IgM antibody capable of degrading IgG

Abstract

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

Description

[0001] The present invention relates to a glycosylated IgM antibody that cross-specifically binds to an IgG antibody and a complexing molecule such as DNA, wherein the binding to the IgG antibody and the complexing molecule induces the degradation of the IgG antibody. d Preferably in 10 -5 Up to 10 -8 The present invention also relates to the medical use of glycosylated IgM antibodies, such as use in the treatment of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis.

[0002] The process of antibody generation leads to the formation of unlimited antigen binding sites through random rearrangement of gene segments, i.e. variable (V), diversity (D) and connection (J) segments. The random nature of antibody specific generation ensures the recognition of almost unlimited antigens, but inevitably leads to the generation of autoreactive specificity. Most early B cells have autoreactive BCRs, and it is believed that highly autoreactive cells are eliminated from the library by central tolerance, which induces receptor editing by secondary immunoglobulin (Ig) gene recombination, thereby changing the specificity of autoreactive B cells. If receptor editing fails to replace autoreactive specificity, the corresponding autoreactive B cells are eliminated by clonal deletion. If autoreactive B cells escape central tolerance, they are considered to be functionally silenced by peripheral anergy as mature B cells. Defects in the elimination of autoreactive B cells are believed to lead to the occurrence 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 earliest discovered and most studied autoantibodies, which has been described as a class of Ig that can bind to the Fc part of IgG in the late 1940s (Volkov, Mikhail, Karin Anna Schie and Diane Woude. 2020, Immunological Reviews 294 (1): 148-63). Although RF-IgM is one of the most characteristic autoantibodies, its role in the pathogenesis of immune diseases remains elusive. Among different RF isotypes, IgM-RF is the most commonly used indicator for clinically estimating the prognosis of rheumatoid arthritis (RA), a chronic autoimmune disease characterized by the infiltration of B cells and T cells in the synovial membrane of the joints. However, the biological function of RF in the pathogenesis of the disease is still unknown (Volkov, Mikhail, Karin Anna Schie and Diane Woude. 2020, Immunological Reviews 294 (1): 148-63).

[0004] An important feature of RA is the presence of anti-citrullinated protein-IgG (ACPA-IgG), which causes inflammation in the synovial fluid. Here, the amino group (NH) of arginine residues is removed by protein arginine deaminase (PAD4). 3+ ) will generate citrullinated proteins that are mainly localized in the joints (Darrah, Erika and Felipe Andrade. 2018, Current Opinion in Rheumatology 30(1):72-78). The binding of ACPA-IgG to citrullinated proteins appears to lead to the deposition of immune complexes in the joints, thereby activating innate immune cells and triggering inflammation.

[0005] In this case, it is conceivable that RF acquires pathogenic properties by forming immune complexes with autoreactive ACPA-IgG antibodies, thereby causing inflammation by stimulating the secretion of proinflammatory cytokines. Interestingly, RA patients are classified as RF positive (RF+) and RF negative (RF-), where 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] Therefore, there is a need for improved means and methods to control IgG antibodies, particularly to control autoreactive IgG antibodies in immune diseases.

[0007] The above technical problems are solved by the embodiments disclosed herein and as defined in the claims.

[0008] Therefore, the present invention relates in particular to the following embodiments:

[0009] 1. A glycosylated IgM antibody that cross-specifically binds to an IgG antibody and a complexing molecule, wherein the binding to the IgG antibody and the complexing molecule induces degradation of the IgG antibody.

[0010] 2. The antibody according to embodiment 1, wherein the K of the binding affinity of the IgM antibody to the IgG antibody is d In 10 -5 Up to 10 -8 In the range of 10 -7 .

[0011] 3. The 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 wherein a glycosylated portion of the IgM antibody binds to the complexing molecule.

[0012] 4. The antibody according to any one of embodiments 1 to 3, wherein the IgG antibody is an autoreactive IgG antibody.

[0013] 5. The antibody according to any one of embodiments 1 to 4, wherein the complexing molecule is DNA.

[0014] 6. The antibody according to any one of embodiments 1 to 5, wherein the autoreactive IgG antibody is an anti-citrullinated protein-IgG antibody.

[0015] 7. The antibody of any one of embodiments 1 to 6, wherein the first chain comprises CDRs that specifically bind to IgG and the second chain comprises CDRs that polyreactively bind to IgG.

[0016] 8. The antibody according to any one of embodiments 1 to 7 for use in medicine.

[0017] 9. The antibody according to any one of embodiments 1 to 7 for use in the treatment of a subject with increased IgM levels, preferably serum IgM levels above 1500 hIgM μm / ml.

[0018] 10. The antibody of any one of embodiments 1 to 7 for use in treating a subject with an increased high affinity rheumatoid factor: low affinity rheumatoid factor ratio.

[0019] 11. The antibody according to any one of embodiments 1 to 7 for use in treating an autoimmune disease or disorder.

[0020] 12. The 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.

[0021] 13. The antibody for use according to embodiment 12, wherein the autoimmune disease or disorder is rheumatoid arthritis.

[0022] 14. A method for diagnosing an autoimmune disease or condition, the method comprising the steps of:

[0023] a) determining a high affinity rheumatoid factor fraction and a low affinity rheumatoid factor fraction based on the affinity of the rheumatoid factor to IgG antibodies in a sample from a subject; and

[0024] b) diagnosing the subject as having an autoimmune disorder based on said high affinity rheumatoid factor portion and said low affinity rheumatoid factor portion and / or their ratio determined in a).

[0025] 15. The IgM antibody according to any one of embodiments 1 to 7 or the IgM antibody use according to any one of embodiments 8 to 13, wherein the antibody comprises:

[0026] 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.

[0027] 16. The IgM antibody according to embodiment 15 or the use of the IgM antibody according to embodiment 15, wherein the antibody comprises:

[0028] A variable heavy (VH) chain sequence comprising an amino acid sequence encoded by a sequence as defined by SEQ ID NO: 4, or an amino acid sequence encoded by 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

[0029] A variable light (VL) chain sequence comprising an amino acid sequence encoded by a sequence as defined in SEQ ID NO: 1, or an amino acid sequence encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 1, preferably at least 95% sequence identity to SEQ ID NO: 1.

[0030] 17. A host cell comprising a polynucleotide having

[0031] a) a sequence as 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

[0032] b) a sequence as 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;

[0033] wherein the polynucleotide further encodes an IgM constant region and / or wherein the host cell comprises an additional polynucleotide encoding an IgM constant region.

[0034] 18. A method for producing an IgM antibody, the method comprising the steps of:

[0035] a) cultivating the host cell according to embodiment 17,

[0036] b) Isolation of IgM antibodies.

[0037] Thus, in one embodiment, the present invention relates to a glycosylated IgM antibody that cross-specifically binds to an IgG antibody and a complexing molecule, wherein the binding to the IgG antibody and the complexing molecule induces degradation of the IgG antibody.

[0038] In one embodiment, the present invention relates to a glycosylated IgM antibody bound to an IgG antibody and to a complexing molecule, wherein preferably, the binding to the IgG antibody and to the complexing molecule induces degradation of the IgG antibody.

