WO2016179579A2 - Compositions d'anticorps anti-cytoplasme et antinucléaires et procédés d'utilisation associés - Google Patents

Compositions d'anticorps anti-cytoplasme et antinucléaires et procédés d'utilisation associés Download PDF

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WO2016179579A2
WO2016179579A2 PCT/US2016/031425 US2016031425W WO2016179579A2 WO 2016179579 A2 WO2016179579 A2 WO 2016179579A2 US 2016031425 W US2016031425 W US 2016031425W WO 2016179579 A2 WO2016179579 A2 WO 2016179579A2
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igm
antibodies
cells
igg
disease associated
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WO2016179579A3 (fr
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Marilyn Diaz
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Therabs Corp
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Therabs Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • Embodiments described herein relate to cytoplasmic, Fc region and nuclear antibody compositions and methods of use thereof, particularly wherein the compositions are used alone or as an enrichment product for intravenous immunoglobulins. More particularly, embodiments described herein relate to methods of use of the disclosed compositions for treating or diagnosing blood related cancers, immune complex-mediated diseases, and certain autoimmune disorders. BACKGROUND
  • Autoimmune disorders are a heterogeneous group of chronic immune disorders that afflict over 20 million Americans. These disorders are characterized by an inappropriate immune response to self-antigens wherein an individual's own cells or tissues are attacked by components of the immune system. Autoimmune diseases vary in the degree of organ involvement, ranging from organ specific autoimmunity such as the thyroid in autoimmune thyroiditis or where multiple organ systems are compromised such as systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disorder (MCTD), and scleroderma (SSc).
  • SLE systemic lupus erythematosus
  • MCTD mixed connective tissue disorder
  • SSc scleroderma
  • SLE is a clear example of a systemic autoimmune disorder with a strong B cell component, and therefore a good model to examine how B cells can impart autoimmunity. It is characterized by the circulation of autoantibodies and immune complex deposition in various tissues, particularly the kidney glomeruli causing nephritis, a common fatal complication of SLE (Yung & Chan (2008)
  • these autoantibodies can trigger an autoimmune cascade characterized by inflammation and tissue destruction.
  • certain autoreactive antibodies of the IgM type are protective from development of lupus nephritis and likely other immune-complex mediated disorders (type III hypersensitivity) .
  • lupus is primarily a disease of young women. There are approximately 16,000 new cases of lupus diagnosed each year, with 70% of them developing the systemic form. Of that group, up to 50% develop lupus nephritis and up to 30% of those progress to end-stage renal disease, and require dialysis or a kidney transplant. Over 30% of all SLE deaths occur between the ages of 15-44.
  • Standard treatment aims to suppress immune system.
  • immunosuppressants such as high dose cyclophosphamide, mycophenolate mofetil, azathioprine are used. Immunosuppressants especially in the context of the immune dysfunction of SLE can lead to severe complications such as sepsis. Belimumab, the newest FDA approved treatment for lupus nephritis, is only marginally effective and a pivotal study increased deaths in the treatment group and was associated with an increased risk of serious infection. Clearly, a novel approach for lupus nephritis is critically needed.
  • autoantibodies can bind and selective kill tumor cells.
  • a composition comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti- cytoplasmic specificity.
  • at least one anti-nuclear specificity is selected from the group consisting of anti-single stranded DNA (anti-ssDNA), anti-double stranded DNA (anti-dsDNA), anti-histone, anti-RNP, anti-Smith (anti-Sm), anti-Ro, anti-La, anti-DNP, anti-Scl- 70, and anti-PM/Scl).
  • at least one anti-cytoplasmic specificity selected from the group consisting of anti-Jo-1, anti-phospholipid, anti-phosphocholine, anti- phosphatidylcholine, and anti-Ribosomal-P.
  • composition comprising IgM antibodies with at least one anti- nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity further comprises IgM antibodies with at least one anti-Fc receptor (of IgG) specificity.
  • composition comprising IgM antibodies with at least one anti- nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity further comprises at least one IgM antibody capable of activating mesangial cells to induce phagocytosis and at least one IgM antibody capable of activating mesangial cells to secrete cytokines.
  • composition comprising IgM antibodies with at least one anti- nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity further comprises at least one IgM antibody capable of binding apoptotic cells and at least one IgM antibody capable of binding immune complexes.
