IgM antibody that degrades IgG

Abstract

The present invention relates to a glycosylated IgM antibody that cross-specifically binds to a complexed molecule such as an IgG antibody and DNA, wherein binding to the IgG antibody and the complexed molecule induces degradation of the IgG antibody. The Kd for the binding affinity of the IgM antibody to the IgG antibody is preferably in the range of 10 -5 to 10 -8 . The present invention further relates to the medical use of the glycosylated IgM antibody, such as use in the treatment of autoimmune diseases, for example, systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

Description

Technical Field

[0001] The present invention relates to a glycosylated IgM antibody that binds cross - specifically to a complexed molecule such as an IgG antibody and DNA, wherein binding to the IgG antibody and the complexed molecule induces degradation of the IgG antibody. The K d for the binding affinity of the IgM antibody to the IgG antibody is preferably in the range of 10 -5 to 10 -8 . The present invention further relates to the medical use of glycosylated IgM antibodies, such as their use in the treatment of autoimmune diseases, for example, systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

Background Art

[0002] The process of antibody generation results in the formation of an infinite number of antigen - binding sites by the random rearrangement of gene segments, namely variable (V), diversity (D), and joining (J) segments. The random nature of antibody specificity generation ensures the recognition of almost infinitely diverse antigens, but inevitably results in the generation of autoreactive specificities. Most early B cells have autoreactive BCRs, and highly autoreactive cells are thought to be eliminated from the repertoire by central tolerance, which induces receptor editing by secondary immunoglobulin (Ig) gene recombination, thereby modifying the specificity of autoreactive B cells. If receptor editing cannot replace the autoreactive specificity, each autoreactive B cell is eliminated by clonal deletion. When autoreactive B cells escape central tolerance, they are thought to be functionally silenced as mature B cells by anergy in the periphery. Defects in the elimination of autoreactive B cells are thought to lead to the development of autoimmune diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), which are characterized and diagnosed by the presence of autoantibodies.

[0003] Rheumatoid Factor (RF) is one of the first and most studied autoantibodies and was already described in the late 1940s as a class of Ig that can bind to the Fc portion of IgG (Volkov, Mikhail, Karin Anna Schie, and Diane Woude. 2020, Immunological Reviews 294(1):148 - 63). Although it is one of the most characterized autoantibodies, the role of RF-IgM in the etiology of immune diseases remains unclear. Among different RF isotypes, IgM-RF is the most clinically used to estimate disease prognosis in rheumatoid arthritis (RA), a chronic autoimmune disease characterized by infiltration of B and T cells in the synovium of joints. However, the biological function of RF in the etiology remains unknown (Volkov, Mikhail, Karin Anna Schie, and Diane Woude. 2020, Immunological Reviews 294(1):148 - 63).

[0004] An important characteristic of RA is the presence of anti-citrullinated protein-IgG (ACPA-IgG), which causes inflammation in the synovium. Here, the removal of the amino group (NH 3+ ) of arginine residues by protein arginine deaminase (PAD4) mainly generates citrullinated proteins localized in joints (Darrah, Erika, and Felipe Andrade. 2018, Current Opinion in Rheumatology 30(1):72 - 78). The binding of ACPA-IgG to citrullinated proteins seems to result in the deposition of immune complexes in joints, thereby activating innate immune cells and initiating inflammation.

[0005] In this scenario, it is thought that RF acquires pathogenic properties through the formation of immune complexes with auto-reactive ACPA-IgG antibodies, thereby causing inflammation by stimulating the secretion of inflammatory cytokines. Interestingly, RA patients are classified as RF positive (RF+) and RF negative (RF-), and the presence of RF indicates a poor prognosis (Smolen, Josef S., Daniel Aletaha, Anne Barton, Gerd R. Burmester, Paul Emery, Gary S. Firestein, Arthur Kavanaugh, Iain B. McInnes, Daniel H. Solomon, Vibeke Strand, and Kazuhiko Yamamoto. 2018. Nature Reviews Disease Primers 4(1):18001).

[0006] Accordingly, there is a need for improved means and methods for controlling IgG antibodies, particularly auto-reactive IgG antibodies in immune diseases.

[0007] The above technical problem is solved by the embodiments disclosed herein and defined in the claims.

Summary of the Invention

[0008] Accordingly, the present invention relates, inter alia, to the following embodiments. 1. A glycosylated IgM antibody that cross-specifically binds to an IgG antibody and a complexing molecule, wherein binding to the IgG antibody and the complexing molecule induces degradation of the IgG antibody. 2. K for the binding affinity of the IgM antibody to the IgG antibody d is in the range of 10 -5 to 10 -8 and preferably 10 -7 The antibody according to embodiment 1. 3. An antibody according to embodiment 1 or 2, wherein at least one complementarity-determining region (CDR) of the IgM antibody binds to the IgG antibody, and the glycosylated portion of the IgM antibody binds to the complexed molecule. 4. An antibody according to any one of embodiments 1 to 3, wherein the IgG antibody is an auto-reactive IgG antibody. 5. An antibody according to any one of embodiments 1 to 4, wherein the complexed molecule is DNA. 6. An antibody according to any one of embodiments 1 to 5, wherein the auto-reactive IgG antibody is an anti-citrullinated protein-IgG antibody. 7. An antibody according to any one of embodiments 1 to 6, wherein the first chain comprises a CDR that specifically binds to IgG, and the second chain comprises a CDR that binds to IgG in a poly-reactive manner. 8. An antibody according to any one of embodiments 1 to 7, for use in a medicament. 9. An antibody according to any one of embodiments 1 to 7, for use in the treatment of a subject having an increased IgM level, preferably a serum IgM level exceeding 1500 hIgM μm / ml. 10. An antibody according to any one of embodiments 1 to 7, for use in the treatment of a subject having an increased high-affinity rheumatoid factor: low-affinity rheumatoid factor ratio. 11. An antibody according to any one of embodiments 1 to 7, for use in the treatment of an autoimmune disease or disorder. 12. An antibody for use according to embodiment 11, wherein the autoimmune disease or disorder is at least one selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. 13. An antibody for use according to embodiment 12, wherein the autoimmune disease or disorder is rheumatoid arthritis. 14. A method for diagnosing an autoimmune disease or disorder, the method comprising: a) determining a high-affinity rheumatoid factor portion and a low-affinity rheumatoid factor portion based on the affinity of the rheumatoid factor for the IgG antibody in a sample from a subject; b) A step of diagnosing the subject with an autoimmune disorder based on the high-affinity rheumatoid factor portion and the low-affinity rheumatoid factor portion determined in a) and / or their ratio, A method comprising: 15. The antibody is A variable heavy (VH) chain comprising a CDR1 sequence encoded by SEQ ID NO: 5, a CDR2 sequence encoded by SEQ ID NO: 6, and a CDR3 sequence encoded by SEQ ID NO: 7, and a CDR1 sequence encoded by SEQ ID NO: 2, GATGCATCC Use of the IgM antibody according to any one of Embodiments 1 to 7 or the IgM antibody according to any one of Embodiments 8 to 13, comprising a variable light (VL) chain comprising a CDR2 sequence encoded by SEQ ID NO: 3 and a CDR3 sequence encoded by SEQ ID NO: 3. 16. The antibody is A variable heavy (VH) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 4 or a sequence having at least 90% sequence identity to SEQ ID NO: 4, preferably at least 95% sequence identity to SEQ ID NO: 4, and Use of the IgM antibody according to Embodiment 15 or the IgM antibody according to Embodiment 15, comprising a variable light (VL) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 1 or a sequence having at least 90% sequence identity to SEQ ID NO: 1, preferably at least 95% sequence identity to SEQ ID NO: 1. 17. a) The sequence defined by SEQ ID NO: 4, or a sequence having at least 90% sequence identity to SEQ ID NO: 4, preferably at least 95% sequence identity to SEQ ID NO: 4, and / or b) A host cell comprising a polynucleotide having the sequence defined by SEQ ID NO: 1, or a sequence having at least 90% sequence identity to SEQ ID NO: 1, preferably at least 95% sequence identity to SEQ ID NO: 1, The polynucleotide further encodes an IgM constant region and / or the host cell comprises a further polynucleotide encoding an IgM constant region. A host cell. 18. A method for producing an IgM antibody, comprising: a) culturing the host cell according to Embodiment 17; and b) isolating the IgM antibody.