[0039] As used herein, the term "IgM antibody" refers to its general meaning in the art, and refers to an immunoglobulin with a heavy m chain. Serum IgM exists in mammals as a pentamer (or hexamer) and accounts for about 10% of the normal human serum Ig content. It dominates the primary immune response to most antigens and is the most effective complement fixation immunoglobulin. IgM is also expressed on the plasma membrane of B lymphocytes in the form of membrane-associated immunoglobulins (which can be organized as multiprotein clusters in the membrane). In this form, it is a B cell antigen receptor, in which each H chain contains an additional hydrophobic domain for anchoring in the membrane. The monomers of serum IgM are bound together by disulfide bonds and connecting (J) chains. Each of the five monomers in the pentamer structure consists of two light chains (κ or λ) and two heavy chains. Unlike IgG (and the general structure shown in the figure above), the heavy chain in the IgM monomer consists of a variable region and four constant regions, in which the additional constant domain replaces the hinge region. IgM can recognize epitopes on invading microorganisms, thereby causing cell agglutination. Then, macrophages destroy this antibody-antigen immune complex by complement fixation or receptor-mediated endocytosis. IgM is the first class of immunoglobulins to be synthesized by newborns, and plays a role in the pathogenesis of some autoimmune diseases. Immunoglobulin M is the third most common serum Ig, and takes one of two forms: pentamer (or hexamer in some cases), in which all heavy chains are identical and all light chains are identical. The membrane-associated form is a monomer that can form a multimer cluster on the membrane (for example, a monomer found on a B lymphocyte as a B cell receptor). In some embodiments, IgM antibodies are monomeric IgM or oligomeric IgM. In some embodiments, oligomeric IgM described herein is selected from the group consisting of: monomeric IgM antibodies, dimeric IgM antibodies, trimeric IgM antibodies, tetrameric IgM antibodies, pentamer IgM antibodies and hexameric IgM antibodies.

[0040] As used herein, the term "glycosylated IgM antibody" refers to an IgM antibody that has glycosylation on at least one glycosylation site (such as a J chain and / or an N-glycosylation site), preferably an N-linked glycosylation site. In some embodiments, the IgM antibody has glycosylation on at least one Asn-linked glycosylation site. In some embodiments, the IgM antibody has glycosylation on 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 antibody described herein is a blood-derived antibody. In some embodiments, the glycosylated IgM antibody described herein is recombinantly produced.

[0041] The term "binding" as used in the context of the present invention defines the binding (interaction) of at least two "antigen interaction sites" with each other.

[0042] As used herein, the term "cross-specific binding" refers to binding to at least two binding partners, preferably, the at least two binding partners are different, such as an IgG antibody and a complexing molecule. Cross-specificity can also be extended to a) multiple complexing molecules and / or b) multiple IgG antibodies or all IgG antibodies. In some embodiments, the glycosylated IgM antibody binds to the constant region of one / multiple IgG antibodies.

[0043] As used herein, the term "complexing molecule" refers to a molecule capable of forming an immunodegradable complex when bound to the glycosylated IgM antibody described herein, preferably when bound to the glycosylated IgM antibody, which is bound to the IgG antibody described herein.

[0044] As used herein, in the context of IgG antibodies, the term "degradation" refers to a reduction in functionality, preferably neutralization by immune cells, for example. Preferably, IgG degradation means a reduction or neutralization of IgG measured in vivo or in vitro, as described in the Examples herein.

[0045] As used herein, the term "IgG" has its general meaning in the art, and refers to an immunoglobulin with a heavy g chain. This type of immunoglobulin produced as a part of the secondary immune response to an antigen accounts for about 75% of total serum Ig. IgG is the only class of Ig that can pass through the human placenta, and it is primarily responsible for protecting newborns in the first few months of life. IgG is the main immunoglobulin in blood, lymph, cerebrospinal fluid, and peritoneal fluid, and is a key player in humoral immune response. Serum IgG in healthy people accounts for about 15% of total protein, in addition to albumin, enzymes, other globulins, and more. Four IgG subclasses (e.g., human IgG1, IgG2, IgG3, and IgG4) have been described in humans, mice, and rats. These subclasses differ in the number of disulfide bonds and the length and flexibility of the hinge region. Except for the variable region, all immunoglobulins in the same class share about 90% homology, but the homology between different classes is only 60%. IgG1 accounts for 60% to 65% of the total major subclass IgG and is primarily responsible for thymus-mediated immune responses against protein and polypeptide antigens. IgG1 binds to the Fc receptors of phagocytes and can activate the complement cascade by binding to the C1 complex. IgG1 immune responses are already measurable in newborns and reach their typical concentrations during infancy. The second largest IgG isotype, IgG2, accounts for 20% to 25% of the major subclasses and is the prevalent immune response against carbohydrate / polysaccharide antigens. "Adult" concentrations are usually reached by 6 or 7 years of age. IgG3 accounts for approximately 5% to 10% of total IgG and plays a major role in immune responses against protein or polypeptide antigens. The affinity of IgG3 can be higher than that of IgG1. IgG4, which usually accounts for less than 4% of total IgG, does not bind to polysaccharides. In the past, testing for IgG4 was associated with food allergies, and recent studies have shown that elevated IgG4 serum levels are found in patients with sclerosing pancreatitis, cholangitis, and interstitial pneumonia caused by infiltrating IgG4-positive plasma cells. In some embodiments, the IgG antibody described herein is an antibody of at least one subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0046] The inventors have found that glycosylated IgM antibodies as rheumatoid factors (RFs) show neutralization to IgG, thereby causing IgG to degrade and reduce faster in vivo. These effector functions are usually unrelated to the pathogenic or beneficial properties of target IgG. Without being bound by theory, it seems that degradable RFs (also present in healthy humans) can regulate the half-life of IgG and control IgG homeostasis, and defects in generating degradable RFs may be important triggers for the development of autoimmune diseases. In this case, it is conceivable that degradable RFs neutralize IgG antibodies by forming large immune complexes with complexing molecules such as nucleic acids, thereby promoting the uptake of IgG by immune cells such as phagocytes. Degradable RFs may act as general regulators of IgG by identifying their constant regions. Alternatively or in addition thereto, degradable RFs may regulate specific IgG idiotypes by identifying individual variable regions, thereby playing a unique role. In the case of IgG-related autoimmune diseases, this shows that highly diverse antibody libraries are important for regulating the broad spectrum IgG antibodies targeting a single idiotype.

[0047] This includes the presence of polyreactive neutralizing IgMs as opposed to protective regulatory IgMs (Amendt, Timm and Hassan Jumaa. 2021. The EMBO Journal 40 (17)). These findings suggest that one way to potentially reduce the levels of harmful IgG antibodies in the circulation is to use low-affinity RF or total IgM antibodies from healthy individuals as therapeutic antibodies. Interestingly, the generation of idiotype-specific anti-IgG IgMs will allow the manipulation of individual IgGs in a specific manner without affecting the entire IgG pool.

[0048] Current thinking suggests that autoantibodies develop due to defects in central and peripheral tolerance mechanisms that, under healthy conditions, should prevent the development of autoreactive B cells (see, e.g., Zikherman, Julie, Ramya Parameswaran, and Arthur Weiss. 2012. Nature 489(7414):160–64), which teachings depart from the present invention.

[0049] Thus, the present invention is based, at least in part, on the discovery that glycosylated IgM antibodies can induce degradation of IgG antibodies as described herein.

[0050] In some embodiments described herein, the RF or IgM antibody described herein is a self-reactive antibody or an autoantibody.

[0051] In some embodiments, the RF of the present invention is an IgM antibody, preferably a glycosylated IgM antibody.

