Methods and Means for Enhancement of Therapeutic Antibodies

Abstract

The present invention relates to a pharmaceutical composition comprising an IgM antibody or a fragment thereof and a therapeutic antibody, said IgM antibody specifically binding to said therapeutic antibody.The present invention further relates to a method of treatment of a disease or disorder, said method comprising the steps of a) administering an effective dose of a therapeutic antibody and b) administering a corresponding dose of an IgM antibody or a fragment thereof, said IgM antibody specifically binding to said therapeutic antibody.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition comprising an IgM antibody or a fragment thereof and a therapeutic antibody, said IgM antibody specifically binding to said therapeutic antibody.The present invention further relates to a method of treatment of a disease or disorder, said method comprising the steps of a) administering an effective dose of a therapeutic antibody and b) administering a corresponding dose of an IgM antibody or a fragment thereof, said IgM antibody specifically binding to said therapeutic antibody.

[0002] explanation Self-tolerance is important to maintain physiological integrity by avoiding autoimmune responses. Currently, it is believed that absolute central and peripheral tolerance controls the B cell receptor (BCR) repertoire during B cell development, thereby preventing the positive selection of autoreactive B cells [1, 2, 4]. Central tolerance is hypothesized to enforce the deletion of autoreactive B cells during early B cell development in the bone marrow [2, 5–7]. Furthermore, autoreactive B cells that escape clonal deletion are subjected to receptor editing, resulting in non-autoreactive BCR specificity [8–10]. Autoreactive B cells that escape central tolerance and migrate to the periphery are counterbalanced by clonal anergy (peripheral tolerance) that results in unresponsiveness, mainly through the inhibitory regulation of IgM BCR expression [1, 11–13]. However, the finding that the majority of serum IgM is autoreactive seems to contrast with the concept of a generalized elimination of autoreactivity

[14] . Indeed, so-called natural polyreactive IgM plays an important role in homeostasis

[15] , arguing against the absolute elimination of autoreactive antibodies.

[0003] Interestingly, it has been shown that disease-specific autoreactive B cells exist within the pre-immune repertoire and germinal centers (GCs) specific for the common autoantigen insulin can form in wild-type mice, which is inconsistent with the concept of central B cell tolerance [ 16 , 17 ].

[0004] For the past decades, B cell autoimmunity research has focused mainly on transgenic mouse models [1, 2, 5, 18, 19]. The usefulness of these models to study autoimmunity is heavily debated for several reasons

[20] . The replacement of the germline configuration by high-affinity mutant autoreactive BCRs not only leads to an atypical situation during B cell development but also generates a monospecific repertoire [1, 5, 19]. Moreover, the characterization of these antigens with regard to their availability, valency and form (soluble vs. membrane-bound) has not been adequately addressed [5, 18]. Moreover, the antigens themselves have no association with any known autoimmune disease [21, 22].

[0005] Epidemiological studies have shown that up to 5% of the population in developed countries suffers from autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or type 1 diabetes (T1D).

[21] In particular, autoantibodies are present in the majority of autoimmune diseases and are often the driving force in their pathogenesis.

[22]

[0006] Thus, there remains a need to develop approaches for the controllable modulation of antibody-based therapeutics to detect or treat or avoid a condition or disease.

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

[0008] Thus, the present invention relates, inter alia, to the following embodiments: 1. A pharmaceutical composition comprising an IgM antibody or a fragment thereof and a therapeutic antibody, wherein said IgM antibody specifically binds to said therapeutic antibody.

[0009] 2. The pharmaceutical composition of embodiment 1, wherein said IgM antibody and said therapeutic antibody are contained in a molar ratio of 5:1 to 1:10, preferably 2:1 to 1:5.

[0010] 3. A method for treating a disease or disorder, the method comprising: a) administering an effective dose of a therapeutic antibody; b) administering a corresponding dose of an IgM antibody or fragment thereof, wherein said IgM antibody specifically binds to said therapeutic antibody, and preferably said corresponding dose of said IgM antibody or fragment thereof is between 10% and 400% of said effective dose of said therapeutic antibody, more preferably between 20% and 200% of said effective dose of said therapeutic antibody.

[0011] 4. The pharmaceutical composition of embodiment 1 or 2, or the method of treatment of embodiment 3, wherein the half-life of said therapeutic antibody is extended by said binding of said IgM antibody.

[0012] 5. The pharmaceutical composition of any one of embodiments 1, 2 or 4, or the method of treatment of embodiment 3 or 4, wherein the IgM antibody binds to the therapeutic antibody, preferably with a KD of at least 10-8 as measured by biolayer interferometry.

[0013] 6. The pharmaceutical composition of any one of embodiments 1, 2, 4 or 5, or the method of treatment of any one of embodiments 3-5, wherein said therapeutic antibody is an anti-rheumatoid arthritis antibody.

[0014] 7. The pharmaceutical composition of any one of embodiments 1, 2, 4-6, or the method of treatment of any one of embodiments 3-6, wherein said therapeutic antibody is an anti-CD20 antibody.

[0015] 8. The pharmaceutical composition of embodiment 5 or the method of treatment of embodiment 7, wherein the therapeutic antibody is rituximab.

[0016] 9. The pharmaceutical composition of any one of embodiments 1 to 7 for use in treating an autoimmune disease or disorder.

[0017] 10. The pharmaceutical composition of embodiment 8, wherein said autoimmune disease or disorder is multiple sclerosis or rheumatoid arthritis.

[0018] 11. The method of treatment of any one of embodiments 3-8, wherein said disease or disorder is an autoimmune disease or disorder.

[0019] 12. The method of treatment of embodiment 11, wherein said autoimmune disease or disorder is multiple sclerosis or rheumatoid arthritis.

[0020] 13. A method for obtaining a protective regulatory antibody, comprising: (a) providing a blood sample from a subject, said subject having experienced induction of an IgG and oligomeric antibody response by a target antigen; (b) concentrating the mature oligomeric antibodies, (i) the binding of the oligomeric antibody is more specific for the target antigen than an IgG type antibody, preferably the oligomeric antibody is monospecific for the target antigen, and / or (ii) The binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of an IgG antibody, and preferably the protective regulatory antibody is 10 -7 Less than 10 -8 Less than 10, more preferably -9 and most preferably less than about 10 -10 ~about 10 -12 K in the range d and binding to a target antigen by (c) isolating the enriched mature oligomeric antibodies; obtaining a protective regulatory antibody that is protective regulatory for the function of the target antigen.

[0021] 14. The method of treatment of embodiment 13, wherein said subject experienced the induction of said IgG and oligomeric antibody response by said target antigen at least 7 days previously, preferably at least 14 days previously, more preferably at least 27 days previously.

[0022] 15. A method for obtaining a degradable oligomeric antibody, comprising: (a) providing a blood sample from a subject, said subject having experienced induction of an IgG and oligomeric antibody response by a target antigen; (b) concentrating the primary oligomeric antibodies, (i) the binding of said oligomeric antibodies is equally or less specific for said target antigen than an IgG type antibody, preferably said oligomeric antibodies being cross-specific for said target antigen and DNA; and / or (ii) the binding affinity of the oligomeric antibody to the target antigen is lower than that of the IgG antibody, and preferably the protective regulatory antibody is 10 -7 More than K d and binding to said target antigen. Steps and (c) isolating the enriched primary oligomeric antibodies to obtain the degraded antibodies capable of forming an immunodegradative complex with the target antigen.

[0023] 16. The method of any one of embodiments 13 to 15, wherein the blood sample is selected from the group consisting of a whole blood, plasma and serum sample, preferably a serum sample.

[0024] 17. The method of any one of embodiments 13-16, wherein isolating the oligomeric antibodies comprises mass-related and / or affinity-related isolation.

[0025] 18. The method of any one of embodiments 13-17, wherein enriching for oligomeric antibodies comprises immunoprecipitation of said oligomeric antibodies.

[0026] 19. The method of any one of embodiments 13-18, wherein said oligomeric antibody is an IgM antibody.

[0027] 20. The pharmaceutical composition of any one of embodiments 1, 2, 4 to 8, the pharmaceutical composition for use of any one of embodiments 9 to 12, or the method of treatment of any one of embodiments 3 to 8, wherein the IgM antibody comprises a variable heavy chain (VH) comprising a CDR1 sequence encoded by SEQ ID NO: 60, a CDR2 sequence encoded by SEQ ID NO: 61, and a CDR3 sequence encoded by SEQ ID NO: 62, and a variable heavy chain (VL) comprising a CDR1 sequence encoded by SEQ ID NO: 57, a CDR2 sequence encoded by GGTGCATCC, and a CDR3 sequence encoded by SEQ ID NO: 58.

[0028] 21. The pharmaceutical composition of embodiment 20, the pharmaceutical composition for use of embodiment 20, or the method of treatment of embodiment 20, wherein the IgM antibody comprises: a variable heavy chain (VH) sequence comprising an amino acid sequence encoded by a sequence defined by SEQ ID NO:59 or by a sequence having at least 90% sequence identity to SEQ ID NO:59, preferably at least 95% sequence identity to SEQ ID NO:59; and a variable light chain (VL) comprising an amino acid sequence encoded by a sequence defined by SEQ ID NO:56 or by a sequence having at least 90% sequence identity to SEQ ID NO:56, preferably at least 95% sequence identity to SEQ ID NO:56.