[0009] Accordingly, in one embodiment, the invention relates to a glycosylated IgM antibody that binds cross - specifically to an IgG antibody and a complexed molecule, wherein binding to the IgG antibody and the complexed molecule induces degradation of the IgG antibody.

[0010] In one embodiment, the invention relates to a glycosylated IgM antibody that binds to an IgG antibody and a complexed molecule, preferably wherein binding to the IgG antibody and the complexed molecule induces degradation of the IgG antibody.

[0011] The term "IgM antibody", as used herein, refers to its general meaning in the art and refers to an immunoglobulin having a heavy m chain. Serum IgM exists as a pentamer (or hexamer) in mammals and constitutes about 10% of the normal human serum Ig content. It predominates in the primary immune response to most antigens and is the most efficient complement-binding immunoglobulin. IgM is also expressed on the plasma membrane of B lymphocytes as a membrane-bound immunoglobulin, which can be organized as a multi-protein cluster in the membrane. In this form, it is the B cell antigen receptor, and each H chain contains an additional hydrophobic domain for membrane anchoring. The monomers of serum IgM are joined together by disulfide bonds and a joining (J) chain. Each of the five monomers within the pentamer structure is composed of two light chains (either kappa or lambda) and two heavy chains. Unlike IgG (and the generalized structure shown above), the heavy chain in the IgM monomer is composed of one variable region and four constant regions, with an additional constant domain replacing the hinge region. IgM can recognize epitopes on invading microorganisms and cause cell aggregation. This antibody-antigen immune complex is then destroyed by complement binding or receptor-mediated endocytosis by macrophages. IgM is the first immunoglobulin class synthesized by newborns and plays a role in the etiology of some autoimmune diseases. Immunoglobulin M is the third most common serum Ig and takes one of two forms: a pentamer (or hexamer in some situations) in which all heavy chains are identical and all light chains are identical. The membrane-bound form is a monomer that can form a multimeric cluster on the membrane (e.g., found on B lymphocytes as the B cell receptor). In some embodiments, the IgM antibody is a monomeric IgM or an oligomeric IgM. In some embodiments, the oligomeric IgM antibodies described herein are antibodies selected from the group consisting of monomeric IgM antibodies, dimeric IgM antibodies, trimeric IgM antibodies, tetrameric IgM antibodies, pentameric IgM antibodies, and hexameric IgM antibodies.

[0012] As used herein, the term "glycosylated IgM antibody" refers to an IgM having glycosylation at at least one glycosylation site, preferably an N-linked glycosylation site, such as a J chain and / or an N-glycosylation site. In some embodiments, the IgM antibody has glycosylation at at least one Asn-linked glycosylation site. In some embodiments, the IgM antibody has glycosylation at at least one glycosylation site selected from the group consisting of ASN-46, ASN-209, ASN-272, ASN-279, ASN-440. In some embodiments, the glycosylated IgM antibodies described herein are blood-derived antibodies. In some embodiments, the glycosylated IgM antibodies described herein are produced recombinantly.

[0013] As used in the context of the present invention, the term "binds to" defines the binding (interaction) of at least two "antigen interaction sites" with each other.

[0014] As used herein, the term "binds cross-specifically" refers to binding to at least two binding partners, preferably at least two binding partners are different, for example, an IgG antibody and a complexed molecule. Cross-specificity can also be extended to a) multiple complexed molecules and / or b) multiple IgG antibodies or all IgG antibodies. In some embodiments, the glycosylated IgM antibody binds to the constant region of an IgG antibody / multiple IgG antibodies.

[0015] As used herein, the term "complexed molecule" refers to a molecule that, when bound to the glycosylated IgM antibody described herein, preferably when bound to the glycosylated IgM antibody while the glycosylated IgM antibody is bound to the IgG antibody described herein, enables the formation of an immunodegradable complex.

[0016] As used herein, the term "degradation" in the context of an IgG antibody refers to a reduction in functionality, preferably neutralization by, for example, immune cells. Preferably, IgG degradation means a decrease or neutralization of IgG measured in vivo or in vitro as described in the examples herein.

[0017] As used herein, the term "IgG" has its general meaning in the art and refers to immunoglobulins having heavy g chains. When produced as part of the secondary immune response to an antigen, this class of immunoglobulins constitutes approximately 75% of total serum Ig. IgG is the only class of Ig that can pass through the placenta in humans and is greatly involved in the protection of newborns during the first few months of life. IgG is the major immunoglobulin in blood, lymph, cerebrospinal fluid, and ascites and plays an important role in the humoral immune response. Serum IgG in healthy humans represents approximately 15% of total protein, in addition to albumin, enzymes, and other globulins. Four IgG subclasses have been described in humans, mice, and rats (e.g., IgG1, IgG2, IgG3, and IgG4 in humans). The subclasses differ in the number of disulfide bonds and the length and flexibility of the hinge region. Except for their variable regions, all immunoglobulins within a class share approximately 90% homology, but only 60% between classes. IgG1 constitutes 60 - 65% of total major subclass IgG and is mainly involved in the thymus-mediated immune response to protein and polypeptide antigens. IgG1 can bind to Fc receptors on phagocytic cells and activate the complement cascade through binding to the C1 complex. The IgG1 immune response can already be measured in newborns and reaches its typical concentration during infancy. IgG2, the second largest IgG isotype, constitutes 20 - 25% of the major subclass and is the dominant immune response to carbohydrate / polysaccharide antigens. "Adult" concentrations are usually reached by 6 or 7 years of age. IgG3 constitutes approximately 5 - 10% of total IgG and plays a major role in the immune response to protein or polypeptide antigens. The affinity of IgG3 may be higher than that of IgG1. IgG4, which usually constitutes less than 4% of total IgG, does not bind to polysaccharides. In the past, tests on IgG4 have been associated with food allergies, and recent studies have shown an increase in serum levels of IgG4 in patients suffering from sclerosing pancreatitis, cholangitis, and interstitial pneumonia caused by infiltration of IgG4-positive plasma cells.In some embodiments, the IgG antibodies described herein are antibodies of at least one subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0018] The inventors have found that glycosylated IgM antibodies that act as rheumatoid factor (RF) exhibit a neutralizing effect on IgG, thereby resulting in faster degradation and reduction of IgG in vivo. These effector functions are typically independent of the pathogenic or beneficial properties of the target IgG. Without being bound by theory, catabolic RF, which is also found in healthy individuals, may regulate the half-life of IgG, control IgG homeostasis, and defects in the production of catabolic RF may be an important trigger for the development of autoimmune diseases. In this scenario, catabolic RF is thought to neutralize IgG antibodies by forming large immune complexes together with complexing molecules such as nucleic acids, thereby promoting the uptake of IgG by immune cells such as phagocytes. Catabolic RF may act as a general regulator of IgG by recognizing its constant region. Alternatively or additionally, catabolic RF may act in a unique manner by regulating specific IgG idiotypes through the recognition of individual variable regions. In the context of IgG-related autoimmune diseases, this suggests that a highly diverse antibody repertoire is important for the regulation of a wide range of IgG antibodies targeting individual idiotypes.