  • composition comprising IgM antibodies with at least one anti- nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity further comprises at least two autoreactive IgM specificities equivalent to specificities of the pathogenic IgG spectra of a disease, selected from the group consisting of a disease associated with Type III hypersensitivity reactions, a disease associated with excessive apoptotic debris and inflammation, an autoimmune disease associated with pathogenic IgG autoantibodies, and a blood cancer.
  • composition comprising IgM antibodies with at least one anti- nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity further comprises: a) at least one IgM antibody capable of activating mesangial cells to induce
  • any of the compositions described above further comprise Ivlg. In another embodiment, any of the compositions described above comprise IgM antibodies that are humanized and/or in chimeric form.
  • a method for treating a disease by administering a therapeutically effective amount of any of the compositions described above to a subject in need thereof, wherein the disease is selected from the group consisting of a disease associated with Type III hypersensitivity reactions, a disease associated with excessive apoptotic debris and
  • the disease associated with Type III hypersensitivity reactions is selected from the group consisting of nephritis, vasculitis, and some forms of arthritis.
  • the disease associated with excessive apoptotic debris and inflammation or the autoimmune disease associated with pathogenic IgG autoantibodies is selected from the group consisting of SLE, scleroderma, rheumatoid arthritis, atherosclerosis, and ischemia.
  • the blood cancer is selected from the group consisting of leukemia, B cell lymphoma, and T cell lymphoma.
  • a method for predicting the likelihood of developing nephritis in a subject with SLE comprising the steps of: a) obtaining a sample from the subject; b) measuring levels of protective IgM antibodies and of pathogenic IgG in the sample; and c) determining the ratio of protective IgM antibodies to pathogenic IgG in the sample; wherein a higher level of protective IgM antibodies as compared the level of pathogenic IgG is indicative of a lower likelihood of developing nephritis in the subject with SLE.
  • the protective antibodies as a group have at least three characteristics selected from the group consisting of: 1) being of the IgM type; 2) having the ability to bind antinuclear and/or cytoplasmic antigens; and 3) having the ability to bind apoptotic cells and stimulate cytokine secretion by mesangial cells.
  • the pathogenic antibodies are of the IgG subtype and bind nuclear and/or certain cytoplasmic antigens.
  • the protective antibodies and the pathogenic antibodies are measured by a method selected from the group consisting of immunofluorescence techniques with antinuclear antigen assays, mesangial cell cultures, apoptotic binding assays, a specific antigen binding assay, and heavy chain immunoglobulin variable region family determination.
  • the specific antigen binding assay is ELISA.
  • FIG. 1 shows that passive transfer of certain autoreactive IgM antibodies protected lupus- prone mice from development of lupus nephritis as measured by levels of protein in the urine.
  • FIG. 2 shows that IgM autoreactive antibodies with some level of protection display a variety of specificities. It is expected that specific combinations of protective autoreactive IgM antibodies, with or without an anti-IgG specificity, is an effective treatment for SLE patients with nephritis.
  • FIG. 3 shows that protective antibodies also vary dramatically in their ability to stimulate IL-6 production by mesangial cells. All depicted treatments were with autoreactive igM antibodies but only two, Hom-1 and WT-1 induced secretion. This was associated with increased proliferation of mesangial cells. Although counterintuitive because of the pro-inflammatory properties of IL-6, antibodies with strongest stimulation of IL-6 production are expected to be a critical component of the combination therapy.
  • FIG 4. shows that antibodies vary in their ability to stimulate phagocytosis by mesangial cells. We expect that combination therapy is enhanced by inclusion of antibodies with the best ability to induce mesangial cell phagocytosis.
  • FIG. 5 illustrates that protective antibodies efficiently bind apoptotic cells.
  • FIG. 6. illustrates that treatment with autoreactive IgM antibodies (high IgM group) can effectively reduce the severity of T cell lymphoma in lymphoma-prone mice. The onset was delayed as well. It is expected that combinations of antibodies with autoreactive specificities is an effective treatment for patients with lymphoma.
  • 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.
  • administration of an agent or combination of agents to stimulate the production of germline IgM antibodies that specifically bind dsDNA is understood as administration of an agent or combination of agents to stimulate the production of germline IgM antibodies that specifically bind dsDNA.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • antibody is understood as a globular plasma protein having a molecular weight of about 150kDa, 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.
