti-dsDNA IgM from AID-deficient MRL/lpr mice could be affinity to dsDNA. However, apparent affinity to dsDNA did not correlate with protection. For example, the anti-dsDNA IgM derived from wild type MRL/lpr mice that had the highest apparent affinity value at 12.96 (clone 4A5) if anything conferred less protection than the AID-deficient MRL/lpr mouse derived anti-dsDNA IgM's with an apparent affinity of 0.76 and 0.21 (clones 12H5 and 13D2). Example 13 — Screening for germline human IgM anti-dsDNA antibodies

Human germline IgM antibodies can be used for the methods of the invention. Methods of isolating antibodies having specificity and binding properties similar to the mouse antibodies described herein can be readily identified from human subjects. Humans suffering from SLE, particularly those not suffering from lupus nephritis, or having less severe symptoms of lupus nephritis than would be expected for the stage of the disease of the subject, are identified. IgM⁺ memory B cells that bind dsDNA are isolated from SLE patients through single cell sorting using a selecting reagent such as a tetramer with a mimetope of dsDNA. The V_H and V_L of each cell in the plates are amplified using univeral human primers for V_H and Vkappa families. The sequences are used to generate expression vectors for transfectomas wherein each V_H and corresponding V_L is expressed and secreted and tested for dsDNA specifity. Those with dsDNA specificity are tested for protection. Antibodies identified as providing protection against lupus nephritis are used as therapeutics.

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

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All references, patents, patent publications, and sequence reference numbers as of the date of the filing of the instant application cited herein are incorporated herein by reference.

Claims

We claim:

1. Use of an IgM germline antibody for preparation of a medicament for prevention, amelioration, or treatment of lupus nephritis wherein the antibody specifically binds double stranded DNA whereby lupus nephritis is prevented, ameliorated, or treated.

2. The use of claim 1 , wherein the germline antibody is administered as an isolated population of antibodies enriched for an IgM germline antibody.

3. The use of claim 1 or 2, wherein the antibody comprises a monoclonal IgM germline antibody.

4. The use of any of claims 1-3, wherein the IgM germline antibody comprises an antibody produced by a hybridoma cell line selected from the group consisting of Horn 12H5, Homl3D2,

Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3,

15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12, defined by an Accession Numbers deposited at an appropriate cell repository.

5. The use of any of claims 1-4, wherein the IgM antibody comprises the variable heavy chain (V_H) of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn

12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12; and variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

6. The use of any of claims 1-5, wherein the IgM antibody comprises the CDR sequences of the V_H antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12; and the CDR sequences of the V_L of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5,

Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

7. The use of any of claims 1- 6, wherein the IgM antibody is selected from the group consisting of an antibody comprising:

a V_H sequence at least 80% identical to the amino acid sequence Homl2H5_VH (SEQ ID

NO: 2) and a V_L sequence at least 80% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4); a V_H sequence at least 80% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a variable light chain sequence at least 80% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8);

a V_H sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

8. The use of any of claims 1-7, wherein the IgM antibody is selected from the group consisting of antibodies that comprise:

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VH (SEQ ID NO: 2) and a V_L sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4);

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); and

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

9. The use of any of claims 1-8, wherein the IgM antibody comprises a monoclonal antibody.

10. The use of any of claims 1-3 and 5-9, wherein the IgM antibody comprises a humanized IgM antibody.

11. The use of any of claims 1 and 3-10 wherein the IgM germline antibody comprises a cell expressing a therapeutically effective amount of an IgM germline antibody.

12. The use of any of claims 1 and 3-10 wherein providing an IgM germline antibody comprises administration of an agent to promote expression of a therapeutically effective amount of an IgM germline antibody.

13. The use of any of claims 1 to 12, wherein the CDR regions of the V_H and the CDR regions of the V_L are from an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

14. The use of any of claims 1-13, wherein the germline IgM antibody comprises a medicament having at least one effect selected from the group consisting of: significantly reducing a level of circulating immune complexes or a level of at least one inflammatory cytokine in serum of the subject; significantly increasing a level of at least one anti-inflammatory cytokine in a subject; and significantly decreasing proteinurea in a subject as compared to administration of an equivalent dose of a mature IgM that has undergone hypermutation.