[0019] This includes the presence of polyreactive neutralizing IgM, in contrast to protective regulatory IgM (Amendt, Timm, and Hassan Jumaa. 2021. The EMBO Journal 40(17)). These findings indicate that one way to potentially reduce the levels of harmful IgG antibodies in circulation is the use of low-affinity RF or total IgM antibodies from healthy individuals as therapeutic antibodies. Interestingly, the generation of idiotypic-specific anti-IgG IgM enables the manipulation of individual IgG in a specific manner without affecting the entire IgG repertoire.

[0020] The current view (see, e.g., Zikherman, Julie, Ramya Parameswaran, and Arthur Weiss. 2012. Nature 489(7414):160-64) that autoantibodies develop as a result of defects in central and peripheral tolerance mechanisms that should prevent the development of autoreactive B cells in a healthy state is teaching away from the present invention.

[0021] Accordingly, the present invention is based at least in part on the finding that glycosylated IgM antibodies can induce the degradation of IgG antibodies as described herein.

[0022] In some embodiments described herein, the RF or IgM antibodies described herein are autoantibodies or autoantibodies.

[0023] In some embodiments, the RF of the present invention is an IgM antibody, preferably a glycosylated IgM antibody.

[0024] In some embodiments described herein, the IgM antibodies described herein are monoclonal antibodies. In some embodiments, the antibodies described herein are human antibodies, humanized antibodies, or chimeric antibodies. Antibody production can be based on immunization of an animal such as a mouse. However, other animals for antibody / antiserum production are also envisioned within the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, camels, etc. Methods for producing and / or modifying antibodies are known in the art and are described, inter alia, in laboratory manuals (Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., 1994, Methods for General and Molecular Bacteriology ASM Press; Lefkovits, 1997, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press); Golemis, 2002, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press).

[0025] In certain embodiments, the present invention relates to an antibody according to the present invention, wherein the K for the binding affinity of the IgM antibody to the IgG antibody d is in the range of about 10 -5 to about 10 -8 .

[0026] In certain embodiments, the present invention relates to an antibody according to the present invention, wherein the K for the binding affinity of the IgM antibody to the IgG antibody d is about 10 -7 .

[0027] As used herein, the term "low affinity" or "binds with low affinity" refers to a binding affinity in the range of about 10 -5 to about 10 -8 , preferably 10 -5 to 10 -8 , more preferably 10 -6 to 10 -8 , even more preferably 10 -6 to 10 -7 of K d . In a highly preferred embodiment, low affinity refers to a K -7 of 10 d for binding affinity.

[0028] As used herein, the term "high affinity" or "binds with high affinity" refers to a binding affinity in the range of about 10 -9 of K d or lower K d . The term "K d " as used herein refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Those skilled in the art are well aware of the various methods and assays suitable for determining the K d of an antibody or antigen-binding fragment thereof provided herein and encompassed by the present invention. In some embodiments, K d is determined by biolayer interferometry. Preferably, K d is determined by biolayer interferometry as described herein, particularly in the Examples and Figures of the present invention.

[0029] The inventors have found that low affinity rheumatoid factors exhibit an opposing neutralizing effect (compared to high affinity rheumatoid factors) against IgG, thereby resulting in faster degradation and reduction of IgG in vivo. These effector functions depend on the affinity of RF-IgM. RF-IgM contributes to faster degradation when their affinity for IgG is low and when they are polyreactive.

[0030] As used herein, the term "polyreactive" refers to an antibody that binds to an antigen with low affinity. Polyreactive antibodies preferably bind to a variety of structurally unrelated antigens such as free double-stranded DNA.

[0031] Accordingly, the present invention is based at least in part on the finding that IgM antibodies contribute to the more rapid degradation of IgG when their affinity for IgG is low.

[0032] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody binds to an IgG antibody.

[0033] In certain embodiments, the invention relates to an antibody according to the invention, wherein the glycosylated portion of the IgM antibody binds to a complexing molecule.

[0034] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody binds to an IgG antibody and the glycosylated portion of the IgM antibody binds to a complexing molecule.

[0035] In certain embodiments, the invention relates to an antibody according to the invention, wherein the first chain of the IgM antibody contains a CDR that binds to IgG and the second chain binds to a complexing molecule, preferably via a glycosylated chain, preferably via the glycosylation of the IgM antibody.

[0036] The inventors have found that the glycosylated portion, for example glycosylation itself, is particularly efficient in the binding of the complexing molecule when it binds to the complexing molecule.

[0037] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody binds to an IgG antibody.

[0038] In certain embodiments, the invention relates to an antibody according to the invention, wherein the glycosylated portion of the IgM antibody, preferably the glycosylated amino acid sequence, binds to a complexing molecule or is involved in binding to a complexing molecule.

[0039] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody binds to an IgG antibody and the glycosylated portion of the IgM antibody binds to a complexing molecule.

[0040] In certain embodiments, the invention relates to an IgM antibody according to the invention, wherein the IgG antibody is an autoreactive IgG antibody.

[0041] As used herein, the term "autoreactive IgG antibody" refers to an antibody produced by the immune system that is directed against one or more of the subject's own proteins or antigens.

[0042] The autoreactive IgG antibodies described herein may be involved in the regulation of endogenous proteins or may be characteristic of many autoimmune diseases. In some embodiments, the IgM antibody of the invention binds to autoreactive IgG antibodies among other IgG antibodies. In some embodiments, the IgM antibody of the invention binds predominantly to autoreactive IgG antibodies.

[0043] Autoreactive IgG antibodies are likely retained in circulation because they play a specific role in maintaining physiological homeostasis. The IgM antibodies described herein can restore this maintenance upon dysregulation.

[0044] Accordingly, the invention is based at least in part on the finding that glycosylated IgM antibodies can regulate and induce the degradation of autoreactive IgG antibodies as described herein.