[0029] 22. A host cell comprising: a) a sequence defined by SEQ ID NO: 59 or a sequence having at least 90% sequence identity to SEQ ID NO: 59, preferably at least 95% sequence identity to SEQ ID NO: 59; and / or b) comprising a polynucleotide having a sequence defined by SEQ ID NO: 56 or a sequence having at least 90% sequence identity to SEQ ID NO: 56, preferably at least 95% sequence identity to SEQ ID NO: 56; The polynucleotide further encodes an IgM constant region and / or the host cell comprises a further polynucleotide encoding an IgM constant region.

[0030] 23. A method for providing an IgM antibody, the method comprising: a) culturing the host cell of embodiment 22; b) isolating the IgM antibodies.

[0031] The elements of the present invention are described below. Although these elements are listed in certain embodiments, it should be understood that they can be combined in any manner and in any number to form additional embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments, or combine one or more of the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless otherwise specified by context.

[0032] Thus, in one embodiment, the invention relates to a pharmaceutical composition comprising an IgM antibody or a fragment thereof and a therapeutic antibody, wherein said IgM antibody specifically binds to said therapeutic antibody.

[0033] The inventors have identified IgM antibodies and methods for obtaining IgM antibodies that stabilize IgG and enhance its effect in vivo when bound to it. This stabilizing effect depends on the affinity of the IgM antibody for IgG. Without being bound by theory, the effect may be independent of the pathogenic or beneficial properties of the target IgG. Indeed, the inventors have found that high affinity IgM stabilizes autoreactive insulin-specific IgG, inducing higher blood glucose levels and prolonging the hyperglycemic state.

[0034] These findings are in stark contrast to current views which propose the development of autoantibodies as a consequence of defects in central and peripheral tolerance mechanisms that, in health, should prevent the development of autoreactive B cells.

[0035] Thus, the present invention is based at least in part on the protective and regulatory properties of IgM antibodies relative to IgG antibodies, which can improve treatment and improve the efficacy of IgG treatments, such as therapeutic antibodies.

[0036] In some embodiments, the IgM antibodies in the pharmaceutical compositions of the invention are recombinantly produced. In some embodiments, the IgM antibodies in the pharmaceutical compositions of the invention are isolated from human blood, e.g., human plasma.

[0037] The fragments in the pharmaceutical compositions of the invention are preferably antigen-binding fragments of IgM antibodies that have similar, identical, or substantially identical binding characteristics to the parent IgM antibody.

[0038] The term "IgM antibody that specifically binds to a therapeutic antibody" as used herein refers to an IgM antibody or fragment thereof that can bind to a therapeutic antibody with sufficient affinity such that the therapeutic antibody is more useful as a prophylactic, diagnostic and / or therapeutic agent for a desired purpose. In some embodiments, the IgM antibody in the pharmaceutical composition of the invention is more than 10 -7 Less than 10 -8 Less than 10, more preferably -9 less than about 10 -10 ~about 10 -12 K in the range d In one embodiment, the therapeutic antibody is bound to the K d is measured by biolayer interferometry.

[0039] The terms "RF" and "rheumatoid factor" are used interchangeably herein and, unless otherwise specified, refer to an IgM antibody that binds to IgG.

[0040] The IgM antibody in the pharmaceutical composition of the present invention is preferably bound to a region that does not interfere or does not substantially interfere with the target binding activity of IgG antibody, such as the Fc region of IgG antibody.In some embodiments, the IgM antibody described herein is a recombinant antibody and is not glycosylated or is substantially not glycosylated.Endogenous IgM antibody is usually glycosylated.However, the present inventors have found that IgM antibody does not require glycosylation for its protective function.

[0041] In some embodiments, the IgM antibody described herein is an antibody selected from the group of a monomeric IgM antibody, a dimeric IgM antibody, a trimeric IgM antibody, a tetrameric IgM antibody, a pentameric IgM antibody, and a hexameric IgM antibody.

[0042] In some embodiments, the present invention relates to a pharmaceutical composition of the present invention, wherein the IgM antibody and the therapeutic antibody are contained in a molar ratio of about 10:1 to about 1:100, about 7:1 to about 1:50, about 5:1 to about 1:10, about 2:1 to about 1:5, or about 1:1.

[0043] The ratio of IgM antibodies to therapeutic antibodies may depend on the number of binding sites of the IgM antibodies.

[0044] In one embodiment, the present invention relates to a pharmaceutical composition of the present invention, wherein the IgM antibody is a monomeric antibody, and the IgM antibody and the therapeutic antibody are contained in a molar ratio of about 10:1 to about 1:10, about 7:1 to about 1:5, about 5:1 to about 1:2, about 2:1 to about 1:1, or about 1:1.

[0045] In one embodiment, the present invention relates to the pharmaceutical composition of the present invention, wherein the IgM antibody is a pentameric antibody, and the IgM antibody and the therapeutic antibody are contained in a molar ratio of about 10:1 to about 1:50, about 3:1 to about 1:20, about 2:1 to about 1:10, about 1:1 to about 1:5, or about 1:1.

[0046] In one embodiment, the present invention relates to a method of treatment of a disease or disorder, said method comprising the steps of a) administering an effective dose of a therapeutic antibody, and b) administering a corresponding dose of an IgM antibody or a fragment thereof, wherein said IgM antibody specifically binds to said therapeutic antibody.

[0047] A variety of factors can affect the actual effective amount used in a particular application, for example, frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition may require that more or less of the actual effective amount be administered.

[0048] The administration of the therapeutic antibody and the IgM antibody can be performed sequentially or simultaneously. Typically, the therapeutic antibody and the IgM antibody are administered in such a way that they are simultaneously present in the subject's body in substantial amounts. For example, the therapeutic antibody and the IgM antibody can be administered within 1, 2, 3 or 4 half-lives of the therapeutic antibody and / or the IgM antibody. In some embodiments, the therapeutic antibody and the IgM antibody described herein are contacted before administration so that protective binding can occur before exposure to the subject's body.

[0049] In one embodiment, the invention relates to a method of treatment of a disease or disorder, said method comprising the steps of a) administering an effective dose of a therapeutic antibody and b) administering a corresponding dose of an IgM antibody or a fragment thereof, said IgM antibody specifically binding to said therapeutic antibody, said corresponding dose of said IgM antibody being between 10% and 400% of said effective dose of said therapeutic antibody, preferably between 20% and 200% of said effective dose of said therapeutic antibody.

[0050] Monomeric IgM antibodies typically benefit from equimolar or molar excess relative to the therapeutic antibody, whereas oligomeric, e.g., pentameric, IgM antibodies typically require less molar IgM antibody, e.g., 20%-100% of the molar amount of the therapeutic antibody.

[0051] In one embodiment the invention relates to a pharmaceutical composition according to the invention or a method of treatment according to the invention, wherein the half-life of said therapeutic antibody is extended by binding of said IgM antibody.

[0052] In certain embodiments, the present invention relates to the pharmaceutical composition of the present invention or the treatment method of the present invention, wherein said therapeutic antibody is an anti-rheumatoid arthritis antibody.

[0053] In one embodiment, the present invention relates to a pharmaceutical composition of the invention or a treatment method of the invention, wherein said therapeutic antibody is an anti-CD20 antibody.

[0054] In certain embodiments, the present invention relates to a pharmaceutical composition of the present invention or a method of treatment of the present invention, wherein said therapeutic antibody is rituximab.

[0055] In one embodiment, the present invention relates to a pharmaceutical composition of the present invention for use in treating an autoimmune disease or disorder.

[0056] The fact that low-affinity RFs are found in healthy individuals and regulate the half-life of IgG suggests that IgG homeostasis is controlled by such RFs and that RF low This suggests that defective production may be an important trigger for the onset of autoimmune disease. The means and methods described herein have an effect in or against homeostatic disturbances and are therefore particularly useful in the context of autoimmune disease.

[0057] In one embodiment, the present invention relates to a pharmaceutical composition for use according to the present invention, wherein said autoimmune disease or disorder is multiple sclerosis or rheumatoid arthritis.

[0058] In one embodiment, the invention relates to a method of treatment according to the invention, wherein said disease or disorder is an autoimmune disease or disorder.

[0059] In one embodiment, the invention relates to a method of treatment according to the invention, wherein said autoimmune disease or disorder is multiple sclerosis or rheumatoid arthritis.

[0060] In certain embodiments, the present invention relates to a composition comprising: (i) a monovalent antigen particle comprising an antigenic moiety comprising no more than one antigenic structure capable of inducing an antibody-mediated immune response against a target antigen; and (ii) a multivalent antigen particle comprising an antigenic moiety comprising two or more antigenic structures capable of inducing an antibody-mediated immune response against a target antigen, wherein the multivalent antigen particle comprises two or more antigenic structures that are cross-linked.

[0061] The term "valent" as used within this application indicates the presence of a certain number of binding sites within an antibody or antigen molecule, respectively. Thus, the binding site of an antibody is a paratope, whereas the binding site within an antigen is generally referred to as an epitope. For example, a natural antibody or a full-length antibody according to the invention has two binding sites and is bivalent. Antigen proteins are monovalent (when present as monomers), but when such antigen proteins are provided as multimers, they may contain two or more identical epitopes and thus may be multivalent, which may be bivalent, trivalent, tetravalent, etc. Thus, the term "trivalent" indicates the presence of three binding sites within an antibody molecule. Thus, the term "tetravalent" indicates the presence of four binding sites within an antibody molecule.