  • antibody also includes fragments, e.g., proteolytic fragments, of antibodies including Fab fragments, Fc fragments, and F(ab) 2 fragments.
  • 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.
  • 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
  • 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).
  • 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.
  • the term "antigen" refers to a molecule that is bound by an antibody paired
  • 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.
  • an "antibody with autoreactive specificity” is understood as an antibody that specifically binds an autoantigen in the absence of proteins.
  • the antibody binds the autoantigen 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 non-specific antigens.
  • 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.
  • class 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.
  • C constant regions of the immunoglobulin heavy chain.
  • naive 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.
  • 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, ⁇ - 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 (or level) and measurement of the change can vary. Determination of statistical significance is within the ability of those skilled in the art.
  • CDR complementarity determining region
  • antigen binding pocket e.g. immunoglobulin and T cell receptor
  • Each polypeptide chain of an antigen receptor contains three CDRs (CDR1, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • detecting 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 (or level) 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.
  • 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.
  • 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.
  • side -effects 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.
  • 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.
  • 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.
  • 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.
  • 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 or relative to the amounts normally present in sera preparations.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.,
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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., 3 H
  • fluorescent labels e.g., FITC, rhodamine, lanthanide phosphors
  • enzymatic labels e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase
  • biotinyl groups e.g., predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • the label is part of the fusion protein, e.g. Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP).
  • GFP Green Fluorescent Protein
  • “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 Weening et al. (2004) Am. Soc. Nephrol. 15:241-250, which is incorporated herein by reference. A summary table reproduced from Weening et al. is provided below as Table 1. Other more simplified classification systems are provided in the Examples.
  • ISN/RPS International Society of Nephrology/Renal Pathology Society
  • IV-S diffuse segmental
  • IV-G diffuse global
  • Active lesions diffuse segmental proliferative lupus nephritis
  • Active lesions diffuse global proliferative lupus nephritis
  • Active and chronic lesions diffuse segmental proliferative and sclerosing lupus
  • Class V lupus nephritis may occur in combination with class III or IV in which case both will be diagnosed
  • “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.
  • 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.
  • phrases "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
  • 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.
  • 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'
  • wetting agents 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.
  • antioxidants examples 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, a-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • 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
  • Formulations of the present invention include those suitable for intravenous, 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.
  • plurality is understood to mean more than one.
  • a plurality refers to at least two, three, four, five, or more.
  • 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.
  • prone to refers to a subject more likely than the general population to develop the disease or condition.
  • 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.
  • 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.
  • sample refers to a biological material that is isolated from its
  • 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.
  • “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 10 2 -fold higher, at least 10 3 -fold higher, at least 10 4 -fold higher, or at least 10 5 - fold higher.
  • a "subject" as used herein refers to living organisms.
  • the living organism is a human.
  • the living organism is an animal.
  • the subject is a mammal such as primate including a non-human primate.
  • the subject is a domesticated mammal. Accordingly, 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.
  • flares periods of illness
  • 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
  • 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.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • biocompatible, biodegradable lactide polymer such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • lactide/glycolide copolymer or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of certain agents.
  • 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 500 ⁇ g/kg/week to 10 mg/kg/week.
  • transfection refers to the introduction of a transgene into a cell.
  • transgene 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.
  • stable transfection or "stably transfected” refers to the introduction and integration of a transgene into the genome of the transfected cell.
  • stable transfectant refers to a cell which has stably integrated one or more transgenes into the genomic DNA.
  • 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.
  • transient transfectant refers to a cell which has transiently integrated one or more transgenes.
  • 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.
  • 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).
  • this process generates a huge number of antibodies, each with different paratopes, and thus different antigen specificities.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • IL-6 refers to interleukin 6, which is an interleukin acting both as a proinflammatory and anti-inflammatory cytokine. It is secreted by T cells and macrophages and other immune cells.
  • phagocytosis refers to the process that cells use to ingest or engulf other cells, particles or immune complexes.
  • immune complex refers to an antigen and antibody complex of interlocking antigens and antibodies forming a network of molecules that can be difficult to clear in certain conditions.
  • immunologists In order to classify the processes by which the immune system produces cellular damage, immunologists have divided immune responses into four broad classes (Type I, II, III and IV) (Roitt, I.M., et aL , Immunology, C.V. Mosby, N.Y., 1985, p. 19.1 ).