[0045] In certain embodiments, the invention relates to an antibody according to the invention, wherein the complexing molecule is a nucleic acid, preferably DNA, more preferably double-stranded DNA.

[0046] As used herein, the term "DNA" typically refers to any complexed molecule, including deoxyribonucleic acid in polymeric form, e.g., double-stranded form. DNA as a complexed molecule can be provided, for example, in the form of extracellular DNA released by immune cells.

[0047] Accordingly, the present invention is based at least in part on the finding that the binding of the IgM antibodies of the present invention to DNA results in the formation of a complex that can be efficiently degraded as described herein.

[0048] In certain embodiments, the present invention relates to an antibody according to the present invention, wherein the autoreactive IgG antibody is an anti-citrullinated protein-IgG antibody.

[0049] As used herein, the term "anti-citrullinated protein-IgG antibody" refers to autoantibodies directed against citrullinated peptides and proteins. These antibodies are typically observed in patients with RA and are thought to play a role in the onset and pathology of RA.

[0050] In certain embodiments, the present invention relates to an antibody according to the present invention, wherein the first chain comprises a CDR that specifically binds to IgG and the second chain comprises a CDR that binds to IgG polyreactively.

[0051] In certain embodiments, the first chain of the antibody according to the present invention comprises a CDR that binds to IgG with high affinity, and the second chain of the antibody according to the present invention comprises a CDR that binds to IgG with low affinity. The term "low affinity" refers to a binding affinity in the range of about 10 -5 ~ about 10 -8 , preferably 10 -5 ~ 10 -8 , more preferably 10 -6 ~ 10 -8 , even more preferably 10 -6 ~ 10 -7 for K d . Most preferably, the low affinity is 10-7 refers to the K of d The term "high affinity" refers to a K in the range of about 10 -9 or lower K of d refers to. d

[0052] RF antibodies are mainly associated with RA. Nevertheless, studies on RF production and incidence have shown that circulating RF can be found in healthy individuals. Interestingly, the RF antibodies studied in RA patients are characterized by extensive somatic mutations and have high antigen-binding affinity and specificity for IgG acquired during the process of affinity maturation. In contrast, RF found in healthy individuals closely resembles natural autoantibodies, which are a class of autoantibodies with restricted epitope specificities encoded mainly by germline variable gene segments. Thus, most natural autoantibodies are polyreactive and bind to self-molecules with low antigen-binding affinity. Similarly, RF in healthy individuals shows no evidence of affinity maturation and isotype switching, suggesting low antigen-binding affinity for IgG (Mageed et al. 1997; Volkov et al. 2020).

[0053] In certain embodiments, the invention relates to an antibody according to the invention for use in a medicament.

[0054] In certain embodiments, the invention relates to an antibody according to the invention for use in the treatment of a subject having increased IgM levels, preferably serum IgM levels exceeding 1500 hIgM μm / ml. Antibody levels are determined as described herein, particularly in the Examples.

[0055] High-titer, high-affinity RF in the synovium of RA patients is thought to acquire a pathogenic role by stabilizing pathogenic IgG, such as anti-citrullinated protein-IgG antibodies, thereby perpetuating the inflammatory state. The exaggerated function of autoreactive IgG in joints continuously induces macrophages and complement activation via Fc receptors, leading to the formation of immune complexes that can prolong inflammation in the synovium.

[0056] Treatment with the IgM antibodies of the present invention can control general IgG isotype, for example, when recognizing the constant region of IgG, and can selectively eliminate pathogenic IgG when acting at the level of individual idiotypes.

[0057] The inventors have found the coexistence of high-affinity RF and low-affinity RF, and that the effect of destructive low-affinity RF is dominant over protective high-affinity RF.

[0058] Accordingly, the present invention is based at least in part on the finding that the IgM antibodies of the present invention can be used for treatment in the presence of (increased) IgG-protective IgM antibodies such as high-affinity IgM.

[0059] In certain embodiments, the present invention relates to an antibody according to the present invention for use in the treatment of a subject having an increased high-affinity rheumatoid factor:low-affinity rheumatoid factor ratio. The term "increased high-affinity rheumatoid factor:low-affinity rheumatoid factor ratio" refers to an increased ratio when comparing a patient suffering from an autoimmune disease or disorder, particularly to a healthy subject not suffering from an autoimmune disease or disorder.

[0060] The inventors have found the coexistence of high-affinity RF and low-affinity RF, and that the effect of destructive low-affinity RF is dominant over protective high-affinity RF. Expanding this finding to a more general level, the ratio between the two RF populations is thought to be relevant in the context of autoimmunity.

[0061] Accordingly, the present invention is based at least in part on the finding that the IgM antibodies of the invention can be used to restore a healthy high / low affinity RF ratio.

[0062] In certain embodiments, the invention relates to an antibody according to the invention for use in the prevention of an autoimmune disease or disorder, preferably a chronic autoimmune disease or disorder.

[0063] In certain embodiments, the invention relates to an antibody according to the invention for use in the treatment of an autoimmune disease or disorder, preferably a chronic autoimmune disease or disorder.

[0064] In certain embodiments, the invention relates to a method for treating or preventing an autoimmune disease or disorder, preferably a chronic autoimmune disease or disorder, the method comprising administering to a patient an antibody according to the invention.

[0065] "Treatment" (and grammatical variations thereof such as "treating" or "treat") as used herein refers to a clinical intervention in an attempt to modify the natural course of an individual being treated, which can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of the development or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, decrease in the rate of disease progression, improvement or alleviation of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or to slow the progression of a disease.

[0066] The term "prevention" as used herein relates to the ability to prevent, minimize, or interfere with the onset or development of a disorder, disease, or condition prior to its occurrence.

[0067] The fact that low-affinity RF is found in healthy individuals and modulates the half-life of IgG suggests that IgG homeostasis is controlled by such RFs and that defects in the production of low-affinity RFs may be an important trigger for the development of autoimmune diseases. The insulin data provided herein show that high- and low-affinity IgM antibodies having opposing effects on those homologous antigens can occur against substantially all self-antigens. Without being bound by theory, it can be hypothesized that the boundary between physiological and pathological autoimmunity is strongly characterized not only by tolerance mechanisms but also by the affinity for self-antigens. Defects in the establishment of these equilibria are most likely to result in the development of an autoimmune response.

[0068] The inventors have found that patients suffering from autoimmune diseases have somewhat higher levels of total serum IgM and IgG antibodies compared to healthy donors. However, the amount of RF-IgM detected in MS patients is significantly lower than the amount observed in healthy individuals. Thus, RA patients are characterized by an increase in the amount of high-affinity protective RF that results in an increase in IgG function including autoreactive antibodies, while MS patients are most likely to lack low-affinity destructive RF. The absence of low-affinity RF modifies the hemostasis of IgG antibodies, resulting in the accumulation and enhancement of IgG function including autoreactive specificities.

[0069] Accordingly, the present invention is based at least in part on the finding that the IgM antibodies of the present invention can be used to restore the high / low-affinity RF ratio to prevent and / or treat autoimmune diseases.