[0062] The term "monovalent antigen particle" refers in the context of the invention disclosed herein to a molecule or molecular complex, such as a protein or protein complex, that is antigenic and thus capable of stimulating an immune response in a vertebrate. Typically, a monovalent antigen particle is composed of an antigenic portion that comprises one or less of the antigenic structures capable of inducing an antibody-mediated immune response against such antigenic structure. As used herein, the term "antigenic structure" refers to a fragment of an antigenic protein that retains the ability to stimulate an antibody-mediated immune response. Such an antigenic structure is understood to provide an antigenic determinant or "epitope", which refers to a region of the molecule that specifically reacts with an antibody, more specifically, with the paratope of the antibody. In a preferred embodiment of the invention, the monovalent antigen particle of the invention comprises one or less copies of one specific epitope of the antigenic structure. Thus, preferably, only one antibody molecule of a certain antibody species with a particular paratope can bind to the monovalent antigen particle according to the invention.

[0063] The term "multivalent antigen particle" refers in the context of the invention disclosed herein to a molecule or molecular complex, such as a protein or protein complex, that is antigenic and thus capable of stimulating an immune response in a vertebrate. In the present invention, unlike monovalent antigen particles, multivalent antigen particles are composed of an antigenic moiety that comprises two or more of the antigenic structures capable of inducing an antibody-mediated immune response. In a preferred embodiment of the present invention, the multivalent antigen particle of the present invention comprises two or more copies of one specific epitope of the antigenic structure. Thus, preferably, two or more antibody molecules of a certain antibody species having a specific paratope can be bound to the monovalent antigen particle according to the present invention. Such a multivalent antigen particle may have a structure in which two or more of the antigenic structures are covalently or non-covalently cross-linked to each other. Preferably, two or more of the antigenic structures contained in the antigenic moiety of the multivalent antigen particle comprise a plurality of identical antigenic structures.

[0064] In the context of the present invention, monovalent antigen particles of the present invention are often referred to as "soluble" particles or antigens, whereas multivalent antigen particles are referred to as "composite" particles or antigens.

[0065] The term "antigen" may refer to any, preferably disease-associated, molecule or structure that contains an antigenic structure. Preferably, the antigens described herein are autoantigens, cancer-associated antigens, or pathogen-associated antigens. In one very specific exemplary embodiment of the present invention, the antigen is insulin and the associated disease is diabetes. Human insulin protein is produced as proinsulin, which includes c-peptide, insulin B chain, and active insulin peptide. The amino acid sequence and further characteristics are known to those skilled in the art and can be obtained under accession number P01308 (https: / / www.uniprot.org / uniprot / P01308) in the UniProt database in the version of January 27, 2020.

[0066] The target antigen of the present invention is preferably an antigen associated with a disease or condition, preferably a disease or condition that the subject is suffering from or suspected of suffering from. Such diseases may be pathogen-related, autoimmune-related, therapeutic-related, e.g. when the antigenic protein is used as a therapeutic agent such as a therapeutic antibody, cancer-related, etc., as mentioned. The target antigen of the present invention may be a natural or synthetic immunogenic agent, such as a complete, fragmented or partial immunogenic agent, which may be selected from a nucleic acid, a carbohydrate, a peptide, a hapten, or any combination thereof.

[0067] In the context of the present invention, monovalent antigen particles are distinguished in contrast to multivalent antigen particles. Each particle is considered as a single molecular entity, which may contain covalently or non-covalently connected moieties. However, according to the present invention, each particle has immunogenic activity against a specific antigen. Thus, a monovalent antigen particle is understood to contain only a single antigen structure capable of eliciting an immune response against the antigen, whereas a multivalent antigen particle contains multiple copies of such an antigen structure. In the context of the present invention, the term "soluble" antigen is sometimes also used for monovalent antigen particles in contrast to the "complex" antigen of a multivalent antigen particle. In most cases, the antigen structure is understood to comprise or consist of an epitope that elicits an antibody-mediated immune response, whereas it is understood to be the binding site of an antibody produced on the basis of a cell-mediated immune response, as defined elsewhere herein. In other words, the present invention distinguishes between the presentation of an immune-eliciting epitope as a soluble single epitope or the presentation of the same epitope in a complex array.

[0068] The term "crosslinking," as used herein, refers to a bond that links at least two antigen structures together, where the crosslinked complex has different physical properties than the isolated antigen structures. In some embodiments, the crosslinked complex is less soluble than the isolated antigen structures. In some embodiments, the crosslinks described herein comprise at least one covalent bond. In some embodiments, the crosslinks described herein comprise at least one ionic bond.

[0069] The present invention is based on the surprising discovery that antigens can induce different immune responses depending on whether they are presented to immune cells as soluble antigens or as complex multivalent antigens: the latter results in a particularly strong and memory IgG antibody response, whereas the former can suppress such an IgG response and induce a protective IgM (or IgA) antibody response.

[0070] Thus, the present invention is based at least in part on the surprising discovery that the composition of the present invention can regulate the immune response to target antigen, as described herein.Therefore, the present invention proposes to regulate the ratio of soluble and composite immune response to control the focus of B cell immunity.This approach can be used for novel controlled vaccination treatment, or to tackle autoimmune diseases such as diabetes.

[0071] In certain embodiments, the present invention relates to a composition of the invention, wherein the two or more antigenic structures comprise a plurality of identical antigenic structures.

[0072] In a preferred embodiment of the present invention, the multivalent antigen particle of the present invention comprises two or more copies of one specific epitope of an antigen structure. Thus, preferably, two or more antibody molecules of a certain antibody species with a specific paratope can be bound to the monovalent antigen particle according to the present invention. Such a multivalent antigen particle may have a structure in which two or more of the antigen structures are covalently or non-covalently cross-linked to each other. Thus, in a preferred embodiment, the multivalent antigen particle comprises a complex comprising at least two identical, at least three or at least four epitopes, which allows the binding of two antibodies to the multivalent antigen particle at the same time. Preferably, two or more of the antigen structures comprised in the antigenic portion of the multivalent antigen particle comprise a plurality of identical antigen structures.

[0073] Thus, in a preferred embodiment, the multivalent antigen particle comprises a complex that comprises at least two, at least three or at least four identical epitopes, allowing simultaneous binding of two antibodies to the multivalent antigen particle.

[0074] Compositions including such particles (see, eg, FIG. 2a, FIG. 21) invention can modulate immune responses (see, eg, FIG. 18).

[0075] Thus, the present invention is based, at least in part, on the surprising discovery that multiple linked identical structures can modulate the immune response to a target antigen, as described herein.

[0076] In certain embodiments, the present invention relates to a composition of the invention, wherein the monovalent antigen particles further comprise a carrier moiety bound to the antigenic moiety, wherein the carrier does not comprise another copy of the antigen structure.

[0077] In some embodiments of the invention, the monovalent antigen particle further comprises a carrier moiety, optionally linked via a linker, to the antigenic moiety, wherein the carrier, and optionally the linker, do not comprise another copy of the antigen structure, and the carrier moiety, and optionally the linker, are not capable of eliciting a cell-mediated immune response against the target antigen. In another alternative or additional embodiment of the invention, the multivalent antigen particle further comprises a carrier moiety, optionally linked via a linker, to the antigenic moiety. A "linker" in the context of the present invention may include any molecule(s), protein, or peptide that can be used to covalently or non-covalently connect two moieties of the compounds of the invention to each other.

[0078] The term "carrier moiety" in the context of the invention disclosed herein preferably relates to a substance or structure that presents or comprises the antigenic structure of the particle of the invention. The carrier moiety is preferably a substance or structure selected from immunogenic or non-immunogenic polypeptides, immune CpG islands, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), bacteria or bacterial ghosts, liposomes, chitosomes, virosomes, microspheres, dendritic cells, particles, microparticles, nanoparticles or beads.

[0079] Preferably, neither the carrier moiety, and optionally the linker, is capable of eliciting a cell-mediated immune response against a target antigen, such as an antigen associated with an autoimmune disease.

[0080] A "linker" in the context of the present invention is preferably a peptide linker that can have any size and length suitable for a given application in the context of the present invention. The linker can have a length of 1 to 100 amino acids, preferably 2 to 50 amino acids. The linker can be a typical 4GS linker in 2, 3, 4, 5, 6 or more repeats.

[0081] The carrier moiety can facilitate the presentation of the antigen to the immune system and improve the stability of the particle.

[0082] The present invention is therefore based, at least in part, on the surprising discovery that carriers linked to antigenic moieties can improve the antigenic, pharmacological and / or pharmacokinetic properties of monovalent antigen particles and thus affect the modulation of the immune response to the target antigens described herein.

[0083] In certain embodiments, the present invention relates to a composition of the invention, wherein the multivalent antigen particle further comprises a carrier moiety bound to the antigenic moiety.

[0084] The carrier moiety can facilitate the presentation of the antigen to the immune system and improve the stability of the particle.

[0085] The present invention is therefore based, at least in part, on the surprising discovery that carriers linked to antigenic moieties can improve the antigenic, pharmacological and / or pharmacokinetic properties of multivalent antigen particles and thus affect the modulation of the immune response to the target antigens described herein.

[0086] In certain embodiments, the present invention relates to a composition of the present invention, wherein the carrier moiety comprises a structure selected from the group of a polypeptide, an immune CpG island, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), a bacterium or bacterial ghost, a liposome, a chitosome, a virosome, a microsphere, a dendritic cell, a particle, a microparticle, a nanoparticle, or a bead.