15. The use of claim 15, wherein the inflammatory cytokine comprises TNF-alpha and an anti- inflammatory cytokine is selected from a group consisting of IL-4, IL-6, IL-IO.

16. The use of any of claims 1 to 15, wherein the lupus neprhitis is prevented, ameliorated, or treated by reductiuon of macrophage activation by the medicament comprising the germline IgM antibody.

17. The use of any of claims 1 to 16, wherein the medicament comprising the antibody comprises a cell expressing the therapeutically effective amount of IgM germline antibody.

18. A composition comprising an isolated population of antibodies enriched for an IgM germline antibody or antibodies wherein the antibodies specifically binds double stranded DNA.

19. The composition of claim 18, wherein the IgM germline antibody comprises an antibody produced by a hybridoma cell line selected from the group consisting of Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3,

15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12 defined by an Accession Numbers deposited at an appropriate cell repository.

20. The composition of claim 18 or 19, wherein the IgM antibody comprises the variable heavy chain (V_H) an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12; and variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

21. The composition any of claims 18 to 20, wherein the IgM antibody comprises the CDR sequences the variable heavy chain (V_H) an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ- Hom5G91G12; and the CDR sequences of the V_L of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3,

Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ- Hom5G91G12.

22. The composition any of claims 18 to 21, wherein the IgM antibody is selected from a group of antibodies comprising:

a V_H sequence at least 80% identical to the amino acid sequence Homl2H5_VH (SEQ ID NO: T) and a V_L sequence at least 80% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4); a V_H sequence at least 80% identical to the amino acid sequence Homl3D2_VH (SEQ ID

NO: 6) and a variable light chain sequence at least 80% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); and

a V_H sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

23. The composition any of claims 18 to 22, wherein the IgM antibody is selected from a group of antibodies comprising:

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VH (SEQ ID NO: 2) and a V_L sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4);

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); and

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

24. A composition comprising a single chain antibody wherein the antibodies specifically binds double stranded DNA.

25. The composition of claim 24, wherein the antibody comprises the variable heavy chain (V_H) an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ- Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12; and variable light chain (V_L) sequence of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

26. The composition any of claims 24 or 25, wherein the antibody comprises the CDR sequences the variable heavy chain (V_H) an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12; and the CDR sequences of the V_L of an antibody produced by a hybridoma cell line selected from the group consisting of: Horn 12H5, Homl3D2, Hom5G9, HomlE21H3, Hom5C3, Homl2E2, Hom6H2, Homl2C12, Homl2El l, 13CJ-Homl3E3, 15CJ-HomlE21H3, Horn, and 16CJ-Hom5G91G12.

27. The composition any of claims 24 to 26, wherein the antibody is selected from a group of antibodies comprising:

a V_H sequence at least 80% identical to the amino acid sequence Homl2H5_VH (SEQ ID NO: T) and a V_L sequence at least 80% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4); a V_H sequence at least 80% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a variable light chain sequence at least 80% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); and

a V_H sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 80% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

28. The composition any of claims 24 to 27, wherein the antibody is selected from a group of antibodies comprising:

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VH (SEQ ID NO: 2) and a V_L sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl2H5_VL (SEQ ID NO: 4);

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VH (SEQ ID NO: 6) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Homl3D2_VL (SEQ ID NO: 8); and

a V_H sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 9) and a variable light chain sequence at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence Hom5G9 (SEQ ID NO: 10).

29. A composition of any of claims 18 to 28 in a pharmaceutically acceptable carrier.

30. A method for providing a prognosis for a subject suffering from or suspected of suffering from lupus nephritis comprising: obtaining a serum sample from the subject; and

detecting a germline anti-dsDNA IgM germline antibody in the subject serum sample, wherein detection of at least one germline anti-dsDNA IgM germline antibody in the subject sample is indicative of a positive prognosis.