[0070] In certain embodiments, the present invention relates to an antibody for use of the present invention, wherein the autoimmune disease or disorder is at least one selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

[0071] In certain embodiments, the present invention relates to an antibody for use of the present invention, wherein the autoimmune disease or disorder is rheumatoid arthritis.

[0072] The RF found in RA is significantly different from the RF found in healthy individuals, because the latter is polyreactive and shows few signs of affinity maturation. Conversely, the RF expressed by RA patients is highly somatically mutated, is monospecific, and has a high affinity for IgG.

[0073] In certain embodiments, the invention is a method for diagnosing an autoimmune disease or disorder, the method comprising: a) determining a high-affinity rheumatoid factor moiety and a low-affinity rheumatoid factor moiety based on the affinity of rheumatoid factor for IgG antibodies in a sample from a subject, preferably an ex vivo sample; and b) diagnosing the subject as having an autoimmune disorder based on the high-affinity rheumatoid factor moiety and low-affinity rheumatoid factor moiety determined in a) and / or their ratio.

[0074] Based on the limited mutation rate and reduced affinity, the inventors propose that natural autoantibodies are primary IgM antibodies secreted during the process of early B cell activation before affinity maturation. Indeed, previous studies have shown that, despite the similar usage of V light chain and V heavy chain genes, RF in healthy individuals shows a significantly reduced mutation pattern in their CDRs compared to RF in RA patients. Thus, the low-affinity RF found in the healthy population is most likely the result of a regulated selection mechanism that limits affinity maturation in healthy individuals, thereby preventing low-affinity RF autoantibodies from becoming pathogenic.

[0075] In certain embodiments, the present invention relates to an IgM antibody or the use of an IgM antibody of the present invention, wherein the IgM antibody comprises a variable heavy (VH) chain comprising a CDR1 sequence encoded by SEQ ID NO: 5, a CDR2 sequence encoded by SEQ ID NO: 6, and a CDR3 sequence encoded by SEQ ID NO: 7, and a variable light (VL) chain comprising a CDR1 sequence encoded by SEQ ID NO: 2, a CDR2 sequence encoded by GATGCATCC, and a CDR3 sequence encoded by SEQ ID NO: 3.

[0076] In certain embodiments, the present invention relates to an IgM antibody or the use of an IgM antibody of the present invention according to claim 15, wherein the IgM antibody comprises a variable heavy (VH) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 4 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 4, and a variable light (VL) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 1 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 1.

[0077] In certain embodiments, the present invention relates to a host cell comprising a polynucleotide having 1) a) the sequence defined by SEQ ID NO: 4 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 4, and / or b) the sequence defined by SEQ ID NO: 1 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 1, and 2.) the polynucleotide further encoding an IgM constant region and / or the host cell comprising a further polynucleotide encoding an IgM constant region.

[0078] In certain embodiments, the invention relates to a method for producing an IgM antibody, the method comprising: a) culturing a host cell according to the invention; and b) isolating the IgM antibody.

[0079] As used herein, the terms "a", "an", and "the" are used to refer to one or more of the grammatical objects of the articles (i.e., at least one, or one or more).

[0080] "Or" should be understood to mean any one of the alternatives, both, or any combination thereof.

[0081] "And / or" should be understood to mean either one or both of the alternatives.

[0082] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" are used to mean the inclusion of the stated step or element or group of steps or elements and do not imply the exclusion of any other step or element or group of steps or elements.

[0083] The terms "include" and "comprise" are used synonymously. Unless the context requires otherwise, the terms "comprise" or "include", and variations such as "comprises / includes" and "comprising / including" are to be understood in an inclusive sense, i.e., they are meant to imply the inclusion of elements, integers, steps, or groups thereof but not the exclusion. "Consisting of" means including and limited to what follows the phrase "consisting of".

[0084] "Preferably" means one of a series of alternatives that does not exclude other alternatives. "For example (e.g.)" means an example that is not limited to the examples mentioned.

[0085] As used herein, the terms "about" or "approximately" refer to "within 20%", more preferably "within 10%", and even more preferably "within 5%" of a given value or range.

[0086] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "a particular embodiment", "additional embodiments", "some embodiments", "specific embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that a clear description of features in one embodiment may serve as a basis for excluding features in a particular embodiment.

[0087] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and / or materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0088] The general methods and techniques described in this specification can be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), as well as Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

[0089] Embodiments of the present invention are illustrated and described in detail in the drawings and the foregoing specification, but such illustration and description should be considered illustrative or exemplary and not restrictive. It is understood that changes and modifications can be made by those skilled in the art within the scope and spirit of the following claims. In particular, the present invention encompasses further embodiments having any combination of features from the different embodiments described above and below.

Brief Description of the Drawings

[0090]

Figure 1

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Figure 8

Example

[0091] Example 1: Recombinant low-affinity anti-insulin IgM destroys insulin in vivo To confirm the inventors' hypothesis that IgM affinity and specificity determine the outcome of the interaction with the recognized homologous antigen, the inventors used a recombinant anti-insulin antibody as a model. Since the inventors proposed that affinity and single specificity for the target are the main requirements for determining the effector function of autoreactive antibodies, the inventors predicted that reversion of each of the variable regions of anti-insulin IgM to their respective germline (gl) versions would result in a reduction in its affinity for the target. For this purpose, the inventors reverted the heavy chain (HL) and light chain (LC) sequences to the germline and tested the reverted HC / LC combinations for their insulin-binding affinity. Most combinations lost insulin binding, but a recombinant insulin-specific antibody (anti-insulin IgM 低 ) consisting of the original LC and the germline-reverted HC version of the anti-insulin antibody showed a reduction in affinity for insulin compared to the original antibody (Figure 1A). Indeed, the K D of the germline-reverted anti-insulin IgM 低 was in the range of 10 -7 (Figure 1C) and was thus significantly lower than the affinity of the original anti-insulin IgM 高 . In addition, decreased insulin binding was observed for anti-insulin IgM 低 by ELISA (Figure 7). To test whether two antibodies, namely anti-insulin IgM 低 and its high-affinity counterpart IgM 高 , have different effects on glucose metabolism, the inventors injected the same molar amount of anti-insulin IgM 高 and anti-insulin IgM 低 into WT mice. Higher blood glucose levels (hyperglycemia) were observed in mice receiving anti-insulin IgM 低 within 2 hours after injection, while anti-insulin IgM 高 did not modify blood glucose and was able to protect insulin from IgG-dependent degradation (Figure 1D).

[0092] Interestingly, the revertant version of anti-insulin IgM differs only in two point mutations in complementarity-determining region 2 (CDR2), which is thought to be involved in affinity maturation (Figure 1A). Importantly, when evaluating the quality of antibodies produced in vitro, it was revealed that there were no structural differences between purified IgM 高 antibodies and IgM 低 antibodies (Figure 1B).

[0093] These data suggest that by reverting the immunoglobulin heavy chain variable region (IGHV) to its germline version (low affinity), high-affinity autoantibodies with a protective role can be converted into autoantibodies with a destructive role. This confirms the inventors' hypothesis of the regulatory role of IgM antibodies and suggests that mutations acquired during the affinity maturation process can convert destructive IgM antibodies into protective IgM antibodies.