[0087] The carrier moiety is preferably a substance or structure selected from an immunogenic or non-immunogenic polypeptide, an immune CpG island, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxin subunit B (CTB), bacteria or bacterial ghosts, liposomes, chitosomes, virosomes, microspheres, dendritic cells, particles, microparticles, nanoparticles, or beads.

[0088] Certain carrier moieties are particularly useful in improving the presentation of antigens to the immune system and / or the stability of the particle, while being biologically tolerable.

[0089] The present invention is therefore based, at least in part, on the surprising discovery that certain carriers linked to antigenic moieties can improve the antigenic, pharmacological and / or pharmacokinetic properties of monovalent antigen particles and thus affect the modulation of the immune response to the target antigens described herein.

[0090] In certain embodiments, the present invention relates to a composition of the invention, wherein the multivalent antigen particle comprises a conjugate of the following formula ALA, where A is a moiety comprising a target antigen and L is a cross-linking linker, preferably L is a bismaleimide, and most preferably the conjugate has the following structure (I), and R is a moiety comprising a target antigen.

[0091] [ka]

[0092] Preferably, neither the carrier moiety, and optionally the linker, is capable of eliciting an antibody-mediated immune response against the target antigen.

[0093] The carrier moiety can facilitate the presentation of the antigen to the immune system and improve the stability of the particle.

[0094] The present invention is therefore based, at least in part, on the surprising discovery that carriers linked to antigenic moieties can improve the antigenic, pharmacological and / or pharmacokinetic properties of multivalent antigen particles and thus affect the modulation of the immune response to the target antigens described herein.

[0095] In certain embodiments, the present invention relates to a composition of the invention, wherein the multivalent antigen particles comprise a linker having a cross-linking reactive group for protein conjugation.

[0096] The term "crosslinking reactive group for protein conjugation" as used herein refers to any chemical group or structure that allows for the formation of a bond between the antigen particles and a protein as described herein. Such crosslinking reactive groups and their preparation are well known to those skilled in the art (see, for example, Brinkley, M., 1992, Bioconjugate chemistry, 3(1), 2-13; Kluger, R., & Alagic, A, 2004, Bioorganic chemistry 32.6(2004):451-472.; Stephanopoulos, N.; Francis, MB, 2011, Nature Chemical Biology.7(12):876-884).

[0097] The inventors have found that linkers that are linked to antigen particles (e.g., multivalent antigen particles) described herein and that contain cross-linking reactive groups for binding to endogenous proteins in a subject can enhance the immune response (see, e.g., Figures 34-36, Examples 12, 13, 15).

[0098] In certain embodiments, the present invention relates to compositions of the invention, wherein the multivalent antigen particles comprise a linker with cross-linking reactive groups for stable protein conjugation.

[0099] The term "stable protein conjugation" as used herein refers to a covalent protein conjugation that is not a SS bond. In some embodiments, the stable protein conjugation described herein is hydrolytically stable. In some embodiments, the stable protein conjugation described herein is an irreversible bond.

[0100] The present inventors have found that stable conjugation to endogenous proteins can enhance the immune response to the antigen particles described herein (Example 14).

[0101] In certain embodiments, the present invention relates to a composition of the invention, wherein the cross-linking reactive group is attached to the protein at least one selected from the group consisting of a lysine amino acid residue, a cysteine ​​residue, a tyrosine residue, a tryptophan residue, the N-terminus, and the C-terminus.

[0102] In certain embodiments, the present invention relates to compositions of the invention, wherein the crosslinking reactive groups are groups selected from carboxyl-amine reactive groups, amine reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, and photoreactive groups.

[0103] In certain embodiments, the present invention relates to a composition of the present invention, wherein the crosslinking reactive group is a group selected from carbodiimides, NHS esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphines, maleimides, haloacetyls, hydrazides, alkoxyamines, diazirines, and arylazides.

[0104] Thus, the present invention is based, at least in part, on enhancing the immune response by binding to endogenous proteins.

[0105] In certain embodiments, the present invention relates to a composition of the invention, wherein the multivalent antigen particles are linked to the adjuvant, preferably the multivalent particles are covalently linked to the adjuvant.

[0106] The term "adjuvant" as used herein refers to an agent that does not contain a target antigen and can enhance the immune response to the antigen particles described herein. In some embodiments, the adjuvant described herein comprises at least one adjuvant selected from the group of oils (e.g., paraffin oil, peanut oil), bacterial products, saponins, cytokines (e.g., IL-1, IL-2, IL-12), squalene, and IgG, and preferably, the adjuvant comprises a free SH group.

[0107] The inventors have found that the immune response, particularly that induced by multivalent antibodies, can be enhanced by linking the antigen particles described herein to an adjuvant (Figures 36D and E, Figure 34). This linking to an adjuvant substantially reduces the need to formulate the antigen particles described herein with more unlinked adjuvant. Furthermore, the adjuvant can increase the stability of the antigen particles described herein.

[0108] Thus, the present invention is based, at least in part, on the discovery that by linking the antigenic particles described herein to an adjuvant, the immune response elicited can be enhanced.

[0109] In certain embodiments, the present invention relates to a composition of the invention, wherein the multivalent antigen particle comprises at least two copies of an antigen structure in spatial proximity to each other.

[0110] The multivalent antigen particles of the present invention preferably comprise at least two copies of an antigen structure in spatial proximity to each other, preferably within a nanometer range selected from the ranges of 1 nm to 10 μm, more preferably 1 nm to 5 μm, 1 nm to 1000 nm, 1 nm to 500 nm, 1 nm to 100 nm, 1 nm to 50 nm and 1 nm to 10 nm.

[0111] The term "spatial proximity" as used herein refers to being on the same antigen particle and close enough to modulate an immune response. "Close enough" depends on the size and structure of the multivalent antigen particle itself, as well as the size of the antigen structure. In some embodiments, the distance between two copies of the antigen structure is within the range of 3 nm to 20 nm.

[0112] In some embodiments, at least two copies of the antigen structure are spatially close to each other within a range of about 1 nm to about 1000 nm, preferably about 1 nm to about 500 nm, preferably about 1 nm to about 100 nm, preferably about 1 nm to about 50 nm, preferably about 1 nm to about 20 nm, or preferably about 3 nm to about 20 nm.

[0113] Methods for measuring spatial proximity are known to those of skill in the art (see, e.g., F. Schueder et al., 2021, Angew. Chem. Int. Ed. 2021, 60, 716; Erickson, D. et al., 2008, Microfluidics and nanofluidics, 4(1-2), 33-52; Turkowyd, B., et al., 2016, Anal Bioanal Chem 408, 6885-6911).

[0114] The inventors have found that multivalent particles of certain size ranges are particularly effective at selecting specific immune responses.

[0115] Thus, the present invention is based, at least in part, on the surprising discovery that the size and / or spatial proximity of antigen particles can influence the modulation of the immune response to a target antigen, as described herein.

[0116] In certain embodiments, the present invention relates to a composition of the invention, wherein the target antigen comprises at least one substance selected from the group of nucleic acids, carbohydrates, peptides, and haptens.

[0117] The term "hapten" as used herein refers to a small molecule that elicits a detectable immune response when bound to a carrier moiety. Haptens as described herein may also include an immunogenic group. In some cases, the immunogenic group includes a fluorescent group, an enzyme or fragment thereof, a peptide or fragment thereof, or biotin. In some cases, the immunogenic group is selected from the list including biotin, fluorescein, digoxigenin, or dinitrophenyl.

[0118] Nucleic acids, carbohydrates, peptides, and / or haptens are useful structures for copying or mimicking endogenous or pathological antigenic patterns, and furthermore, they can be designed to induce specific immune responses without substantial side effects.

[0119] Thus, the present invention is based, at least in part, on the surprising discovery that certain antigen types can affect the modulation of the immune response to the target antigens described herein.

[0120] In certain embodiments, the present invention provides a monovalent antigen particle:multivalent antigen particle ratio of greater than 1, preferably greater than 10 1 More than 10, more preferably 2 More than 10, more preferably 3 More than 10, more preferably 4 The present invention relates to a composition of the present invention having a viscosity of 1000:1 or more.

[0121] In the context of the present invention, it has been found that a specific ratio of monovalent and polyvalent antigens can modulate the antibody-mediated immune response mediated by B cells. Therefore, it is a preferred embodiment of the present invention that the composition comprising monovalent and polyvalent antigen particles comprises a specific antigen ratio, preferably a ratio of monovalent antigen particles to polyvalent antigen particles. In particular, among such preferred embodiments, the modulation of the cell-mediated target antigen-specific immune response in a subject can be achieved by increasing one or more of the subject's B cells by more than 1, preferably 10 1 Super, 10 2 Super, 10 3 Super, 10 4In another embodiment of the invention, contacting one or more of the subject's B cells with a composition comprising more than 1, preferably 10 specific antigen ratios constitutes the control of IgG-type (and / or IgM) target antigen-specific B cell responses in a subject. In another embodiment of the invention, contacting one or more of the subject's B cells with a composition comprises more than 1, preferably 10 specific antigen ratios in a subject. 1 Super, 10 2 Super, 10 3 Super, 10 4 The method involves administering to a subject an amount of monovalent antigen particles effective to produce a particular antigen ratio that is greater than or equal to that of the individual.

[0122] The present inventors have found that the ratio of monovalent antigen particles:multivalent antigen particles can be used to regulate immune responses (see, for example, FIG. 18). A higher ratio of monovalent antigen particles:multivalent antigen particles can reduce the IgG antibody production induced by multivalent antigen particles (see, for example, FIG. 1b, d) and improve the generation of protective regulatory IgM antibody production (see, for example, FIG. 7, 11). A higher ratio of monovalent antigen particles:multivalent antigen particles can protect the function of the target antigen against the immune response (see, for example, FIG. 16).