  • Type I responses are also called immediate hypersensitivity reactions and include those diseases which produce the symptoms classically associated with “allergies” or the “allergic syndrome” including allergic rhinitis (hay fever), allergic asthma, allergic conjunctivitis, and allergic reactions to insect stings or foods.
  • allergic rhinitis hay fever
  • allergic asthma allergic conjunctivitis
  • allergic reactions to insect stings or foods a specialized sequence of events within mast cells and basophils must be triggered by
  • immunoglobulin E (IgE) antibodies that have been manufactured within the body.
  • the hypersensitivity states characterized by types II, III and IV hypersensitivity are distinguished from type I hypersensitivity by many distinct and diverse features.
  • the sensitizing agent allergen
  • Types II, III and IV hypersensitivity may have immune responses directed towards antigens located on ceils and molecules that are normal constituents of the body. Such immune responses toward normal constituents of the body are termed "autoimmune diseases" and constitute a medically important class of diseases distinct from allergic diseases.
  • Type II hypersensitivity occurs when IgG or IgM antibodies bind to antigens located on the surfaces of cells. Such binding is mediated by the antibodies' Fab arms which contain specific structures that recognize cell surface antigens. Upon binding, the Fc regions of IgG or IgM interact with the complement system (a family of inflammatory and cell-killing molecules) or immune system "killer" cells bearing IgG or IgM Fc receptors.
  • complement system a family of inflammatory and cell-killing molecules
  • IgG or IgM Fc receptors include transfusion reactions, hemolytic disease of the newborn, autoimmune hemolytic anemias, hyperacute graft rejection, Goodpasture's syndrome, myesthenia gravis and other conditions.
  • Type III hypersensitivity is produced when complexes or aggregates of antibodies (usually IgG or IgM) and soluble antigens form in abnormally large amounts and activate the complement inflammatory system.
  • Type III hypersensitivity reactions refers to the process whereby insoluble immune complexes form in the blood and are deposited in various tissues which can trigger an immune response and inflammation.
  • diseases in which type III hypersensitivity reactions are pathogenically important include nephritis, vasculitis, and some forms of arthritis.
  • Type IV hypersensitivity (delayed-type hypersensitivity), by contrast to the other three hypersensitivity reactions, is triggered primarily by T cells having specialized T cell receptors able to recognize and bind to the specific sensitizing antigen on a cell's surface.
  • Diseases in which type IV hypersensitiviiy is believed to play an important pathogenic role are frequently termed "T-cell mediated" to reflect the unique role played by the T-cell in recognizing the sensitizing antigen. These diseases include multiple sclerosis, rheumatoid arthritis, juvenile onset diabetes mellitus, ulcerative colitis, and regional enteritis (Crohn's disease), among others.
  • the term "mesangial” refers to kidney myofibroblasts cells at the glomerulus and along the vascular pole of the glomerulus with contractile and phagocytic properties.
  • compositions comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity.
  • at least one anti-nuclear specificity is selected from the group consisting of anti- single stranded DNA (anti- ssDNA), anti-double stranded DNA (anti-dsDNA), anti-histone, anti-RNP, anti-Smith (anti-Sm), anti-Ro, anti-La, anti-DNP, anti-Scl-70, and anti-PM/Scl.
  • At least one anti- cytoplasmic specificity is selected from the group consisting of anti-Jo-1, anti-phospholipid, anti- phosphocholine, anti-phosphatidylcholine, or anti-Ribosomal-P.
  • the combination further comprises Ivlg.
  • IgM antibodies are humanized and/or in chimeric form.
  • compositions comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity, further comprising IgM antibodies with at least one anti-Fc receptor of IgG specificity.
  • the combination further comprises Ivlg.
  • IgM antibodies are humanized and/or in chimeric form.
  • compositions comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity, further comprising at least one IgM antibody capable of activating mesangial cells to induce phagocytosis and at least one IgM antibody capable of activating mesangial cells to secrete cytokines.
  • the combination further comprises Ivlg.
  • IgM antibodies are humanized and/or in chimeric form.
  • compositions comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity, further comprising at least one IgM antibody capable of binding apoptotic cells and at least one IgM antibody capable of binding immune complexes.
  • the combination further comprises Ivlg.
  • IgM antibodies are humanized and/or in chimeric form.