[0052] 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. The production of antibodies can be based on, for example, the immunity of animals (such as mice). However, other animals used to produce antibodies / antisera are also within the scope of the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, etc. Methods for producing and / or altering antibodies are known in the art and are described, inter alia, in laboratory manuals (see 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).

[0053] In certain embodiments, the invention relates to an antibody according to the invention, wherein the K of the binding affinity of the IgM antibody to the IgG antibody is d In about 10 -5 to about 10 -8 within the range.

[0054] In certain embodiments, the invention relates to an antibody according to the invention, wherein the K of the binding affinity of the IgM antibody to the IgG antibody is d About 10 -7 .

[0055] As used herein, the term "low affinity" or "binding with low affinity" refers to a binding affinity that is between about 10 -5 to about 10 -8 , preferably 10 -5 Up to 10 -8 , more preferably 10 -6 Up to 10 -8 , even more preferably 10 -6 Up to 10 -7 K in the range dIn a highly preferred embodiment, low affinity means that for binding affinity, 10 -7 K d .

[0056] As used herein, the term "high affinity" or "binding with high affinity" refers to a binding affinity of about 10 -9 K in the range d or lower K d As used herein, the term "K d " refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Those skilled in the art are familiar with methods suitable for determining the K of antibodies or antigen-binding fragments thereof as provided herein and encompassed by the present invention. d In some embodiments, K is determined by biolayer interferometry. d Preferably, K d Determined by bio-layer interferometry as described herein, particularly in the examples and figures of the invention.

[0057] The inventors have found that low-affinity rheumatoid factor demonstrates opposite neutralization to IgG (compared with high-affinity rheumatoid factor), thereby causing IgG to degrade and reduce faster in vivo. These effector functions depend on the affinity of RF-IgM. If RF-IgM has low affinity to IgG and if they are polyreactive, they contribute to faster degradation.

[0058] As used herein, the term "polyreactive" refers to an antibody that binds to an antigen with low affinity. Polyreactive antibodies preferentially bind to multiple structurally unrelated antigens, such as free double-stranded DNA.

[0059] Thus, the present invention is based, at least in part, on the discovery that IgM antibodies contribute to faster degradation of IgG if their affinity for IgG is low.

[0060] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody is bound to an IgG antibody.

[0061] In certain embodiments, the invention relates to an antibody according to the invention, wherein the glycosylated portion of the IgM antibody is bound to a complexing molecule.

[0062] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody is bound to the IgG antibody and wherein a glycosylated portion of the IgM antibody is bound to the complexing molecule.

[0063] In certain embodiments, the invention relates to an antibody according to the invention, wherein a first chain of the IgM antibody comprises a CDR binding to an IgG and a second chain binding to a complexing molecule, preferably via a glycosylated chain, preferably via glycosylation of the IgM antibody.

[0064] The inventors have discovered that a glycosylation moiety is particularly effective at binding to a complexing molecule if the glycosylation moiety (eg, the glycosylation itself) is bound to the complexing molecule.

[0065] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody is bound to an IgG antibody.

[0066] In certain embodiments, the present invention relates to an antibody according to the present invention, wherein the glycosylated part, preferably the glycosylated amino acid sequence, of the IgM antibody is bound or participates in the binding of the complexing molecule.

[0067] In certain embodiments, the invention relates to an antibody according to the invention, wherein at least one CDR of the IgM antibody is bound to the IgG antibody and wherein a glycosylated portion of the IgM antibody is bound to the complexing molecule.

[0068] In certain embodiments, the invention relates to an IgM antibody according to the invention, wherein the IgG antibody is an autoreactive IgG antibody.

[0069] As used herein, the term "autoreactive IgG antibodies" refers to antibodies produced by the immune system against one or more of the subject's own proteins or antigens.

[0070] The autoreactive IgG antibodies described herein can participate in the regulation of endogenous proteins, or can be the characteristics of many autoimmune diseases. In some embodiments, the IgM antibodies of the present invention bind to the autoreactive IgG antibodies in other IgG antibodies. In some embodiments, the IgM antibodies of the present invention mainly bind to the autoreactive IgG antibodies.

[0071] Autoreactive IgG antibodies remain in the circulation, probably because they play a specific role in maintaining physiological homeostasis. The IgM antibodies described here may restore this maintenance when dysregulation occurs.

[0072] Thus, the present invention is based, at least in part, on the discovery that glycosylated IgM antibodies can modulate and induce degradation of autoreactive IgG antibodies as described herein.

[0073] 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.

[0074] As used herein, the term "DNA" refers to any complex molecule comprising deoxyribonucleic acid, usually in polymeric form, such as double-stranded form. DNA as a complex molecule can be provided, for example, in the form of extracellular DNA released by immune cells.

[0075] Thus, the present invention is based, at least in part, on the discovery that 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.

[0076] In certain embodiments, the invention relates to an antibody according to the invention, wherein the autoreactive IgG antibody is an anti-citrullinated protein-IgG antibody.

[0077] As used herein, the term "anti-citrullinated protein-IgG antibodies" refers to autoantibodies against citrullinated peptides and proteins. These antibodies are commonly observed in patients with RA and are believed to play a role in the development and pathology of RA.

[0078] In certain embodiments, the invention relates to an antibody according to the invention, wherein the first chain comprises CDRs that specifically bind to IgG and the second chain comprises CDRs that polyreactively bind to IgG.

[0079] In certain embodiments, the first chain of an antibody according to the invention comprises a CDR that binds to IgG with high affinity, and the second chain of an antibody according to the invention comprises a CDR that binds to IgG with low affinity. The term "low affinity" refers to a binding affinity that is between about 10 -5 to about 10 -8 , preferably 10 -5 Up to 10 -8 , more preferably 10 -6 Up to 10 -8 , even more preferably 10 -6 Up to 10 -7 K in the range d Most preferably, low affinity means 10 -7 K d The term "high affinity" refers to a binding affinity of about 10 -9 K in the range d or lower K d .

[0080] RF antibodies are primarily associated with RA, however studies on RF production and incidence have shown that circulating RF can be found in healthy individuals. Interestingly, RF antibodies that have been studied in RA patients are characterized by extensive somatic mutations and have high antigen-binding affinity and specificity for IgG acquired during affinity maturation. In contrast, RF found in healthy individuals are very similar to natural autoantibodies, a class of autoantibodies with limited epitope specificity that are primarily encoded by germline variable gene segments. Therefore, most natural autoantibodies are polyreactive and bind to self-molecules with low antigen-binding affinity. Similarly, RF in healthy individuals show no evidence of affinity maturation and isotype switching, indicating low antigen-binding affinity for IgG (Mageed et al. 1997; Volkov et al. 2020).

[0081] In certain embodiments, the invention relates to an antibody according to the invention for use in medicine.

[0082] In certain embodiments, the invention relates to an antibody according to the invention for use in treating a subject with increased IgM levels, preferably serum IgM levels above 1500 hIgM μm / ml. Antibody levels are determined as described herein, especially in the Examples.

[0083] It is conceivable that high titers of high-affinity RF in the synovium of RA patients acquire a pathogenic role because they perpetuate the inflammatory state by stabilizing pathogenic IgG (such as anti-citrullinated protein-IgG). Excessive function of autoreactive IgG in the joints leads to the formation of immune complexes that can continuously trigger macrophage and complement activation through Fc receptors, thereby prolonging the duration of synovial inflammation.