[0123] Thus, the present invention is based, at least in part, on the surprising discovery that modulation of the immune response to a target antigen is dependent on the monovalent:multivalent antigen particle ratio.

[0124] In certain embodiments, the present invention relates to a composition of the present invention further comprising a pharma- ceutically acceptable carrier and / or excipient.

[0125] The term "pharmaceutically acceptable carrier" as used herein refers to an ingredient in a composition, other than an active ingredient, that is nontoxic to a recipient at the dosage and concentration used.

[0126] Pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum alcohols, such as ethanol, ethanolamine, ethanolamines ... Examples of suitable pharmacokinetic and / or therapeutic agents include proteins such as bumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacokinetic and therapeutic agents include interstitial drug dispersion agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in US2005 / 0260186 and US2006 / 0104968.

[0127] Pharmaceutically acceptable carriers and / or excipients may enhance the stability, delivery and / or pharmacokinetic / pharmacodynamic properties of the compositions of the invention.

[0128] In certain embodiments, the present invention relates to a method of inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response, the method comprising the steps of a) contacting one or more B cells with a composition of the present invention, and b) inducing and / or modulating the humoral target antigen-specific immune response and / or the B cell-mediated target antigen-specific immune response.

[0129] A "cell-mediated target antigen-specific immune response" in the context of the present invention refers to an immune response that involves one or more B lymphocytes (B cells), preferably a B cell-mediated immune response.

[0130] The term "B lymphocyte" or "B cell" as used herein refers to a lymphocyte that plays a role in humoral immunity of the adaptive immune system and is characterized by the presence of a B cell receptor (BCR) on the cell surface. B cell types include plasma cells, memory B cells, B-1 cells, B-2 cells, marginal zone B cells, follicular B cells, and regulatory B cells (B reg). Furthermore, the term "B cells" (also known as "B lymphocytes") refers to immune cells that express cell surface immunoglobulin molecules and ultimately differentiate into antibody-secreting cells upon activation. It therefore includes, for example, conventional B cells, CD5 B cells (also known as B-1 cells and transitional CD5 B cells). "B cells" should also be understood to encompass references to B cell mutants. "Mutants" include naturally or non-naturally modified B cells, such as, but not limited to, genetically modified cells. References to "B cells" should also be understood to extend to B cells that exhibit commitment to the B cell image. These cells may be at any differentiation stage of development and therefore may not necessarily express surface immunoglobulin molecules. B cell commitment may be characterized by the onset of immunoglobulin gene rearrangement, or it may correspond to an earlier stage of commitment characterized by some other phenotypic or functional characteristic, such as cell surface expression of CD45R, MHCII, CD10, CD19 and CD38. Examples of B cells at various stages of differentiation include early B cell precursors, early pro-B cells, late pro-B cells, pre-B cells, immature B cells, mature B cells, plasma cells, and memory (B) cells. In the context of the present invention, B cells can be viewed as immature B cells that express mainly IgM type B cell receptors, mature B cells that express mainly IgD type B cell receptors, or memory B cells that express IgG type B cell receptors. The difference between IgM type B cell receptors and IgD type B cell receptors is the type of heavy chain sequence, which is either μ type or δ type. Methods for obtaining genetically modified B cells are known to those skilled in the art (see, for example, Johnson, MJ, et al., 2018, Scientific reports, 8(1), 1-9). Further methods for obtaining cells that show commitment to the B cell image are known to those skilled in the art (see, for example, Brudno, JN, 2018, Journal of Clinical Oncology, 36(22), 2267).

[0131] In the context of the present invention, the term "cell-mediated target antigen-specific immune response" preferably relates to a cellular immune type response involving immune cells, such as lymphocytes, preferably B lymphocytes (B cell-mediated immune response), preferably comprising and / or expressing one or more antibodies, or variants thereof, and / or B cell receptors specific for a target antigen, and / or variants thereof. Preferably, the cell-mediated target antigen-specific immune response involves B cells expressing immunoglobulin (Ig)M, IgD, IgA or IgG type antibodies and / or B cell receptors.

[0132] In general, the term "contacting" should be understood as presenting such antigenic particles to the subject's immune system, preferably to induce a B cell-mediated immune response.

[0133] In some embodiments, the present invention relates to a method of inducing and / or modulating a cell-mediated target antigen-specific immune response in a subject, the method comprising: (i) a monovalent antigen particle composed of an antigenic moiety comprising one or less of an antigenic structure capable of inducing an antibody-mediated immune response against a disease-associated antigen; (ii) a multivalent antigen particle, which is comprised of an antigenic moiety comprising two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, wherein two or more of the antigenic structures are covalently or non-covalently cross-linked; with a composition comprising:

[0134] In some embodiments alternative to the first aspect, the invention relates to a composition for use in eliciting and / or modulating a cell-mediated target antigen-specific immune response in a subject, the composition comprising: (iii) a monovalent antigen particle composed of an antigenic moiety that includes one or less of an antigenic structure capable of inducing an antibody-mediated immune response against a disease-associated antigen; (iv) a multivalent antigen particle, comprising an antigenic moiety comprising two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, wherein two or more of the antigenic structures are covalently or non-covalently cross-linked; The compositions are used by contacting one or more immune cells of a subject with the composition.

[0135] In a preferred embodiment of the invention, contacting one or more immune cells of a subject or patient with a composition comprising monovalent and multivalent antigen particles involves (i) administering monovalent antigen particles to the subject, (ii) administering multivalent antigen particles to the subject, or (iii) administering monovalent and multivalent antigen particles to the subject, and in (i), (ii) and (iii) the immune cells of the subject are monovalent and multivalent antigen particles as a result of the administration of the composition. Preferably, in (i), the subject is characterized by the presence of multivalent antigen particles prior to administration of the monovalent antigen particles, and in (ii), the subject is characterized by the presence of monovalent antigen particles prior to administration of the multivalent antigen particles.

[0136] In further specific embodiments of the invention, the method is preferred when contacting one or more of the subject's B cells with an amount of monovalent antigen particles is administered either in direct combination with or without administering multivalent antigen particles to the subject.

[0137] In the context of the present invention, modulation of a cell-mediated target antigen-specific immune response in a subject preferably involves reducing one or more of the subject's B cells by less than 1, preferably 10 -1 , 10 -2 , 10 -3 , 10 -4 The present invention constitutes an increase in an IgG-type target antigen-specific B cell response in a subject by contacting the subject with a composition comprising a specific antigen ratio below 1. Preferably, contacting one or more of the subject's B cells with the composition provides an increase in an IgG-type target antigen-specific B cell response in a subject by contacting the subject with a composition comprising a specific antigen ratio below 1. -1 , 10 -2 , 10 -3 , 10 -4It involves administering to a subject an amount of multivalent antigen particles effective to produce a specified antigen ratio below:

[0138] Contacting one or more of the subject's B cells with an amount of multivalent antigen particles is preferably administered either with or without a direct composition that administers monovalent antigen particles to the subject.

[0139] In some embodiments, the methods described herein are non-therapeutic and non-surgical methods. In this embodiment, the method of the invention is not for treating a subject, but for inducing an immune response for the production and isolation of novel antibodies, which are isolated in the next step. In this embodiment, the subject is a generally healthy subject not suffering from any disease that is treated by carrying out the method. In this aspect, the subject is preferably a non-human vertebrate.

[0140] In some embodiments, the methods described herein are methods for diagnosis.

[0141] Thus, the present invention is based, at least in part, on the surprising discovery that the compositions of the invention can be used in methods of modulating B cell immune responses.

[0142] In certain embodiments, the present invention relates to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention, wherein the B cell-mediated target antigen-specific immune response comprises one or more antibodies and / or B cell receptors, and / or variants thereof, that are specific for the target antigen.

[0143] As used herein, the term "antibody" may be understood in the broadest sense as any immunoglobulin (Ig) capable of binding to its epitope. Antibodies themselves are a species of ABP. Full-length "antibodies" or "immunoglobulins" are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (VH) followed by three carboxy-terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy chain antibodies, which consist of only two heavy chains and lack the two light chains typically found in antibodies. Heavy chain antibodies include hcIgG (IgG-like) antibodies from camelids, such as dromedaries, camels, llamas, and alpacas, and IgNAR antibodies from cartilaginous fishes (e.g., sharks). Still other forms of antibodies include single domain antibodies (sdAbs, called nanobodies by developer Ablynx), which are antibody fragments consisting of a single monomeric variable antibody domain. Single domain antibodies are typically produced from heavy chain antibodies, but may be derived from conventional antibodies.

[0144] Exemplary antibody Ig variants discussed in the context of the present invention include IgG, IgM, IgE, IgA, or IgD antibodies.

[0145] The term "B cell receptor," as used herein, refers to a transmembrane protein on the surface of a B cell, such as a membrane-bound antibody.

[0146] The term "variant" as used herein refers to a first agent (e.g., a first molecule) related to a second agent (e.g., a parent molecule). A variant molecule (e.g., a variant antibody, a variant of a B cell receptor) can be derived from, isolated from, based on, or homologous to a parent molecule. The term variant can be used to describe either a polynucleotide or a polypeptide.