[0094] Example 2: Recombinant low-affinity RF is polyreactive and binds to DNA To confirm the inventors' findings regarding the role of low-affinity RF in the interaction with target antigens, the inventors reviewed available reports that describe the extent of somatic mutations of RF in patients with rheumatoid arthritis (RA) (Randen et al. 1992; Youngblood et al. 1994). Most of the rheumatoid factors (RFs) isolated from the synovium of RA patients are highly affinity for the Fc portion of IgG and not reactive to other test antigens, but the inventors identified one RF (RF-IgM) isolated from an RA patient that is thought to be polyreactive and binds to other antigens such as tetanus toxoid, DNA, and bovine serum albumin (BSA) (Youngblood et al. 1994). Interestingly, detailed analysis of the IGHV and IGLV sequences of the selected RF revealed a high degree of homology to the germline gene counterparts. In fact, the selected antibody variable heavy chain shared residues 96.9% identical to IGHV3-30-3 * 01 (allele 1), and the light chain was IGKV3-11 *had 99.3% identity (Figure 3A). Due to the high identity to the germline gene and previously published data showing the polyreactivity of this RF, the inventors predicted that this antibody was a low-affinity RF (RF 低 ). Therefore, the inventors cloned and expressed RF 低 as recombinant IgM (Figure 3B). The biolayer interferometry assay revealed that the IgG binding affinity of RF 低 was in the range of 10 -7 , while the Kof RF 高 D was 10 -9 (Figure 3C).

[0095] The ability of RF 低 to bind IgG was also tested by ELISA, and it was revealed that recombinant RF 低 bound IgG to a lesser extent than RF 高 , which is most likely a result of the reduced IgG affinity of RF 低 (Figure 3D). Additionally, the inventors confirmed previously published data showing that, in contrast to RF 高 , recombinant RF 低 bound to double-stranded DNA (Figure 3E) and was reactive on HEp2 slides (Figure 3F).

[0096] These data confirm available data suggesting that low-affinity RF is multispecific / polyreactive as it binds to DNA in addition to IgG, in contrast to typical high-affinity RF from RA patients (Figure 3G).

[0097] Example 3: RF 低 controls IgG in vivo function by enhanced degradation Using the above monoclonal low-affinity RF (RF 低 ), the inventors tested whether low-affinity RF neutralizes its target in vivo. For this purpose, the inventors used equimolar amounts of RF 低Anti-insulin IgG was injected into WT mice together with non-specific monoclonal IgM (mIgM) as a control. As expected, mice injected with anti-insulin IgG alone or with anti-insulin IgG together with control mIgM showed an equivalent increase in blood glucose levels. In contrast, mice receiving RF 低 showed constant blood glucose levels, suggesting that RF 低 controls the function of autoreactive IgG (Figure 4A).

[0098] Next, the inventors investigated whether the protective or destructive effects of RF could be observed with other IgGs such as therapeutic antibodies. For this purpose, the inventors used rituximab as a well-known therapeutic IgG antibody targeting CD20. This monoclonal anti-CD20 antibody consisting of the human constant region and the murine variable domain (Pierpont, Limper, and Richards 2018; Tobinai 2001) is approved for the treatment of B cell malignancies and autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) (Aletaha and Smolen 2018; Malmstrom et al. 2017; Taylor and Lindorfer 2007). The inventors intravenously injected equimolar amounts of anti-CD20 IgG alone or in combination with either RF 高 or mIgM into WT mice and monitored the human IgG (hIgG) concentration over time. Our data showed that mice injected with rituximab together with RF 高 exhibited significantly higher levels of hIgG compared to mice receiving anti-CD20 IgG alone or in combination with mIgM (Figure 4B). Together with the above data, these results led the inventors to the hypothesis that if the higher hIgG titer was due to the presence of RF 高 , co-injection of RF 低 and anti-CD20 IgG should show the opposite effect, i.e., a reduction in hIgG levels over time. Therefore, the inventors used equimolar amounts of recombinant RF 低or anti-CD20 IgG in combination with control monoclonal mIgM. We observed a significant difference in hIgG levels between the two groups already one day after injection. Indeed, RF 低 Animals injected with RF 高 showed significantly lower concentrations of hIgG for mice receiving IgG (Figure 4C).

[0099] Our results show that high-affinity RF can stabilize IgG in vivo, thereby dramatically extending its half-life, whereas low-affinity RF exhibits the opposite, destructive effect in vivo. Collectively, these data indicate that RFs have different effects on the half-life of IgG depending on their affinity for their target. Interestingly, this is effective not only for autoreactive antibodies but also for therapeutic antibodies.

[0100] Example 4: RF 低 is RF 高 More dominant To better understand the dynamics of RF interaction with IgG in vivo, we investigated the effect of the combined presence of low- and high-affinity RF on IgG function. To this end, we used equimolar amounts of RF. 高 and RF 低 We injected anti-insulin IgG bearing mIgM into WT mice and subsequently monitored their blood glucose levels. As expected, the blood glucose levels of mice injected with anti-insulin IgG combined with mIgM increased within 2 hours after injection. Interestingly, insulin-specific IgG as well as RF 高 and RF 低 Combination of anti-insulin IgG and RF 高 +RF 低 ) injected mice showed significantly higher blood glucose levels than RF 低 RF levels were not different from those of mice injected with anti-insulin IgG alone. 高 is RF 低 These results suggest that anti-insulin IgG+RF is unable to exert its protective role in the presence of 高+RF 低 In these mice that received 低 , the blood glucose concentration was significantly lower than that in mice that received anti-insulin IgG alone with RF 高 (Figure 5).

[0101] In summary, our data suggest that the presence of RF 低 counteracts the stabilizing activity of RF 高 and results in target disruption comparable to the effects observed with RF alone 低 .

[0102] Example 5: Unregulated ratio of high-affinity RF and low-affinity RF in autoimmune diseases From the above results suggesting that the effects observed in the presence of low-affinity RF are dominant over those of high-affinity RF, we hypothesized that the inability to maintain the balance between the two classes of RF may contribute to the development of autoimmune diseases. To gain a deeper understanding, we collected sera from young and old healthy donors, as well as from patients suffering from two well-known autoimmune diseases, namely rheumatoid arthritis (RA) and multiple sclerosis (MS). We characterized these samples for the total serum levels of IgM and IgG. Interestingly, the total serum IgM levels in MS and RA patients appear to be increased compared to healthy individuals (Figure 6A). Furthermore, the total serum IgG concentrations in young and old healthy individuals were in a similar range, but the IgG levels in MS patients were significantly increased compared to old healthy individuals, and a similar albeit non-significant trend was shown by the total IgG levels in RA patients (Figure 6B).

[0103] Next, we evaluated whether the higher circulating levels of IgG in MS correlate with an altered amount of circulating RF-IgM. Interestingly, MS patients show a significantly lower amount of RF-IgM than young and old healthy individuals (Figure 6C). These data suggest that low-affinity RF is reduced in MS patients compared to healthy individuals, and thus, the regulation of IgG homeostasis, including autoreactive antibodies, is altered.