[0084] For example, when recognizing the constant region of IgG, treatment with the IgM antibodies of the invention can control general IgG homeostasis, whereas when acting at the individual specific type level, they can selectively eliminate pathogenic IgG.

[0085] The present inventors have found that when high-affinity RF and low-affinity RF exist simultaneously, the destructive low-affinity RF has a better effect than the protective high-affinity RF.

[0086] Thus, the present invention is based, at least in part, on the discovery that the IgM antibodies of the invention can be used for treatment in the presence of (increased) IgG protective IgM antibodies, such as high affinity IgM.

[0087] In certain embodiments, the invention relates to an antibody according to the invention for use in treating a subject with 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 with an autoimmune disease or disorder to a healthy subject, particularly a healthy subject not suffering from an autoimmune disease or disorder.

[0088] The inventors found that when high-affinity RFs and low-affinity RFs coexist, the destructive low-affinity RFs outperform the protective high-affinity RFs. Extending this finding to a more general level, it is conceivable that the ratio between the two RF populations is relevant in the context of autoimmunity.

[0089] Thus, the present invention is based, at least in part, on the discovery that the IgM antibodies of the present invention can be used to restore a healthy high / low affinity RF ratio.

[0090] In certain embodiments, the invention relates to an antibody according to the invention for use in preventing an autoimmune disease or disorder, preferably a chronic autoimmune disease or disorder.

[0091] 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.

[0092] 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, wherein the method comprises administering an antibody according to the invention to a patient.

[0093] As used herein, the term "treatment" (and grammatical variants such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be used preventively or during clinical pathological processes. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0094] As used herein, the term "prevent" or "preventing" relates to the ability to prevent, minimize or hinder the onset or development of a disorder, disease or condition before its onset.

[0095] The fact that low-affinity RF is present in healthy individuals and regulates the half-life of IgG indicates that IgG homeostasis is controlled by such RF, and defects in generating low-affinity RF may be important triggers for the development of autoimmune diseases. The insulin data provided herein show that high-affinity and low-affinity IgM antibodies with opposite effects to their cognate antigens can be produced for almost every autoantigen. Without being bound by theory, it can be assumed that the boundary between physiological autoimmunity and pathological autoimmunity is strongly marked by affinity to autoantigens rather than only by tolerance mechanisms. Defects in establishing these balances are likely to lead to the development of autoimmune responses.

[0096] The inventors found that patients affected by autoimmune diseases had slightly higher levels of total serum IgM and IgG antibodies compared to healthy donors. However, the amount of RF-IgM detected in MS patients was significantly lower than that observed in healthy individuals. Therefore, although RA patients are characterized by an increase in the amount of high-affinity protective RFs, which leads to an upgrade of IgG functions (including autoreactive antibodies), MS patients are likely to lack low-affinity destructive RFs. The lack of low-affinity RFs leads to hemostatic changes in IgG antibodies, resulting in the accumulation and strengthening of IgG functions (including autoreactive specificity).

[0097] Thus, the present invention is based, at least in part, on the discovery 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.

[0098] In certain embodiments, the invention relates to the antibodies for use in the 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.

[0099] In certain embodiments, the invention relates to an antibody for use in the invention, wherein the autoimmune disease or disorder is rheumatoid arthritis.

[0100] The RF found in RA differs significantly from that found in healthy individuals, as the latter are polyreactive and show little evidence of affinity maturation. In contrast, the RF expressed by RA patients is highly somatically mutated, monospecific, and has a high affinity for IgG.

[0101] In certain embodiments, the present invention relates to a method for diagnosing an autoimmune disease or disorder, the method comprising the following steps: a) determining a high-affinity rheumatoid factor portion and a low-affinity rheumatoid factor portion based on the affinity of rheumatoid factor to IgG antibodies in a sample (preferably an ex vivo sample) of a subject; and b) diagnosing a subject with an autoimmune disorder based on the high-affinity rheumatoid factor portion and the low-affinity rheumatoid factor portion and / or their ratio determined in a).

[0102] Based on the limited mutation rate and reduced affinity, we proposed that natural autoantibodies are primary IgM antibodies that are secreted during early B-cell activation prior to affinity maturation. Indeed, earlier studies have shown that despite similar use of V light and V heavy genes, RFs in healthy individuals display significantly reduced mutation patterns in their CDRs compared with RFs in RA patients. Therefore, the low-affinity RFs found in healthy populations are most likely the result of regulated selection mechanisms that limit affinity maturation in healthy individuals, thereby preventing low-affinity RF autoantibodies from becoming pathogenic.

[0103] In certain embodiments, the present invention relates to the IgM antibody of the present invention or the use of the 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.

[0104] In certain embodiments, the present invention relates to the IgM antibody of claim 15 or the use of the IgM antibody of the present invention, wherein the IgM antibody comprises: a variable heavy (VH) chain sequence comprising an amino acid sequence encoded by a sequence as defined by SEQ ID NO:4, or an amino acid sequence encoded by 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 a variable light (VL) chain sequence comprising an amino acid sequence encoded by a sequence as defined by SEQ ID NO:1, or an amino acid sequence encoded by a sequence having at least 90% sequence identity to SEQ ID NO:1, preferably at least 95% sequence identity to SEQ ID NO:1.

[0105] In certain embodiments, the present invention relates to a host cell comprising a polynucleotide having 1) a) a sequence as defined by SEQ ID NO: 4 or a sequence having at least 90% sequence identity to SEQ ID NO: 4, preferably a sequence having at least 95% sequence identity to SEQ ID NO: 4; and / or b) a sequence as defined by SEQ ID NO: 1 or a sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0106] SEQ ID NO: 1 has at least 90% sequence identity, preferably a sequence having at least 95% sequence identity to SEQ ID NO: 1; and 2.) wherein the polynucleotide further encodes an IgM constant region and / or wherein the host cell comprises an additional polynucleotide encoding an IgM constant region.

[0107] In certain embodiments, the present invention relates to a method for producing an IgM antibody, the method comprising the steps of: a) culturing a host cell according to the present invention; and b) isolating the IgM antibody.

[0108] "A," "an," and "the" refer to one or more than one (ie, to at least one, or one or more than one) the grammatical object of the article.

[0109] "Or" should be understood to mean any one, two, or any combination of the alternatives.

[0110] "And / or" should be understood to mean either or both of the alternatives.

[0111] Throughout the specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0112] 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 a non-exhaustive sense, i.e., they imply inclusion but not exclusion of elements, integers, steps or groups thereof, etc. "Consisting of" means including and limited to what follows the phrase "consisting of".

[0113] "Preferably" means one option in a series of options not excluding other options. "For example" means one example without being limited to the mentioned example.

[0114] As used herein, the term "about" or "approximately" means "within 20%", more preferably "within 10%", and even more preferably "within 5%" of a given value or range.

[0115] References throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "additional embodiments," "some embodiments," "specific embodiments," or "another embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the foregoing phrases appearing in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that positive recitation of a feature in one embodiment serves as a basis for excluding the feature in a particular embodiment.