[0147] The method of the present invention allows to induce immune responses involving different antibodies and / or B cell receptors and / or variants. Depending on the characteristics of the compositions of the present invention (e.g., antigen type, particle size, particle ratio, priming / boosting) and on the characteristics of the B cells (cell type, maturation stadium, mutation), the immune response can be altered (see, for example, Figures 1, 2, 3, 7, 8, 19, 20A).

[0148] Thus, the present invention is based, at least in part, on the surprising discovery that the compositions of the invention can be used in methods of modulating antibody-mediated, B cell receptor-mediated, and / or variant-mediated immune responses to a target antigen.

[0149] In certain embodiments, the present invention relates to a method of inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention, wherein the B cell-mediated target antigen-specific immune response comprises immunoglobulin (Ig)M, IgD, IgA or IgG type antibodies and / or B cells expressing a B cell receptor.

[0150] In certain embodiments, the present invention relates to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention, wherein the B cell-mediated target antigen-specific immune response comprises immunoglobulin (Ig)M, IgA and / or IgG type antibodies and / or B cells expressing a B cell receptor.

[0151] In certain embodiments, the present invention relates to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention, wherein the B cell-mediated target antigen-specific immune response comprises B cells expressing immunoglobulin (Ig)M and / or IgG type antibodies.

[0152] As used herein, the term "IgG" has its general meaning in the art and refers to an immunoglobulin with a heavy g chain. This class of immunoglobulins, produced as part of the secondary immune response to antigens, constitutes about 75% of total serum Ig. IgG is the only class of Ig that can cross the human placenta and is largely responsible for protecting the newborn during the first few months of life. IgG is the major immunoglobulin in blood, lymph, cerebrospinal and peritoneal fluids and is the linchpin of the humoral immune response. Serum IgG in healthy humans represents about 15% of the total protein, besides albumin, enzymes, other globulins and many more. 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 the variable region, all immunoglobulins within a class share about 90% homology, but only 60% between classes. IgG1 represents 60-65% of all major subclass IgG and is primarily involved in thymus-mediated immune responses to protein and polypeptide antigens. IgG1 can bind to the Fc receptors of phagocytes and activate the complement cascade through binding to the C1 complex. IgG1 immune responses can already be measured in newborns and reach their typical concentrations during infancy. IgG2, the second largest of the IgG isotypes, constitutes 20-25% of the major subclasses and is the general immune response to carbohydrate / polysaccharide antigens. "Adult" concentrations are usually reached by 6 or 7 years of age. IgG3 constitutes about 5-10% of total IgG and plays a major role in immune responses to protein or polypeptide antigens. The affinity of IgG3 can be higher than that of IgG1. IgG4, which usually constitutes less than 4% of total IgG, does not bind polysaccharides. In the past, IgG4 testing has been associated with food allergies, but recent studies have shown that elevated serum levels of IgG4 are found in patients with sclerosing pancreatitis, cholangitis, and interstitial pneumonia caused by infiltration of IgG4-positive plasma cells.

[0153] As used herein, the term "IgM" has its general meaning in the art and refers to an immunoglobulin with a heavy m chain. Serum IgM exists as a pentamer (or hexamer) in mammals and constitutes about 10% of normal human serum Ig content. It predominates in the primary immune response to most antigens and is the most efficient complement-fixing immunoglobulin. IgM is also expressed as a membrane-associated immunoglobulin on the plasma membrane of B lymphocytes (it may be organized as a multiprotein cluster within the membrane). In this form, it is the B cell antigen receptor, and the H chains each contain an additional hydrophobic domain for anchoring in the membrane. Monomers of serum IgM are bound together by disulfide bonds and a joining (J) chain. Each of the five monomers in the pentameric 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, resulting in cell agglutination. This antibody-antigen immune complex is then destroyed by complement fixation or receptor-mediated endocytosis by macrophages. IgM is the first immunoglobulin class synthesized by newborns and plays a role in the pathogenesis of several autoimmune diseases. Immunoglobulin M is the third most common serum Ig and takes one of two forms: a pentamer (or under some circumstances a hexamer) in which all heavy chains are identical and all light chains are identical. The membrane-associated form is a monomer that can form multimeric clusters on the membrane (e.g., found on B lymphocytes as the B cell receptor).

[0154] IgM is the first antibody constructed during an immune response. In theory, its pentameric structure not only has high affinity but also provides 10 free antigen-binding sites, so it is involved in agglutination and cytolysis reactions. Due to conformational constraints between the 10 Fab moieties, IgM only has a valency of 5. Furthermore, IgM is not as versatile as IgG. However, it is crucial in complement activation and agglutination. IgM is found primarily in lymph and blood and is a very effective neutralizing agent in the early stages of disease. Elevated levels can be a sign of recent infection or exposure to antigens.

[0155] As used herein, the term "IgA" has its common meaning in the art and refers to immunoglobulins with a heavy a chain. IgA constitutes about 15% of all immunoglobulins in healthy serum. IgA in serum is primarily monomeric, whereas in secretions such as saliva, tears, colostrum, mucus, sweat, and gastric juices, IgA is found as a dimer connected by a linking peptide. Most IgA exists in a secretory form. This is thought to be due to its properties that prevent pathogens from invading by adhering to and penetrating epithelial surfaces. IgA does not induce bacterial cell lysis via the complement system, as it is a very weak complement-activating antibody. However, secretory IgA works together with lysozyme (also present in many secretory fluids) that can hydrolyze carbohydrates within bacterial cell walls, thereby allowing the immune system to clear the infection. IgA is found on the epithelial cell surface where it acts primarily as a neutralizing antibody. There are two IgA subtypes in humans, IgA1 and IgA2, while mice have only one subclass. They differ in the molecular weight of the heavy chain and their concentration in serum. IgA1 constitutes about 85% of the total IgA concentration in serum. IgA1 shows broad resistance to several proteases, although some can affect / splice out the hinge region. IgA1 shows a good immune response to protein antigens and, to a lesser extent, polysaccharides and lipopolysaccharides. IgA2, which accounts for only ~15% of the total IgA in serum, plays an important role in the mucosa of the respiratory tract, eyes, and gastrointestinal tract to combat polysaccharide and lipopolysaccharide antigens. It also shows good resistance to proteolysis and many bacterial proteases, confirming the importance of IgA2 in fighting bacterial infections.

[0156] As used herein, the term "IgD" has its general meaning in the art and refers to an immunoglobulin with a heavy d chain. IgD is an immunoglobulin that constitutes about 1% of the proteins in the plasma membrane of immature B lymphocytes and is usually co-expressed with another cell surface antibody, IgM. IgD is also produced in a secretory form found in very small amounts in serum, accounting for 0.25% of serum immunoglobulins. Secretory IgD is produced as a monomeric antibody with two heavy chains of the delta (δ) class and two Ig light chains.

[0157] The method of the present invention allows to induce and / or modulate an immune response that includes a specific antibody type and / or a certain ratio of antibody types. Depending on the characteristics of the composition of the present invention (e.g., antigen type, particle size, particle ratio, priming / boosting) and depending on the characteristics of the B cells (cell type, maturation stadium, mutation), the immune response can be modified (see, for example, Figures 1, 2, 3, 7, 8).

[0158] Thus, the present invention is based, at least in part, on the surprising discovery that the compositions of the invention can be used in methods of modulating an immune response to a target antigen mediated by IgM, IgD, IgA or IgG type antibodies and / or B cell receptors.

[0159] In certain embodiments, the present invention relates to a method of inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention, wherein the induced B cell-mediated target antigen-specific immune response comprises inducing at least one IgG type antibody and at least one oligomeric antibody.

[0160] The method of the present invention allows to induce and / or modulate immune responses, including IgG and IgM antibodies. Depending on the characteristics of the composition of the present invention (e.g., antigen type, particle size, particle ratio, priming / boosting) and on the characteristics of B cells (cell type, maturation stadium, mutation), the immune response can be modified, for example, in that IgG antibodies are suppressed and IgM antibodies are increased (see, for example, Figures 1, 2, 3, 7, 8).

[0161] Thus, the present invention is based, at least in part, on the surprising discovery that the compositions of the invention can be used in methods of modulating immune responses to target antigens that are mediated by IgM and IgG type antibodies.

[0162] In certain embodiments, the present invention relates to a method for obtaining a protective regulatory antibody, comprising the steps of: (a) inducing at least one IgG type antibody and at least one oligomeric antibody according to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b) isolating mature oligomeric antibodies, the binding affinity of the oligomeric antibody to the target antigen being equal to or greater than that of the IgG type antibody, to obtain a protective regulatory antibody that is protectively regulatory with respect to the function of the target antigen.

[0163] The methods in such embodiments are preferably non-medical methods, such as in vitro methods.

[0164] The term "protective regulatory with respect to the function of a target antigen" as used herein refers to modulating the function of a target antigen. In some embodiments, the invention relates to a method for obtaining a protective regulatory antibody, where the function of the target antigen is extended by the protective regulatory antibody (e.g., by preventing a degrading immune response). In some embodiments, the invention relates to a method for obtaining a protective regulatory antibody, where the function of the target antigen is extended by the protective regulatory antibody by extending the half-life of the target antigen.

[0165] In certain embodiments, the present invention relates to a method for obtaining a protective regulatory antibody, comprising the steps of: (a) inducing at least one IgG type antibody and at least one oligomeric antibody according to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b)(i) isolating mature oligomeric antibodies, in which the binding of the oligomeric antibody to the target antigen is more specific than the IgG type antibody, to obtain a protective regulatory antibody that is protective regulatory with respect to the function of the target antigen.

[0166] The methods in such embodiments are preferably non-medical methods, such as in vitro methods.