[0104] In summary, these findings suggest that the increase in protective IgG RF in RA patients or the decrease in pathogenic IgG RF in MS patients may be important pathogenic mechanisms associated with the development of autoimmune diseases. 高 or the decrease in pathogenic IgG RF in MS patients 低 may be important pathogenic mechanisms associated with the development of autoimmune diseases.

[0105] Mouse Female C57BL / 6 mice aged 8 - 15 weeks were used in all experiments reported in this study. For antibody stability experiments, 20 - 50 μg of antibody (as detailed in the figure legends for each experiment) was injected intravenously (i.v.) into the lateral tail vein, and blood was collected at the indicated time points to obtain serum.

[0106] For blood glucose monitoring experiments, 100 μg of anti - insulin IgG or anti - insulin IgM was injected i.v. into the lateral tail vein, and blood was collected at the indicated time points to obtain serum.

[0107] Animal experiments were conducted in accordance with the guidelines of German law and approved by the local ethics committee (Tubingen, Germany) under license 1484. All mice used in this study were either bred and housed in the animal facility of the University of Ulm under specific pathogen - free conditions or obtained at 6 weeks of age from Charles River.

[0108] Antibody specificity, host / isotype, conjugate clone, class, supplier catalog number: Anti - human CD20 (rituximab, human IgG1, SelleckChem); rheumatoid factor concentrate (Lee Biosolutions), human IgM (unlabeled, SouthernBiotech, number 0158L - 01), RF 低(Human IgM, self-made in the IgM constant region, from Youngblood, Kathy, Lori Fruchter, Guifeng Ding, Javier Lopez, Vincent Bonagura, and Anne Davidson. 1994. Journal of Clinical Investigation 93(2):852 - 61. heavy and light chain sequences - VH sequence encoded by the sequence defined by SEQ ID NO: 4 (HDCR1 encoded by the sequence defined by SEQ ID NO: 5, HCDR2 encoded by the sequence defined by SEQ ID NO: 6, HCDR2 encoded by the sequence defined by SEQ ID NO: 7) and VL sequence encoded by the sequence defined by SEQ ID NO: 1 (LDCR1 encoded by the sequence defined by SEQ ID NO: 2, LCDR2 encoded by GATGCATCC, LCDR2 encoded by the sequence defined by SEQ ID NO: 3) of RC1; RF 高 (Human IgM, self-made in the IgM constant region, from Youngblood, Kathy, Lori Fruchter, Guifeng Ding, Javier Lopez, Vincent Bonagura, and Anne Davidson. 1994. Journal of Clinical Investigation 93(2):852 - 61. heavy and light chain sequences - VH sequence encoded by the sequence defined by SEQ ID NO: 11 (HDCR1 encoded by the sequence defined by SEQ ID NO: 12, HCDR2 encoded by the sequence defined by SEQ ID NO: 13, HCDR2 encoded by the sequence defined by SEQ ID NO: 14) and VL sequence encoded by the sequence defined by SEQ ID NO: 8 (LDCR1 encoded by the sequence defined by SEQ ID NO: 9, LCDR2 encoded by GGTGCATCC, LCDR2 encoded by the sequence defined by SEQ ID NO: 10) of RO7.

[0109] Anti-insulin IgG (purified from IVIg, see below); total serum IgM (isolated from healthy donor serum, see below); anti-insulin IgM 高and anti-insulin IgM 低 (Human IgM, self-made, sequence from Ikematsu, H., Y. Ichiyoshi, E. W. Schettino, M. Nakamura, and P. Casali. 1994. Journal of Immunology 152(3):1430 - 41. Germline reversion achieved using the online tool IMGT® V-Quest).

[0110] HEK293-6E cell culture and antibody production HEK293-6E cells were cultured in FreeStyle F17 expression medium (Invitrogen) supplemented with 0.1% Kolliphor® P188 (Sigma-Aldrich) and 4 mM L-glutamine (Gibco Life Technologies). Transfection was performed according to the manufacturer's instructions. Briefly, two pTT5 plasmids encoding the heavy and light chains of the antibody of interest were transfected into the cells using polyethyleneimine (Polysciences) (total 1 μg DNA / ml culture). 24 - 48 hours after transfection, Tryptone N1 (TekniScience Inc number 19553) was supplied to the cells up to a final concentration of 0.5%.

[0111] Harvest was performed 120 hours after transfection. The antibody was purified using a HiTrap® IgM column (GE Healthcare, Sigma-Aldrich) as described below.

[0112] Antibody purification and pull-down of total serum IgM For IgM purification from human serum, IgG depletion was performed by incubating the sample with Protein G Sepharose beads (GE Healthcare, Sigma-Aldrich) according to the manufacturer's instructions.

[0113] For the purification of IgM from IgG-depleted human serum and HEK293-6E cell supernatant, a HiTrap® IgM column (GE Healthcare, Sigma-Aldrich) was used according to the manufacturer's protocol, and the eluate was dialyzed overnight in 1×PBS at 300-fold sample volume. Quality control of the isolated immunoglobulins was addressed via SDS-PAGE stained with Coomassie Brilliant Blue R-250 (BIO-RAD), and quantification of the eluted proteins was evaluated via ELISA.

[0114] Isolation of antigen-specific immunoglobulins from IVIg A streptavidin bead column (Thermo Scientific, number 21115) was loaded with 20 μg of biotin-insulin (ibt biosystem). The IVIg preparation was incubated at room temperature for 90 minutes to ensure binding of the antigen-specific antibodies to the beads. Isolation of the antibodies was performed by an acidic pH shift using the manufacturer's elution and neutralization solutions. The quality of the isolated immunoglobulins was examined via SDS-PAGE stained with Coomassie Brilliant Blue R-250 (BIO-RAD) and ELISA. For further in vivo experiments, the isolated antibodies were dialyzed overnight in 1×PBS at 300-fold sample volume.

[0115] Enzyme-linked immunosorbent assay (ELISA) A 96-well plate (Nunc, Thermo Scientific) was coated with either 10 μg / ml anti-human IgM or anti-human IgG antibody (SouthernBiotech), or 10 μg / ml human IgG (SouthernBiotech), or 2.5 μg / ml calf thymus dsDNA (Rockland), or 2.5 μg / ml native insulin (Sigma-Aldrich). Blocking was performed in 1% BSA blocking buffer (SERVA). Serial dilutions of 1:3 of IgM or IgG antibody (SouthernBiotech) were used as standards. Relative concentrations described as arbitrary units (AU) were determined via detection with alkaline phosphatase (AP)-labeled anti-IgM / anti-IgG (SouthernBiotech). p-Nitrophenyl phosphate (pNPP; Genaxxon) in diethanolamine buffer was added, and data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). All samples were measured in duplicate.