  • compositions comprising a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity, further comprising at least two autoreactive IgM specificities equivalent to specificities of the pathogenic IgG spectra of a disease from the group consisting of a disease associated with Type III hypersensitivity reactions, a disease associated with excessive apoptotic debris and inflammation, an autoimmune disease associated with pathogenic IgG autoantibodies, and a blood cancer.
  • the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity
  • IgM antibodies are humanized and/or in chimeric form.
  • compositions comprising: 1) a combination of IgM antibodies with different autoreactive specificities, wherein the combination of IgM antibodies comprises IgM antibodies with at least one anti-nuclear specificity and IgM antibodies with at least one anti-cytoplasmic specificity; 2) at least one IgM antibody capable of activating mesangial cells to induce phagocytosis and at least one IgM antibody capable of activating mesangial cells to secrete cytokines; 3) at least one IgM antibody capable of binding apoptotic cells and at least one IgM antibody capable of binding immune complexes; and 4) at least two autoreactive IgM specificities equivalent to specificities of the pathogenic IgG spectra of a disease selected from the group consisting of a disease associated with Type III hypersensitivity reactions, an autoimmune disease associated with pathogenic IgG autoantibodies, a disease associated with excessive apoptotic debris and inflammation, and a blood cancer.
  • the combination of IgM antibodies comprises IgM antibodies with at
  • the presently disclosed subject matter is directed to methods of treating a disease by administering a therapeutically effective amount of any of the compositions disclosed herein to a subject in need thereof, wherein the disease is selected from the group consisting of a disease associated with Type III hypersensitivity reactions, a disease associated with excessive apoptotic debris and inflammation, an autoimmune disease associated with pathogenic IgG autoantibodies, and a blood cancer.
  • the disease associated with Type III hypersensitivity reactions is selected from the group consisting of nephritis, vasculitis, and some forms of arthritis.
  • the disease associated with excessive apoptotic debris and inflammation or the autoimmune disease associated with pathogenic IgG autoantibodies is selected from the group consisting of SLE, scleroderma, rheumatoid arthritis, atherosclerosis, and ischemia.
  • Blood cancers include, but are not limited to, leukemia, and B and T cell lymphomas.
  • the presently disclosed subject matter is directed to a method for predicting the likelihood of developing nephritis in a subject with SLE, the method comprising the steps of: a) obtaining a sample from the subject; b) measuring levels of protective IgM antibodies and of pathogenic IgG in the sample; and c) determining the ratio of protective IgM antibodies to pathogenic IgG in the sample; wherein a higher level of protective IgM antibodies as compared the level of pathogenic IgG is indicative of a lower likelihood of developing nephritis in the subject with SLE.
  • protective antibodies may be defined as having the following characteristics: 1) being of the IgM type; 2) having the ability to bind antinuclear and/or cytoplasmic antigens; and 3) having the ability to bind apoptotic cells and stimulate cytokine secretion by mesangial cells.
  • pathogenic antibodies may be defined by the following characteristics: 1) being of the IgG subtype; and 2) binding nuclear and/or certain cytoplasmic antigens.
  • Antibodies may be measured either using immunofluorescence techniques with antinuclear antigen assays, mesangial cell cultures, apoptotic binding assays, a specific antigen binding assay such as ELISA, and/or heavy chain immunoglobulin variable region family determination.
  • autoantibodies and the deposition of immune complexes in various tissues, particularly the kidneys, leading to glomerulonephritis Oates et al. (2002) Curr. Opin. Rheumatol. 14:498).
  • Hallmark SLE autoantibodies include antibodies against double- stranded DNA (antidsDNA), histones and ribonucleoproteins (Yung et al. (2008) Autoimmun Rev. 7:317). These pathogenic autoantibodies form immune complexes that trigger inflammatory responses, leading to organ damage.
  • Factors that have been implicated in SLE and in lupus-like syndromes in mice include defective apoptosis, Toll receptor signaling, defects in B and T cell tolerance, complement activation, cytokine regulation, and defects in endothelial cell function. (Peng et al. (1996) Mol. Biol. Rep. 23:247; Pickering et al. (2000) Adv. Immunol. 76:227; Li et al. (2002) J. Exp. Med. 196: 1543, and others).
  • MRL-Faslpr/lpr mice develop an auto-immune syndrome similar to SLE
  • MRL/lpr mice display high levels of autoantibodies to dsDNA and the development of lupus nephritis through immune complex deposition, glomerular disease, and tissue infiltration by inflammatory cells. Many loci contribute to autoimmunity in MRL/lpr mice, leading to defects in cytokine regulation, lymphocyte tolerance, and apoptosis .