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0117] Unless otherwise indicated, the general methods and techniques described herein can be performed 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. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992) and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0118] Although embodiments of the present invention are illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary rather than restrictive. It should be understood that changes and modifications may be made by one of ordinary skill in the art within the scope and spirit of the following claims. In particular, the present invention encompasses additional embodiments having any combination of features from the different embodiments described above and below. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 : Recombinant low-affinity anti-insulin IgM destroys insulin in vivo

[0120] A) Schematic representation of the recombinant, in-house purified anti-insulin IGHV highlighting the two mutations in CDR2 that reverted to the germline form of the IGHV3-74*01 allele. Bright red: α-insulin IgM 高 (WT-IGHV); medium grey: α-insulin IgM 低 (gl-IGHV). B) Coomassie-stained SDS-PAGE showing α-insulin IgM purified under reducing conditions (with β-mercaptoethanol). 高 and α-insulin IgM 低 The image is representative of three independent experiments. C) α-Insulin IgM measured by biolayer interferometry 高 and α-insulin IgM 低 The insulin binding affinity was calculated by software. D (Dissociation constant). The experiments shown are representative of 3 independent experiments. D) Intravenous (iv) injection of 100 μg α-insulin IgM measured at the indicated time points 高 (n=4) or α-insulin IgM 低 Blood glucose concentrations of WT mice (n=4). Statistical significance of mean ± SD was calculated using two-way ANOVA with Tukey's multiple comparison test.

[0121] *p<0,05

[0122] Figure 2 :High-affinity RF enhances the effect of autoreactive IgG

[0123] A) The inflammatory response of 100 μg anti-insulin IgG injected intravenously alone (n=4) or in combination with 20 μg of RF concentrate from patients with rheumatoid arthritis (RF 高，Blood glucose concentrations of WT mice in combination with either 100 μg / mL IgM control (n=4) or monoclonal IgM control (mIgM, n=4). Mean ± SD statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. **p<0.01 B) Scheme depicting the procedure for isolation of total IgM from healthy donor (HD) serum. C) Coomassie stained SDS-PAGE showing the isolation of total IgM from n=2 healthy donors (IgM) under reducing conditions (with β-mercaptoethanol). HD ) Total IgM isolated. The image is representative of three independent experiments. D) IgM, RF isolated from healthy donors measured by biolayer interferometry 高 The binding affinity of IgG to mIgM is calculated by software. D (dissociation constant). The experiments shown are representative of 3 independent experiments. E) Hep-2 slide showing anti-nuclear structure reactive IgM (ANA) for total IgM separation (IgM HD ), RF 高 and monoclonal IgM control. Scale bar 65 μm. Green fluorescence indicates IgM binding to Hep-2 cells. Images are representative of three independent experiments. F) Blood glucose concentrations of WT mice injected intravenously (iv) with 100 μg anti-insulin IgG in combination with 20 μg total IgM purified from healthy donors (n=4) or monoclonal IgM control (n=4) measured at the indicated time points. Mean ± SD statistical significance was calculated using two-way ANOVA with Sidak's multiple comparison test. *p<0.01

[0124] Figure 3 : Recombinant low-affinity RF is polyreactive and binds DNA

[0125] A) Schematic representation of the immunoglobulin heavy and light variable genes (IGHV and IGLV, respectively) of the recombinant purified low-affinity RF compared to the closest germline corresponding alleles. Mutations are highlighted in bold.

[0126] IGHM: immunoglobulin constant weight mu; IGVK: immunoglobulin variable kappa

[0127] B) Coomassie-stained SDS-PAGE showing the recombinant monoclonal (in-house purified) low affinity RF (RF 低 ), commercial RF from patients with rheumatoid arthritis (RF 高 ) and monoclonal control IgM (mIgM). The image is representative of three independent experiments. C) RF measured by biolayer interferometry 低 (purple line), RF 高 The binding affinity of IgG (green line) and monoclonal IgM (blue line) was calculated by software. D(dissociation constant). The experiments shown are representative of 3 independent experiments. D) Dissociation constant of purified RF measured by ELISA 低 (n=3), RF 高 Anti-IgG IgM concentrations detected in the mIgM control (n=3) and mIgM control (n=3) (coating: human IgG). Mean ± SD statistical significance was calculated using ordinary one-way ANOVA with Tukey's multiple comparison test. **p<0.01 E) RF measured by ELISA 低 (n=3), RF 高 Anti-dsDNA-IgM concentrations (coating: calf thymus dsDNA) of (n=3) and IgM control (n=3). Mean ± SD. Results represent three independent measurements. F) Hep-2 slide showing anti-nuclear structure reactive IgM (ANA). Scale bar 65 μm. Green fluorescence indicates IgM binding to Hep-2 cells. Images represent three independent experiments. G) RF 高 and RF 低 Schematic overview of the characteristics of .

[0128] Figure 4 :RF 低 Controlling IgG function in vivo through enhanced degradation

[0129] The intravenous (iv) injection of 100 μg anti-insulin IgG alone (n=4) or with 20 μg RF was measured at the indicated time points. 低 Blood glucose concentrations of WT mice in combination with either IgG (n=4) or mIgM control (n=4). Mean ± SD statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. **p<0.01

[0130] B As measured by ELISA, the expression of α-CD20 human IgG (rituximab) was detected after a single iv injection of 20 μg alone (n=4) or with RF 高 Serum human IgG concentrations in WT mice on day 0 and day 1 after combination of 100 μM (n=4) or mlgM control (n=4). Mean ± SD statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test.

[0131] ****p<0,0001

[0132] C As measured by ELISA, a single iv injection of 20 μg α-CD20 human IgG (rituximab) with RF 高 (n=4), RF 低Serum human IgG concentrations in WT mice on day 0 and day 1 after combination of 100 mg / kg (n=4) or IgM control (n=5). Mean ± SD statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. *p<0.05; ****p<0.0001

[0133] Figure 5 :RF 低 Better than RF 高

[0134] Intravenous (iv) injection of 100 μg anti-insulin IgG and 20 μg RF was measured at the indicated time points. 高 (n=7), 20 μg RF 低 (n=6), 20 μg RF 低+ 20 μg RF 高 Blood glucose concentrations of WT mice in combination with either IgG (n=7) or IgM control (n=5). Mean ± SD statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

[0135] Figure 6 : Deregulated ratios of high- and low-affinity RFs in autoimmune diseases

[0136] A Total amount of IgM detected in sera from young (n=20) and old (n=17) healthy donors (HD), rheumatoid arthritis (RA) patients (n=15) and multiple sclerosis (MS) patients (n=28, red bars) measured by ELISA. Bars depict mean ± SD, individual values ​​are represented by single points. Statistical significance was calculated using the Kruskal Wallis test. *p<0,05; **p<0,01

[0137] The mean values ​​of IgM (μg / ml) were as follows: young HD 1517.55; old HD 1258.02; MS patients 2143.72; RA patients 2361.29.

[0138] B Total IgG amounts detected in sera from young (n=20) and old (n=17) healthy donors (HD), rheumatoid arthritis (RA) patients (n=15) and multiple sclerosis (MS) patients (n=28) measured by ELISA. Bars depict mean±SD, individual values ​​are represented by single points. Statistical significance was calculated using the Kruskal Wallis test. **p<0,01

[0139] The average values ​​of IgG (μg / ml) were as follows: young HD 7733,22; old HD 6856,48; MS patients 10419,28; RA patients 10345,23.

[0140] C Total RF-IgM (coating: human IgG) detected in sera from young (n=20) and old (n=17) healthy donors (HD), rheumatoid arthritis (RA) patients (n=15) and multiple sclerosis (MS) patients (n=28) measured by ELISA. Bars depict mean±SD, individual values ​​are represented by single points. Values ​​from RA patients are plotted separately for simplified visualization. Statistical significance was calculated using the Kruskal Wallis test. *p<0,05; **p<0,01; ****p<0,0001

[0141] The mean values ​​of RF-IgM (AU) were as follows: young HD 4,71; old HD 2,31; MS patients 1,72; RA patients 737,58.