[0167] In certain embodiments, the present invention relates to a method for obtaining a protective regulatory antibody, comprising the steps of: (a) inducing at least one IgG type antibody and at least one oligomeric antibody according to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b) isolating mature oligomeric antibodies, in which (i) the binding of the oligomeric antibody to the target antigen is more specific than that of the IgG type antibody and (ii) the binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of the IgG type antibody, to obtain a protective regulatory antibody that is protectively regulatory with respect to the function of the target antigen.

[0168] The methods in such embodiments are preferably non-medical methods, such as in vitro methods.

[0169] In certain embodiments, the present invention relates to a method for obtaining a protective regulatory antibody, comprising the steps of: (a) inducing at least one IgG type antibody and at least one oligomeric antibody according to a method for inducing and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b) isolating mature oligomeric antibodies, (i) in which the binding of the oligomeric antibody to the target antigen is more specific than that of an IgG type antibody and the oligomeric antibody is monospecific for the target antigen, and (ii) in which the binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of an IgG type antibody, to obtain a protective regulatory antibody that is protectively regulatory with respect to the function of the target antigen.

[0170] The methods in such embodiments are preferably non-medical methods, such as in vitro methods.

[0171] In some embodiments, the "oligomeric" antibody is an IgM type antibody or an oligomeric antibody derived therefrom. In some embodiments, the "oligomeric" antibody is an IgM type antibody.

[0172] In certain embodiments, the present invention provides a method for obtaining protective regulatory antibodies, comprising the steps of: (a) eliciting at least one IgG type antibody and at least one oligomeric antibody according to a method for eliciting and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b) eliciting at least one IgG type antibody and at least one oligomeric antibody, wherein (i) the binding of the oligomeric antibody to the target antigen is more specific than the IgG type antibody, and (ii) the binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of the IgG type antibody, and the protective regulatory antibody is at least 10 -7 Less than 10 -8 Less than 10, more preferably -9 and most preferably less than about 10 -10 ~about 10 -12 K in the range d and isolating mature oligomeric antibodies that bind to the target antigen with the nucleic acid to obtain protective regulatory antibodies that are protective regulatory for the function of the target antigen.

[0173] In certain embodiments, the present invention provides a method for obtaining protective regulatory antibodies, comprising the steps of: (a) eliciting at least one IgG type antibody and at least one oligomeric antibody according to a method for eliciting and / or modulating a humoral target antigen-specific immune response and / or a B cell-mediated target antigen-specific immune response according to the present invention; and (b) isolating the matured oligomeric antibodies, wherein: (i) the binding of the oligomeric antibody to the target antigen is more specific than that of an IgG type antibody, the oligomeric antibody being monospecific for the target antigen; (ii) the binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of an IgG type antibody; and the protective regulatory antibody is more than 10 -7 Less than 10 -8 Less than 10, more preferably -9 and most preferably less than about 10 -10 ~about 10 -12 and isolating mature oligomeric antibodies that bind to the target antigen with a Kd in the range of 0.1 to 0.5 to obtain protective regulatory antibodies that are protective regulatory for the function of the target antigen.

[0174] In one embodiment, the present invention provides a method for obtaining protective regulatory antibodies, comprising the steps of: (a) providing a blood sample from a subject, the subject having experienced induction of an IgG and oligomeric antibody response by a target antigen; and (b) concentrating mature oligomeric antibodies, wherein (i) the binding of the oligomeric antibodies is more specific for the target antigen than an IgG type antibody, preferably the oligomeric antibody is monospecific for the target antigen, and / or (ii) the binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of the IgG type antibody, preferably the protective regulatory antibody is greater than 10 -7 Less than 10 -8 Less than 10, more preferably -9 and most preferably less than about 10 -10 ~about 10 -12 K in the range dand (c) isolating the enriched mature oligomeric antibodies to obtain the protective regulatory antibodies that are protective regulatory for a function of the target antigen.

[0175] In one embodiment, the present invention relates to a method according to the present invention, wherein said subject has experienced the induction of said IgG and oligomeric antibody response by said target antigen at least 7 days previously, preferably at least 14 days previously, more preferably at least 27 days previously.

[0176] In one embodiment, the invention relates to a method of the invention, wherein said subject is a (healthy) subject having said IgG and oligomeric antibodies in their blood, e.g. in their plasma, thus induction is not actively induced but is visible by the presence of IgG and oligomeric antibodies in the blood, e.g. in the plasma.

[0177] Thus, the present invention is directed, at least in part, to the isolation of new antibody fractions, in particular protective regulatory high affinity IgG, with distinct properties.

[0178] In one embodiment, the present invention provides a method for obtaining degradable oligomeric antibodies, comprising the steps of: (a) providing a blood sample from a subject, the subject experiencing induction of an IgG and oligomeric antibody response by a target antigen; and (b) concentrating primary oligomeric antibodies, wherein (i) the binding of the oligomeric antibodies is equally or less specific for the target antigen than IgG type antibodies, preferably the oligomeric antibodies are cross-specific for the target antigen and DNA, and / or (ii) the binding affinity of the oligomeric antibodies to the target antigen is lower than that of the IgG type antibodies, preferably the protective regulatory antibodies are less than 10 -7 Larger K d and (c) isolating the enriched primary oligomeric antibodies to obtain the degrading antibodies capable of forming an immunodegrading complex with the target antigen.

[0179] Thus, the present invention is directed, at least in part, to the isolation of new antibody fractions, particularly resolvable affinity IgM antibodies, with distinct properties.

[0180] In an embodiment, the present invention relates to a method according to the present invention, wherein the blood sample is selected from the group consisting of a whole blood, a plasma and a serum sample, preferably a serum sample.

[0181] In one embodiment, the invention relates to a method according to the invention, wherein isolating the oligomeric antibodies comprises mass-related and / or affinity-related isolation.

[0182] In one embodiment the invention relates to a method according to the invention, wherein enriching oligomeric antibodies comprises immunoprecipitation of said oligomeric antibodies.

[0183] In one embodiment the invention relates to a method according to the invention, wherein said oligomeric antibody is an IgM antibody.

[0184] "K D The term "dissociation constant," as used herein, is intended to refer to the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). DValues ​​can be determined using methods well established in the art, such as plasmon resonance (BIAcore®), biolayer interferometry (BLI), ELISA, and KINEXA. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably by using a biosensor system such as the BIAcore® system, or by ELISA. The term "Ka" (or "K-assoc"), as used herein, refers broadly to the association rate of a particular antibody-antigen interaction, and the term "Kd" (or "K-diss"), as used herein, refers to the dissociation rate of a particular antibody-antigen interaction. Another preferred method is the use of BLI. The term "biolayer interferometry" or "BLI" refers to an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces, a layer of immobilized protein on a biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time.

[0185] In some embodiments, an antibody is considered "more specific" based on at least one specificity evaluation method herein. The specificity of an antibody, variant or fragment can be tested, for example, by evaluating the binding of the antibody, variant or fragment under conventional conditions (see, for example, Harlow and Lane, 1988 Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and Harlow and Lane, 1999 using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press). These methods can include, among others, binding studies, blocking studies and competition studies with structurally and / or functionally closely related molecules. These binding studies also include FACS analysis, surface plasmon resonance, analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy, or radiolabeled ligand binding assays. (Cross)specificity can be determined experimentally by methods known in the art and described herein. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.

[0186] The term "monospecific" in the context of antibodies, as used herein, refers to an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen. More importantly, the term "monospecific" in the context of the present invention relates to such an antibody that has high affinity for one antigen and does not specifically bind to any other antigen. In this embodiment, a monospecific antibody is one that has more than 10 -7 Less than nM, preferably 10 -8 less than nM, more preferably less than 10 -9 less than nM, most preferably about 10 -10 K in nM DSuch monoclonal IgM binds to antigens associated with autoimmune disorders in a ELISA. Thus, such monoclonal IgM does not bind to unrelated antigens other than the antigens associated with autoimmune diseases, and therefore, preferably, the treatment of the present invention does not include the use of polyspecific antibodies specific to unrelated antigens other than the antigens associated with autoimmune diseases. In some embodiments, the monospecificity of the antibody is defined as not recognizing dsDNA in ELISA and not showing binding in Hep-2 slides (see, for example, Example 4, Figures 16C, 16D, and Materials and Methods).

[0187] The term "mature oligomeric antibody" as used herein refers to an antibody that is a) monospecific for a target antigen, b) has a molecular weight of 10 or more, and c) is oligomeric. -7 Less than nM, preferably 10 -8 less than nM, more preferably less than 10 -9 less than nM, more preferably less than 10 -10 less than nM, more preferably less than 10 -11 less than nM, and most preferably about 10 -12 K in nM Dand / or c) has undergone a maturation process. The maturation process of oligomeric antibodies can be modulated by the B cell development stage, genetic modification (e.g., by the absence of IgD, see Figures 25, 29) and / or contact with maturation modified cells or signaling agents. In some embodiments, the maturation process and / or its completion is defined by several mutations, preferably at least 1, at least 2, at least 3 mutations, at least 4 mutations, at least 50 mutations, at least 100 mutations, or at least 500 mutations, of the matured oligomeric antibody compared to the first generation oligomeric antibody. In some embodiments the maturation process and / or completion thereof is performed over a certain period of time, preferably more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, more than 14 days, more than 15 days, more than 16 days, more than 17 days, more than 18 days, more than 19 days, more than 20 days, more than 21 days, more than 22 days, more than 23 days, more than 24 days, more than 25 days, more than 26 days, more than 27 days, more than 28 days, more than 29 days, more than 30 days, more than 31 days, more than 32 days, more than 33 days, more than 34 days, more than 35 days, more than 36 days, more than 37 days, more than 38 days, more than 39 days, more than 40 days, more than 41 days, more than 42 days, more than 43 days, more than 44 days, more than 45 days, more than 46 days, more than 47 days, more than 48 days, more than 49 days, more than 50 days, more than 51 days, more than 52 days, more than 53 days, more than 54 days, more than 55 days, more than 56 days, more than 57 days, more than 58 days, more than 59 days, more than 60 days, more than 61 days, more than 62 days, more than 63 days, more than 64 days, more than 65 days, more than 66 days, more than 67 days, more than 68 days, more than 69 days, more than 70 days, more than 71 days, more than 72 days, more than 73 days, more than 74 days, more than 75 days, more than 76 days, more than 77 days, more than 78 days, more than 79 days, more than 80 days, more than 81 days, more than 82 days, more than 83 days, more than 84 days, or more than 85 days.