  • AID activation-induced deaminase
  • mice AID-/- MRL/lpr mice were developed by backcrossing C57BL/6 AID-deficient mice onto the MRL/lpr background, using the MRL/MpJ-Faslpr/2J mice (stock no. 006825; The Jackson Laboratory), for 6 generations. They were then backcrossed an additional 9 generations into cryorecovered MRL/lpr mouse strain MRL/MpJ-Faslpr/J (stock no. 000485; The Jackson
  • mice All mice were housed in specific pathogen-free facilities at the National Institute of
  • IgM antidsDNA-producing hybridomas were generated according to standard protocols. Spleens were collected from
  • nonimmunized AID-/-, AID-/-, and AID-/- MRL/lpr mice (10-12 weeks old; 3-6 mice per group).
  • Splenocytes were fused with murine myeloma NS-1 cells (5: 1 ratio) in 50% polyethylene glycol (PEG 1500; Roche).
  • Supernatants were screened for anti-dsDNA specificity by enzyme- linked immunosorbent assay (ELISA).
  • ELISA enzyme- linked immunosorbent assay
  • Cell clones secreting IgM anti-dsDNA were injected (2 x 10 6 cells) into the peritoneal cavity of RAG-l-knockout mice (The Jackson Laboratory) that had been pretreated with 0.2 ml of pristane (Sigma) to induce ascites.
  • MRL/lpr mice ages 8-10 weeks were injected intraperitoneally with pooled sera collected from older MRL/lpr mice, sera from age-matched AID-/- MRL/lpr mice, or with phosphate buffered saline (PBS) at a dosage of 200 ml per mouse, twice a week for 8 weeks.
  • Wild- type mouse sera contained both IgM and IgG autoantibodies, while AID-deficient sera contained high levels of IgM autoantibodies but no IgG.
  • Urine samples were collected weekly for proteinuria testing. After 8 weeks, mice were killed. In antibody-transfer experiments, MRL/lpr mice were treated with IgM anti-dsDNA mAb at a dosage of 100 microg per mouse, twice weekly for 8-15 weeks, depending on the experiment. For therapeutic experiments, treatment was not started until the mice had moderate proteinuria (50-60 mg/dl). Detection of urinary protein. Urinary protein was tested by dipstick using Multistix 10 SG strips (Siemens Healthcare Diagnostics). Results were scored as described previously (Jiang et al. (2007) . Immunol. 178:7422). ELISA.
  • the PCR cycle conditions were as follows: 95°C for 2 minutes, with 35 cycles at 95°C for 15 seconds, 58°C for 1 minute, and 68°C for 1 minute.
  • the amplified VH DNA fragments were purified using a QIAquick Gel Extraction kit (Qiagen).
  • the purified VH fragments were sequenced using a BigDye Terminator vl.l Cycle Sequencing kit (Applied Biosystems) plus MCjJ-R ⁇ MCy-R primer, depending on the VH fragments.
  • spleens were collected from AID wild-type and AID-deficient MRL/lpr mice (3 mice per group, ages 10-14 weeks).
  • CD19+ B cells were purified with anti-CD19 MACS MicroBeads (Miltenyi Biotec) and lysed with TRIzol.
  • the cDNA was synthesized, and the Vh fragments were amplified and sequenced as above.
  • Immunofluorescence and immunohistochemical staining Frozen kidney samples were stained for C3. For each animal, 20 randomly selected glomeruli were evaluated to obtain an average score, and were graded on a scale of 0-3, where 0 means no staining; + means weak staining, with limited localization in the glomerulus (50%); ++ means moderate staining, where localization in the glomerulus was more diffuse (50-75%); and +++ means intense staining, with diffuse and homogeneous stain covering most of the glomerulus (>75%). For F4/80 staining, paraffin-fixed kidney sections were deparaffinized, hydrated, and treated with 3% hydrogen peroxide. Slides were placed in a Decloaking Chamber (BioCare Medical).
  • +++ 20 F4/80-positive cells.
  • Staining for cleaved caspase 3 was performed in a manner similar to that for F4/80 staining, except that rabbit anti-cleaved caspase 3 antibody (Promega) was used at a dilution of 1:500, and biotinylated goat anti-rabbit IgG (Vector) was used at a dilution of 1: 1,000.