[0142] Figure 7 :

[0143] A Recombinant in-house purified anti-insulin IgM as measured by ELISA 高 (WT, n=3) and anti-insulin IgM 低 Anti-insulin IgM concentrations detected in (gl, n=3) (coating: human insulin). Mean ± SD are depicted. Data represent three independent measurements.

[0144] Figure 8 :

[0145] A shows the mean ± SD kinetics of blood glucose levels after injection of 100 μg anti-insulin IgG (black line, n=5) or IgG isotype control (n=5). Statistical significance was calculated using two-way ANOVA with Sidak's multiple comparison test. **p<0.01

[0146] B from healthy donors (RF-IgM as measured by ELISA HD , n=3) and from RA patients (RF-IgM RA IgG binding IgM concentration in RF-IgM elution of (coating: human IgG) (n=3). Mean ± SD statistical significance was calculated using unpaired t-test. *p<0.05.

[0147] C IgG binding affinity of RF-IgM isolated from healthy donors and from RA patients measured by biolayer interferometry. K was calculated by software D (Dissociation constant). Experiments shown are representative of 3 independent experiments.

[0148] D IgG binding IgM concentration in total IgM isolated from healthy donors (n=3) as measured by ELISA and in RF高 Comparison of the amount of IgG binding IgM detected in neutralization (n=3) and monoclonal IgM (coating: human IgG). Mean ± SD statistical significance was calculated using ordinary one-way ANOVA with Tukey's multiple comparison test. ***p<0,001 Example

[0149] Example 1: Recombinant low-affinity anti-insulin IgM destroys insulin in vivo

[0150] To confirm our hypothesis that IgM affinity and specificity determine the outcome of the interaction with the recognized cognate antigen, we used recombinant anti-insulin antibodies as a model. Since we proposed that affinity and monospecificity for the target are the main requirements for determining the effector function of an autoreactive antibody, we expected that reversing the variable regions of anti-insulin IgM to their corresponding germline (gl) forms would result in reduced affinity for its target. To this end, we reversed the heavy chain (HL) and light chain (LC) sequences to the germline and tested the insulin binding affinity of the reversed HC / LC combinations. Although most combinations lost insulin binding, recombinant insulin-specific antibodies (anti-insulin IgM) composed of the original LC and the germline-reversed HC form of the anti-insulin antibody showed a strong affinity for insulin. 低 ) showed reduced affinity for insulin compared to the original antibody ( Figure 1 A) In fact, germline reversed anti-insulin IgM 低 K D In 10 -7 Within the range ( Figure 1 C), and thus much lower than the original anti-insulin IgM 高 In addition, anti-insulin IgM was observed by ELISA 低 Reduced insulin binding ( Figure 7 ). To test two antibodies, anti-insulin IgM 低 and its high affinity counterpart IgM 高 To determine whether there are different effects on glucose metabolism, we used the same molar amount of anti-insulin IgM 高 and anti-insulin IgM 低 injected into WT mice. Within two hours after injection, mice receiving anti-insulin IgM 低 Higher blood sugar levels (hyperglycemia) were observed in mice with insulin resistance, while anti-insulin IgM 高 Does not alter blood glucose and protects insulin from IgG-dependent degradation ( Figure 1 D).

[0151] Interestingly, the reversible forms of anti-insulin IgM differed only in two point mutations in the complementarity determining region 2 (CDR2) that appear to be responsible for affinity maturation ( Figure 1 A) Importantly, the quality of the antibodies produced in vitro was assessed and it was found that the purified IgM 高 and IgM 低 There are no structural differences between antibodies ( Figure 1 B).

[0152] These data suggest that protective, high-affinity autoantibodies can be converted to destructive autoantibodies by reversion of the immunoglobulin heavy chain variable region (IGHV) to its germline form (low affinity). This confirms our hypothesis of a regulatory role for IgM antibodies and suggests that mutations acquired during the affinity maturation process can convert destructive IgM antibodies into protective antibodies.

[0153] Example 2: Recombinant low affinity RF is polyreactive and binds DNA

[0154] To confirm our findings regarding the role of low-affinity RFs in the interaction with target antigens, we reviewed available reports describing the extent of somatic mutations in RFs in patients with rheumatoid arthritis (RA) (Randen et al. 1992; Youngblood et al. 1994). Although most of the rheumatoid factors (RFs) isolated from the synovium of RA patients were highly affinity for the Fc part of IgG and unreactive to other antigens tested, we identified one RF isolated from an RA patient (RF-IgM) that appeared to be polyreactive and bound to other antigens such as tetanus toxoid, DNA and bovine serum albumin (BSA) (Youngblood et al. 1994). Interestingly, in-depth analysis of the IGHV and IGLV sequences of the selected RFs revealed a high degree of homology with their germline gene counterparts. In fact, the variable heavy chain of the selected antibody shared 96.9% of the same residues with IGHV3-30-3*01 (allele 1) and the light chain had 99.3% identity with IGKV3-11*01 ( Figure 3 A). Due to the high identity to the germline gene and previously published data showing the polyreactivity of this RF, we expected this antibody to be a low affinity RF (RF 低 ). Therefore, we set RF 低 Cloned and expressed as recombinant IgM ( Figure 3 B) Biolayer interferometry measurements show that RF 低 The IgG binding affinity is 10 -7 within the range, while RF 高 K D For 10 -9 ( Figure 3 C).

[0155] RF was also tested by ELISA 低The ability to bind to IgG revealed that recombinant RF 低 Binds IgG, although to a lesser extent than RF 高 , which is likely RF 低 The result of reduced IgG affinity ( Figure 3 D) In ​​addition, we confirm previously published data showing that 高 In contrast, recombinant RF 低 Binds to double-stranded DNA ( Figure 3 E) and was reactive in HEp2 slides ( Figure 3 F).

[0156] These data confirm available data showing that, in contrast to typical high-affinity RFs from RA patients, low-affinity RFs are multispecific / polyreactive, as they bind DNA in addition to IgG ( Figure 3 G).

[0157] Example 3: RF 低 Controlling IgG function in vivo through enhanced degradation

[0158] Using the above-mentioned monoclonal low affinity RF (RF 低 ), we tested whether low-affinity RFs would neutralize their targets in vivo. To this end, we injected WT mice with anti-insulin IgG and an equimolar amount of RFs 低 As expected, mice injected with anti-insulin IgG alone or with anti-insulin IgG plus control mIgM showed comparable increases in blood glucose levels. 低 and anti-insulin IgG mice showed constant blood glucose levels, suggesting that RF 低 Control of autoreactive IgG function ( Figure 4 A).