[0116] Antibody specificity, host / isotype, conjugate clone, class, supplier catalog number: anti-human IgM (goat, IgG, unlabeled, polyclonal, SouthernBiotech, number 2020-01), anti-human IgG (goat, IgG, unlabeled, polyclonal, SouthernBiotech, number 2040-01), human IgM (unlabeled, SouthernBiotech, number 0158L-01), human IgG (unlabeled, SouthernBiotech, number 0150-01), anti-human IgM (mouse, AP, monoclonal, SouthernBiotech, number 9020-04), anti-human IgG (goat, AP, polyclonal, SouthernBiotech, number 2040-04).

[0117] HEp-2 slides and fluorescence microscopy Using Kallestad HEp-2 slides (BIO-RAD, number 26101), the reactivity of purified homemade IgM or pull-down serum IgM against nuclear antigen (ANA) was evaluated. Approximately 10 μg per sample was applied onto the HEp-2 slides. Anti-IgM-FITC (Biolegend, number 314506) was used for the detection of ANA-IgM. The stained HEp-2 slides were analyzed using a fluorescence microscope DMi8 (Leica) and Leica Application Suite X (LAS X) software (Leica).

[0118] Monitoring of blood and urine glucose levels The blood glucose levels of mice were measured using an AccuChek (Roche Diagnostics, Mannheim) blood glucose monitor. Blood was collected from the lateral tail vein of ad libitum-fed mice and transferred onto a sterile test strip. Glucose levels were measured in mmol / l at the times described in the drawings for each mouse per group.

[0119] SDS-PAGE, Coomassie Samples were separated on a 10 - 12% SDS-polyacrylamide gel, incubated with Coomassie Brilliant Blue R-250 (BIO-RAD) for 45 minutes, and then decolorized.

[0120] Samples from healthy donors and patients Blood samples from healthy donors were obtained via the Deutsch Rotes Kreuz Ulm (DRK). Samples were divided into young (18 - 35 years old) and old (over 55 years old) according to age. Serum was obtained by Pancoll gradient centrifugation.

[0121] Serum from multiple sclerosis patients was provided by the biobank of the Rehabilitationskrankenhaus of the University Hospital Ulm (RKU).

[0122] Serum from patients with rheumatoid arthritis (RA) was provided by the Clinic of Rheumatology and the clinical Immunology of the University Clinic of Freiburg. RA patients were classified according to symptoms and RF positivity.

[0123] Biolayer interferometry (BLI) The affinity of antigen-antibody interactions was determined using a biolayer interference assay (BLItz device, ForteBio) (Kumaraswamy, Sriram, and Renee Tobias. 2015. 「Label-Free Kinetic Analysis of an Antibody-Antigen Interaction Using Biolayer Interferometry.」 Pp. 165-82 in). Here, the inventors used insulin-specific IgM or RF-IgM and insulin-bio (ibt biosystem) or human IgG-bio (labeled using the LYNX Rapid Biotin Antibody conjugation kit, BIORAD) as targets. The targets were loaded onto streptavidin biosensors (ForteBio). The binding affinity of IgM for insulin or IgG was obtained in relative wavelength shift (nm). Subsequently, the calculated affinity value (K a ) was used to determine the dissociation constant (K D ): K D = 1 / K a was determined. The following protocol was used during measurement: 30-second baseline, 30-second loading, 30-second baseline, 120-second association, 60-second dissociation. The manufacturer's sample buffer (ForteBio) was used for buffering samples, targets, and probes.

Claims

1. A glycosylated IgM antibody that binds cross - specifically to an IgG antibody and a complexed molecule, wherein said binding to said IgG antibody and said complexed molecule induces degradation of said IgG antibody.

2. K for the binding affinity of said IgM antibody to said IgG antibody ｄ is in the range of 10 －５ to 10 －８ and preferably is 10 －７ The antibody according to claim 1

3. The antibody according to claim 1 or 2, wherein at least one complementarity - determining region (CDR) of said IgM antibody binds to said IgG antibody.

4. The antibody according to any one of claims 1 - 3, wherein the glycosylated portion of said IgM antibody binds to said complexed molecule.

5. The antibody according to any one of claims 1 - 4, wherein said IgG antibody is an auto - reactive IgG antibody.

6. The antibody according to any one of claims 1 - 5, wherein said complexed molecule is a nucleic acid, preferably DNA.

7. The antibody according to any one of claims 1 - 6, wherein said auto - reactive IgG antibody is an anti - citrullinated protein - IgG antibody.

8. The antibody according to any one of claims 1 - 7, wherein the first chain of said IgM antibody contains a CDR that specifically binds to IgG, and the second chain of said IgM antibody contains a CDR that binds to IgG poly - reactively.

9. The antibody according to any one of claims 1 - 8, for use in a medicament.

10. The antibody according to any one of claims 1 - 8, for use in the treatment of a subject having an increased IgM level, preferably a serum IgM level exceeding 1500 hIgM μm / ml.

11. The antibody according to any one of claims 1 - 8, for use in the treatment of a subject having an increased high - affinity rheumatoid factor: low - affinity rheumatoid factor ratio.

12. The antibody according to any one of claims 1 - 8, for use in the treatment of an autoimmune disease or disorder, preferably a chronic autoimmune disease or disorder.

13. The antibody for use according to claim 12, wherein said autoimmune disease or disorder is at least one selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.

14. The antibody for use according to claim 13, wherein said autoimmune disease or disorder is rheumatoid arthritis.

15. A method for diagnosing an autoimmune disease or disorder, wherein the method comprises a) determining a high-affinity rheumatoid factor portion and a low-affinity rheumatoid factor portion based on the affinity of rheumatoid factor for IgG antibodies in a sample of interest; b) diagnosing the subject with an autoimmune disorder based on the high-affinity rheumatoid factor portion and the low-affinity rheumatoid factor portion determined in a) and / or their ratio, a method comprising. **Claim 16** wherein the antibody is a variable heavy (VH) chain comprising a CDR1 sequence encoded by SEQ ID NO: 5, a CDR2 sequence encoded by SEQ ID NO: 6, and a CDR3 sequence encoded by SEQ ID NO: 7; and a variable light (VL) chain comprising a CDR1 sequence encoded by SEQ ID NO: 2, a CDR2 sequence encoded by GATGCATCC, and a CDR3 sequence encoded by SEQ ID NO: 3, the IgM antibody according to any one of claims 1 to 8 or the IgM antibody for use according to any one of claims 9 to 14. **Claim 17** wherein the antibody is a variable heavy (VH) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 4 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to SEQ ID NO: 4; and a variable light (VL) chain sequence comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 1 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to SEQ ID NO: 1, the IgM antibody according to claim 16 or the IgM antibody for use according to claim 16. **Claim 18** a) a sequence defined by SEQ ID NO: 4, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to SEQ ID NO: 4, and / or b) a host cell comprising a polynucleotide having a sequence defined by SEQ ID NO: 1, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to SEQ ID NO: 1, preferably, the polynucleotide further encodes an IgM constant region and / or preferably, the host cell comprises a further polynucleotide encoding an IgM constant region, a host cell. **Claim 19** A method for producing an IgM antibody, the method comprising a) culturing the host cell according to claim 18; b) isolating the IgM antibody, a method comprising.

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