  • Cleaved caspase 3-positive cells were counted in 10 randomly selected highpower (200X) microscopic fields per animal.
  • cytokines Serum levels of cytokines (interferon- ⁇ , interleukin- ⁇ , IL-12, tumor necrosis factor a IL-6, IL-4, IL-5, and IL-10) were determined with Bio-Plex mouse cytokine assays (Bio-Rad) and mouse Thl/Th2 9-plex ultra- sensitive kit (Meso Scale Discovery). Splenic macrophages were purified with CD l ib MicroBeads and magnetic-activated cell sorter separation columns (Miltenyi Biotec), and incubated with lipopoly saccharide (1 ⁇ g/ml). A mouse Thl/Th2 9- plex ultra- sensitive kit was used to measure cytokine production.
  • the Mann-Whitney rank sum test was performed to analyze the data among different treatment groups. For survival analysis, the log rank (Mantel-Cox) test was used to compare survival curves. For repertoire analysis, the likelihood ratio test was used.
  • mice receiving sera or IgM anti-dsDNA from AID- deficient MRL/lpr mice To determine whether sera from AID-deficient MRL/lpr mice contained a factor that may contribute to increased survival, we transferred sera from AID-deficient and wild- type MRL/lpr mice into young asymptomatic MRL/lpr mice for at least 8 weeks. These mice were 8-10 weeks of age and did not initially exhibit evidence of proteinuria. There was a significant reduction in levels of proteinuria in mice receiving the sera from AID-deficient MRL/lpr mice compared with PBS or wild-type mouse sera (Jiang et al. (2011) Arth. Rheum. 63: 1086).
  • MRL/lpr mice that received AID-deficient MRL/lpr sera also experienced decreased levels of immune complex deposition in the kidneys, as measured by the amount of C3 complement immunofluorescence in the glomeruli (Jiang et al. (2011) Arth. Rheum. 63: 1086).
  • the group receiving serum from AID wild-type MRL/lpr mice exhibited a trend toward increased kidney damage, but it was not significantly different from that in the group that received PBS.
  • mice secreting IgM anti-dsDNA from AID wild-type and AID-deficient MRL/lpr mice were used in passive transfer experiments.
  • a nonautoreactive, IgM-secreting clone (as determined by antinuclear antibody assay) was used as a control.
  • asymptomatic MRL/lpr mice experienced a significant delay in the onset of lupus nephritis, as indicated by proteinuria ( Figure 1) and IgG immune complex deposition. Accordingly, the kidneys of mice receiving IgM anti-dsDNA treatment were smaller than those from other groups in 3 of the 4 groups receiving IgM anti-dsDNA. In most cases, mice receiving this treatment failed to develop significant kidney disease even after 10 weeks of treatment, a point to which most mice were at least 6 months old and when all of the mice in the group receiving PBS or the group receiving nonautoreactive antibody had developed moderate to severe kidney damage.
  • kidney tissues were stained for immunohistochemistry with either F4/80 antibody (macrophage detection) or anti-cleaved caspase 3 antibody.
  • Mice receiving IgM anti-dsDNA treatment displayed lower levels of F4/80 and, in the case of the 12H5 clone, lower levels of cleaved caspase 3, confirming the electron microscopy observations (Jiang et al. (2011) Arth.
  • mice receiving the protective IgM secreted less IFN-gamma there was a trend of mice receiving the IgM anti-dsDNA to have more of the antiinflammatory cytokines IL-4 and IL-10 in their sera, although this was not consistent across experiments.
  • IgM anti-dsDNA blocks the inflammatory cascade that leads to tissue damage and cell death.
  • the variability of the effects of various IgM anti-dsDNA antibodies on correlates of inflammation shows that, while all of them decrease proteinuria and immune complex deposition, their effectiveness and mechanisms of action are different and may be related to their specificity or polyreactivity.
  • IgM repertoire analysis of splenic B cells from AID-deficient and AID wild-type MRL/lpr mice using the same primers did not reveal an increase in VH7183 usage in AID-deficient mice.
  • the apparent affinities were measured for the antibodies used in the study. The results did not reveal a correlation.