[0159] Next, we investigated whether the protective or destructive effects of RF could be observed with other IgGs, such as therapeutic antibodies. To this end, we used rituximab as a well-known therapeutic IgG antibody targeting CD20. This monoclonal anti-CD20 antibody, composed of human constant regions and murine variable domains (Pierpont, Limper, and Richards 2018; Tobinai 2001), is approved for the treatment of B-cell malignancies as well as autoimmune diseases such as RA and systemic lupus erythematosus (SLE) (Aletaha and Smolen 2018; et al. 2017; Taylor and Lindorfer 2007). We used equimolar amounts of anti-CD20 IgG alone or in combination with RF 高or in combination with mIgM were injected intravenously into WT mice, and we monitored the concentration of human IgG (hIgG) over time. Our data showed that compared with mice receiving anti-CD20 IgG alone or in combination with mIgM, injection of rituximab and RF 高 Mice showed significantly higher levels of hIgG ( Figure 4 B). Together with the above data, these results led us to hypothesize that if the higher hIgG titers were due to RF 高 If , then RF 低 Co-injection with anti-CD20 IgG should show the opposite effect, i.e., a decrease in hIgG levels over time. Therefore, we injected an equimolar amount of recombinant RF 低 We observed significant differences in hIgG levels between the two groups one day after injection. 高 Compared with mice injected with RF 低 Animals that received anti-CD20 IgG showed significantly lower hIgG concentrations ( Figure 4 C).

[0160] Our results show that high-affinity RFs are able to stabilize IgG in vivo, thereby impressively extending its half-life, whereas low-affinity RFs display opposite destructive effects in vivo. Taken together, these data suggest that RFs have different effects on the half-life of IgG, depending on the affinity of these RFs for their targets. Interestingly, this is true not only for self-reactive antibodies, but also for therapeutic antibodies.

[0161] Example 4: RF 低 Better than RF 高

[0162] To better understand the dynamics of the interaction between RF and IgG in vivo, we investigated the effects of the combined presence of low-affinity RF and high-affinity RF on IgG function. To this end, we injected WT mice with equimolar amounts of RF 高 and RF 低 We then monitored blood glucose levels. As expected, blood glucose levels in mice injected with anti-insulin IgG combined with mIgM increased within two hours of injection. Interestingly, injection of insulin-specific IgG as well as RF 高 and RF 低 Combination (anti-insulin IgG + RF 高 +RF 低 ) were compared with the blood glucose levels of mice injected with anti-insulin IgG and RF only. 低 The levels of RF were not different in mice 高 In RF 低In fact, the protective effect of anti-insulin IgG+RF 高 +RF 低 The blood glucose concentrations in these mice were significantly lower than those in mice receiving RF alone. 高 The concentration of anti-insulin IgG in mice ( Figure 5 ).

[0163] In conclusion, our data suggest that RF 低 The presence of RF 高 The stabilizing activity of RF leads to target destruction, which is different from the 低 The observed effects were comparable.

[0164] Example 5: Deregulated ratio of high- and low-affinity RFs in autoimmune diseases

[0165] The above results indicate that the effects observed in the presence of low-affinity RFs are superior to those of high-affinity RFs, which led us to hypothesize that failure to maintain a balance between the two classes of RFs may contribute to the development of autoimmune diseases. To gain a deeper understanding, we collected serum from young and old healthy donors as well as from patients with two well-known autoimmune diseases, namely RA and multiple sclerosis (MS). We characterized the total serum levels of IgM and IgG in these samples. Interestingly, total serum IgM levels appeared to be increased in MS and RA patients compared to healthy individuals ( Figure 6 Furthermore, although total serum IgG concentrations were within a similar range in young and old healthy individuals, IgG levels were significantly increased in MS patients compared with old healthy individuals, and total IgG levels in RA patients showed a similar but nonsignificant trend ( Figure 6 B).

[0166] Next, the inventors evaluated whether higher circulating levels of IgG in MS were associated with altered amounts of circulating RF-IgM. Interestingly, MS patients showed significantly lower amounts of RF-IgM ( Figure 6 C) These data suggest that low-affinity RFs are reduced in MS patients compared to healthy individuals and, therefore, it is conceivable that the regulation of IgG homeostasis, including autoreactive antibodies, is altered.

[0167] Taken together, these findings suggest that IgG protects RF in RA patients. 高 Increased or IgG-destructive RF in MS patients 低 A reduction in β-catenin may be an important pathogenic mechanism associated with the development of autoimmune diseases.

[0168] Mouse

[0169] Female C57BL / 6 mice aged 8 to 15 weeks were used in all experiments reported in this study. For antibody stability experiments, 20 μg to 50 μg of antibody (as detailed in the figure legends for each experiment) were injected intravenously (iv) into the lateral tail vein, and blood was collected at the indicated time points to obtain serum.

[0170] For blood glucose monitoring experiments, 100 μg of anti-insulin IgG or anti-insulin IgM was injected iv into the lateral tail vein, and blood was collected at the indicated time points to obtain serum.

[0171] Animal experiments were performed in accordance with the guidelines of German law and approved by the responsible regional committee in Tübingen, Germany, permission number 1484. All mice used in this study were bred and maintained in the animal facility of Ulm University under specific pathogen-free conditions or obtained from Charles River at 6 weeks of age.

[0172] Antibody Specificity, Host / Isotype, Conjugate Clone, Class, Supplier Catalog Number:

[0173] Anti-human CD20 (rituximab, human IgG1, SelleckChem); rheumatoid factor concentrate (LeeBiosolutions), human IgM (unlabeled, SouthernBiotech, #0158L-01), RF 低 (Human IgM, homemade, with IgM constant region, heavy and light chain sequences from Youngblood, Kathy, Lori Fruchter, Guifeng Ding, Javier Lopez, Vincent Bonagura and Anne Davidson. 1994. Journal of Clinical Investigation 93(2):852–61. -RC1, which has a 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 a 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); RF 高(Human IgM, homemade, with IgM constant region, heavy and light chain sequences from Youngblood, Kathy, Lori Fruchter, Guifeng Ding, Javier Lopez, Vincent Bonagura and Anne Davidson. 1994. Journal of Clinical Investigation 93(2):852–61. -RO7, which has a 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 a 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).

[0174] 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, homemade, sequence from Ikematsu, H., Y. Ichiyoshi, EW Schettino, M. Nakamura and P. Casali. 1994. Journal of Immunology 152(3):1430–41., using online available tools V-Quest achieves germline reversal).

[0175] HEK293-6E cell culture and antibody production

[0176] In the supplement there is 0,1% HEK293-6E cells were cultured in FreeStyle F17 expression medium (Invitrogen) with P188 (Sigma-Aldrich) and 4mM L-glutamine (Gibco LifeTechnologies). Transfection was performed according to the manufacturer's instructions. Briefly, cells were transfected with two pTT5 plasmids encoding the heavy and light chains of the antibody of interest using polyethyleneimine (Polysciences) (1 μg DNA / ml culture in total). 24 to 48 hours after transfection, cells were fed with tryptone N1 (TekniScience Inc#19553) to a final concentration of 0.5%.

[0177] Harvest 120 hours after transfection and use as described below Antibodies were purified using IgM columns (GE Healthcare, Sigma-Aldrich).

[0178] Antibody purification and pull-down of total serum IgM

[0179] For purification of IgM from human serum, IgG depletion was performed by incubating samples with protein G agarose beads (GE Healthcare, Sigma-Aldrich) according to the manufacturer's instructions.

[0180] For purification of IgM from IgG-depleted human serum and from HEK293-6E cell supernatant, use the manufacturer's protocol. IgM columns (GE Healthcare, Sigma-Aldrich) were used and the eluate was dialyzed overnight in 300 times the sample volume of 1x PBS. Quality control of the separated immunoglobulins was performed by SDS-PAGE stained with Coomassie Brilliant Blue R-250 (BIO-RAD), and the quantification of the eluted proteins was assessed by ELISA.

[0181] Isolation of antigen-specific immunoglobulins from IVIg

[0182] A streptavidin bead column (Thermo Scientific, #21115) was loaded with 20 μg of biotin-insulin (ibt Biosystems). The IVIg preparation was incubated at room temperature for 90 min to ensure that the antigen-specific antibodies were bound to the beads. The antibodies were separated by acidic pH conversion using the manufacturer's elution and neutralization solutions. The quality of the separated immunoglobulins was tested by SDS-PAGE stained with Coomassie Brilliant Blue R-250 (BIO-RAD) and ELISA. For further in vivo experiments, the separated antibodies were dialyzed overnight in 300 times the sample volume of 1x PBS.

[0183] Enzyme-linked immunosorbent assay (ELISA)

[0184] 96-well plates (Nunc, ThermoScientific) were coated with 10 μg / ml anti-human IgM or anti-human IgG antibodies (SouthernBiotech) or with 10 μg / ml human IgG (SouthernBiotech) or with 2,5 μg / ml calf thymus dsDNA (Rockland) or with 2,5 μg / ml native insulin (Sigma-Aldrich). Blocking was performed in 1% BSA blocking buffer (SERVA). Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standards. The relative concentrations expressed in arbitrary units (AU) were determined by detection of anti-IgM / anti-IgG (SouthernBiotech) labeled with alkaline phosphatase (AP). p-Nitrophenyl phosphate (pNPP; Genaxon) in diethanolamine buffer was added and data were obtained at 405nm using a Multiskan FC ELISA plate reader (Thermo Scientific). All samples were measured in duplicate.

[0185] Antibody specificity, host / isotype, conjugate clone, class, supplier catalog number: anti-human IgM (goat, IgG, unlabeled, polyclonal, SouthernBiotech, #2020-01); anti-human IgG (goat, IgG, unlabeled, polyclonal, SouthernBiotech, #2040-01); human IgM (unlabeled, SouthernBiotech, #0158L-01); human IgG (unlabeled, SouthernBiotech, #0150-01); anti-human IgM (mouse, AP, monoclonal, SouthernBiotech, #9020-04); anti-human IgG (goat, AP, polyclonal, SouthernBiotech, #2040-04).

[0186] HEp-2 slides and fluorescence microscopy

[0187] The reactivity of purified homemade IgM or pulled-down serum IgM to nuclear antigen (ANA) was evaluated using Kallestad HEp-2 slides (BIO-RAD, #26101). Approximately 10 μg / sample was applied to the HEp-2 slides. ANA-IgM was detected using anti-IgM-FITC (Biolegend, #314506). The stained HEp-2 slides were analyzed using a fluorescence microscope DMi8 (Leica) and Leica ApplicationSuite X (LAS X) software (Leica).

[0188] Monitoring of blood and urine glucose levels

[0189] Blood glucose levels in mice were measured using an AccuChek (Roche Diagnostics, Mannheim) blood glucose monitor. Blood was collected from the lateral tail vein of mice fed ad libitum and transferred to sterile test strips. For each mouse in each group, glucose levels were measured in mmol / l at the hours indicated in the figure.

[0190] SDS–PAGE, Coomassie

[0191] Samples were separated on 10%-12% SDS-polyacrylamide gels, incubated with Coomassie Brilliant Blue R-250 (BIO-RAD) for 45 min, and subsequently destained.

[0192] Healthy donor and patient samples

[0193] Healthy donor blood samples were obtained by Deutsch Rotes Kreuz Ulm (DRK). The samples were divided into young (18 to 35 years) and old (over 55 years) according to age. Serum was obtained by Pancoll gradient centrifugation.

[0194] Sera from multiple sclerosis patients were provided by the Biobank of the Department of Rehabilitation, University Hospital Ulm (RKU).

[0195] Sera from patients with rheumatoid arthritis (RA) were provided by the Department of Rheumatology and Immunology, Clinical Institute, University of Freiburg, Germany. RA patients were categorized according to symptoms and RF positivity.

[0196] Biolayer Interferometry (BLI)

[0197] The affinity of the antigen-antibody interaction was determined using biolayer interferometry (BLItz device, FortéBio) (Kumaraswamy, Sriram and Renee Tobias. 2015. “Label-Free Kinetic Analysis of an Antibody–Antigen Interaction Using Biolayer Interferometry.” Pages 165 to 82). Here, we used insulin-specific IgM or RF-IgM and insulin-bio (ibt biosystems) or human IgG-bio (labeled using LYNX Rapid Biotin Antibody Conjugation Kit, BIORAD) as targets. The targets were loaded onto a streptavidin biosensor (FortéBio). The binding affinity of IgM to insulin or IgG was obtained in nm as a relative wavelength shift. Subsequently, the calculated affinity value (K a) to determine the dissociation constant (K D ): K D =1 / K a The following protocol was used during the measurements: 30 sec baseline, 30 sec loading, 30 sec baseline, 120 sec association, 60 sec dissociation. For buffering of samples, targets and probes, the manufacturer's sample buffer (ForteBio) was used.

Claims

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

2. The antibody according to claim 1, wherein the K of the binding affinity of the IgM antibody to the IgG antibody is d In 10 -5 Up to 10 -8 In the range of 10 -7 .

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

4. The antibody according to the preceding claim, wherein a glycosylated portion of the IgM antibody is bound to the complexing molecule.

5. The antibody according to any one of the preceding claims, wherein the IgG antibody is an autoreactive IgG antibody.

6. The antibody according to any one of the preceding claims, wherein the complexing molecule is a nucleic acid, preferably DNA.

7. The antibody according to any one of the preceding claims, wherein the autoreactive IgG antibody is an anti-citrullinated protein-IgG antibody.

8. The antibody according to any one of the preceding claims, wherein the first chain of the IgM antibody comprises CDRs that specifically bind to IgG and the second chain of the IgM antibody comprises CDRs that polyreactively bind to IgG.

9. The antibody according to any one of the preceding claims for use in medicine.

10. The antibody according to any one of claims 1 to 8 for use in the treatment of a subject with increased IgM levels, preferably serum IgM levels above 1500 hIgM μm / ml.

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

12. The antibody according to any one of claims 1 to 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 the 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 the autoimmune disease or disorder is rheumatoid arthritis.

15. A method for diagnosing an autoimmune disease or condition, the method comprising the steps of: a) determining a high-affinity rheumatoid factor fraction and a low-affinity rheumatoid factor fraction based on the affinity of the rheumatoid factor to the IgG antibody in the subject's sample; as well as b) diagnosing the subject as having an autoimmune disorder based on said high affinity rheumatoid factor portion and said low affinity rheumatoid factor portion and / or their ratio determined in a).

16. 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, wherein the 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.

17. The IgM antibody according to claim 16 or the IgM antibody for use according to claim 16, wherein the antibody comprises: A variable heavy (VH) chain sequence comprising an amino acid sequence encoded by a sequence as defined by SEQ ID NO: 4, or an amino acid sequence encoded by 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 A variable light (VL) chain sequence comprising an amino acid sequence encoded by a sequence as defined in SEQ ID NO: 1, or an amino acid sequence encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 1, preferably at least 95% sequence identity to SEQ ID NO:

1.

18. A host cell comprising a polynucleotide having a) a sequence as 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 sequence as 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, Wherein preferably said polynucleotide further encodes an IgM constant region and / or wherein preferably said host cell comprises a further polynucleotide encoding an IgM constant region.

19. A method for producing an IgM antibody, the method comprising the following steps: a) cultivating the host cell according to claim 18, and b) Isolation of IgM antibodies.

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