  • IgM anti-dsDNA 4A5 had the highest apparent affinity at 12.96 ml ⁇ g but did not protect better than clones 12H5 and 13D2, with apparent affinities of 0.76 ml ⁇ g and 0.21 ml ⁇ g, respectively. Therefore, having a high level of dsDNA affinity, surprisingly, did not predict better protection.
  • AID-deficient MRL/lpr mice experienced a dramatic increase in survival.
  • autoreactive IgM is not only nonpathogenic in lupus nephritis, but may be protective.
  • Treatment with some IgM antidsDNA antibodies from AID deficient and wild-type MRL/lpr mouse hybridomas revealed an improvement in all measures of lupus nephritis, such as proteinuria, immune complex deposition, and glomerulonephritis, in MRL/lpr mice.
  • IgG autoreactive antibodies are thought to be pathogenic in autoimmune disease because of their isotype and their affinity for self antigen. Somatic hypermutation and class- switch recombination tend to co-occur, which means that much of the serum IgG has been fine- tuned to a specific antigen through affinity maturation. In SLE and in MRL/lpr mice, this could mean enhanced affinity for self antigen .
  • AID heterozygous MRL/lpr mice experienced a delay in lupus nephritis onset that correlated with reduced levels of high-affinity IgG anti-dsDNA antibodies and reduced hypermutation .
  • the IgG subclass is known to influence autoantibody pathogenicity (Takahashi et al. (1991) J. Immunol. 147:515), and mice deficient in Fc receptor and in Fc receptor type III were shown to experience a reduction in kidney damage (Hazenbos et al. (1996) Immunity 5: 181). It is thus apparent that both class switching and affinity maturation against self antigens through somatic hypermutation contribute to the formation of pathogenic IgG.
  • the data presented herein point to IgM anti-dsDNA as a protective agent. Recently, it was shown that SLE patients whose disease is in remission have high levels of autoreactive naive B cells (Yurasov (2006) J. Exp. Med. 203:2255), which, in light of our findings, shows that these cells are secreting protective IgM. Also, a positive correlation was found between a high ratio of IgG to IgM anti-dsDNA in SLE and lupus nephritis patients (Forger et al. (2004) Lupus 13:36). Not all autoreactive IgM is protective. Antiphospholipid and anti-Sm IgM did not protect significantly. Furthermore, some autoreactive IgM may contribute to autoimmune disease.
  • AID deficiency can enhance autoreactivity by increasing the population of autoreactive B cells, which are known to play an antibody-independent role in enhancing autoimmunity, but ameliorates it by pathogenic IgG deficiency and elevated levels of protective IgM. It is possible that when lupus nephritis is mild in the parental strain, as in C57BL/6 lpr mice, an AID deficiency-mediated increase in polyreactive B cells enhances autoimmunity.
  • IgM anti-dsDNA antibodies protect against lupus nephritis
  • proinflammatory cytokines such as TNF a and IFN ⁇
  • a correlation between IgG anti-dsDNA antibodies and activation of macrophages to secrete proinflammatory cytokines has previously been demonstrated (Jang et al. (2009) Immunol. Lett. 124:70 ); perhaps IgM has the opposite effect.
  • an effective treatment will require a mixture of humanized or chimerized IgM autoreactive specificities determined by the range of specificities seen for pathogenic IgG in patients with specific autoimmune disorder,.
  • This shows a variety of antigen-immune complexes and it is therefore unlikely that a single specificity such as anti-dsDNA IgM will work as treatment for SLE or other autoimmune disorders.
  • the same principle can be applied to various autoimmune disorders across organisms. For instance, in dogs, immune complexes are more likely to represent anti-histone specificities than other nuclear antigens and an effective mixture needs to consider this.

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Abstract

La présente invention concerne des compositions d'anticorps anti-cytoplasme, anti-région Fc et anti-nucléaires, ainsi que des procédés d'utilisation de ceux-ci, les compositions étant particulièrement utilisées comme produit d'enrichissement pour des immunoglobulines par voie intraveineuse. Plus particulièrement, la présente invention concerne des procédés d'utilisation des compositions décrites pour le traitement ou le diagnostic de cancers associés au sang, de maladies médiées par un complexe immunitaire et certaines maladies auto-immunes.
PCT/US2016/031425 2015-05-07 2016-05-09 Compositions d'anticorps anti-cytoplasme et antinucléaires et procédés d'utilisation associés Ceased WO2016179579A2 (fr)

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