[0188] Methods for the selective isolation of antibodies are known to those of skill in the art (see, e.g., Huang J, Doria-Rose NA, et al., 2013, Nat Protoc. Oct;8(10):1907-15).

[0189] Any method known to those skilled in the art can be used to isolate mature antibodies. In some embodiments, isolating the mature oligomeric antibodies described herein comprises at least one method selected from the group of physicochemical fractionation, class-specific affinity, and antigen-specific affinity. For example, antibodies can be isolated as described in the isolation of insulin-specific serum immunoglobulins in the Materials and Methods section of this specification.

[0190] Thus, the present invention is based at least in part on the surprising discovery that the methods of the invention can be used to obtain antibody variants or fragments that protect and / or modulate the function of an antigen by competing with the binding of an antigen-function-limiting antigen-binding agent.

[0191] In certain embodiments, the invention relates to a protective regulatory antibody obtained according to the method for obtaining a protective regulatory antibody according to the invention, or a variant or fragment thereof, which is protective regulatory with respect to the function of the target antigen.

[0192] The term "fragment" of an antibody, as used herein, refers to an antibody fragment that can bind to the same antigen as its antibody counterpart. Such fragments can be easily identified by those skilled in the art, and include, for example, Fab fragments (e.g., by papain digestion), Fab' fragments (e.g., by pepsin digestion and partial reduction), F(ab')2 fragments (e.g., by pepsin digestion), Facb (e.g., by plasmin digestion), Fa (e.g., by pepsin digestion, partial reduction and reaggregation), and scFv (single chain Fv; e.g., by molecular biology techniques) fragments.

[0193] In some embodiments, the protective regulatory antibodies of the invention are oligomeric antibodies, preferably monospecific IgM type antibodies.

[0194] In another embodiment, the protective regulatory antibody, variant or fragment of the invention, preferably the monospecific IgM type antibody of the invention or variant thereof, is not a polyclonal antibody or the antigen-binding fragment is not a fragment of a polyclonal antibody. In a more specific embodiment, the protective regulatory antibody, variant or fragment of the invention, preferably the monospecific IgM type antibody of the invention or variant thereof, is not a primary (polyspecific) IgM type antibody.

[0195] In an alternative and preferred embodiment, the protective regulatory antibody, variant or fragment of the invention, preferably a monospecific IgM type antibody, or variant thereof, is an antibody or antigen-binding fragment thereof, which antibody is a monoclonal antibody, or which antigen-binding fragment thereof is a fragment of a monoclonal antibody.

[0196] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of antibodies that are substantially identical based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (e.g., epitopes) of an antigen, each mAb is typically directed against a single determinant on the antigen. In addition to their specificity, mAbs are advantageous in that they can be synthesized by cell culture (hybridoma, recombinant cells, etc.) uncontaminated by other immunoglobulins. mAbs herein include, for example, chimeric, humanized or human antibodies, or antibody fragments.

[0197] Monoclonal IgM antibodies according to the invention can be prepared by methods well known to those skilled in the art. For example, mice, rats, goats, camels, alpacas, llamas or rabbits can be immunized with the antigen of interest (or a nucleic acid encoding the antigen of interest) together with an adjuvant. Splenocytes are taken as a pool from several immunized animals at certain intervals, with test bleeding performed to assess serum antibody titers. Splenocytes are prepared either for immediate use in fusion experiments or stored in liquid nitrogen for use in future fusions. Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants from wells with growing hybrids are screened, for example, by enzyme-linked immunosorbent assay (ELISA) for mAb secretors. ELISA-positive cultures are cloned either by limiting dilution or by fluorescence-activated cell sorting, typically resulting in established hybridomas from single colonies. The ability of an antibody, including an antibody fragment or subfragment, to bind to a specific antigen can be determined by binding assays known in the art, for example, by using the antigen of interest as a binding partner. Alternatively, splenic B cells that bind to the immunizing antigen are selected as single cells, and then the cDNAs encoding the heavy and light chains are cloned from the single cells. The cloned cDNAs are then used to produce monoclonal recombinant antibodies in vitro, and these antibodies are further characterized based on their specificity and affinity to the immunizing antigen.

[0198] Monospecific oligomeric antibodies or variants thereof according to the present invention may be prepared by genetic immunization methods in which the natural protein is expressed in vivo with normal post-transcriptional modifications, avoiding the isolation or synthesis of the antigen. For example, hydrodynamic tail or leg vein delivery of naked plasmid DNA expression vectors can be used to produce the antigen of interest in vivo in mice, rats, and rabbits, thereby inducing antigen-specific antibodies (Tang et al, Nature 356:152 (1992); Tighe et al, Immunol. Today 19:89 (1998); Bates et al, Biotechniques, 40:199 (2006); Aldevron-Genovac, Freiburg DE). This allows for the efficient generation of high titers of antigen-specific antibodies, which may be particularly useful for diagnostic and / or research purposes. For such genetic immunization, a variety of gene delivery methods can be used, including direct injection of naked plasmid DNA into skeletal muscle, lymph nodes, or dermis, electroporation, ballistic (gene gun) delivery, and viral vector delivery.

[0199] In a further preferred embodiment, the monospecific oligomeric antibody or variant thereof of the invention is an antibody or an antigen-binding fragment thereof, which antibody is a human antibody, a humanized antibody or a chimeric human antibody, or the antigen-binding fragment thereof is a fragment of a human antibody, a humanized antibody or a chimeric human antibody.

[0200] Human antibodies can also be derived by in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Yumab, Symphogen, Alexion, Affimed), etc. In phage display, polynucleotides encoding single Fab or Fv antibody fragments are expressed on the surface of phage particles (see, for example, Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J Mol Biol 222:581 (1991); U.S. Pat. No. 5,885,793). Phages are "screened" to identify those antibody fragments that have affinity for a target. Thus, certain such processes mimic immune selection through the display of antibody fragment repertoires on the surface of filamentous bacteriophage and subsequent selection of phage by binding to their target. In certain such procedures, high affinity functional neutralizing antibody fragments are isolated. Thus, a complete repertoire of human antibody genes can be generated by cloning naturally rearranged human V genes from peripheral blood lymphocytes (see, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87:8095-8099 (1990)) or by generating fully synthetic or semi-synthetic phage display libraries with human antibody sequences (see, e.g., Knappik et al 2000; J Mol Biol 296:57; de Kruif et al, 1995; J Mol Biol 248:97).

[0201] Alternatively, the antibodies described herein may be prepared by utilizing XenoMouse® technology. Such mice are capable of producing human immunoglobulin molecules and antibodies, which are deficient in the production of mouse immunoglobulin molecules and antibodies. In particular, preferred embodiments of transgenic production of mice and antibodies are disclosed in U.S. Patent Application No. 08 / 759,620, filed December 3, 1996, and International Patent Application No. 98 / 24893, published June 11, 1998, and WO 00 / 76310, published December 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Through the use of such technology, fully human monoclonal antibodies against a variety of antigens have been produced. Essentially, XenoMouse® strains of mice are immunized with an antigen of interest, e.g., IGSF11 (VSIG3), lymphocytes (such as B cells) are harvested from the hyperimmunized mice, and the harvested lymphocytes are fused with a myeloid cell line to prepare immortal hybridoma cell lines. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. Other "humanized" mice are also commercially available, e.g., Medarex-HuMab mice, Kymab-Kymouse, Regeneron-Velocimmune mice, Kirin-TC mice, Trianni-Trianni mice, OmniAb-OmniMouse, Harbour Antibodies-H2L2 mice, Merus-MeMo mice. The following other "humanized" species are also available: Rats: OmniAb-OmniRat, OMT-UniRat. Chicken: OmniAb-OmniChicken.

[0202] The term "humanized antibody" according to the present invention refers to an immunoglobulin chain or fragment thereof (e.g., Fab, Fab', F(ab')2, Fv, or other antigen-binding subsequence of an antibody) that contains minimal sequence (but typically still at least a portion) derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the CDR residues of the recipient antibody have been replaced with CDR residues from a non-human species immunoglobulin (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. Thus, at least a portion of the framework sequences of the antibody or fragment thereof may be human consensus framework sequences. In some cases, Fv framework residues of the human immunoglobulin need to be replaced with corresponding non-human residues to increase specificity or affinity. Furthermore, a humanized antibody may also comprise residues that are present neither in the recipient antibody nor in the introduced CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, and typically at least two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human immunoglobulin, and all or substantially all of the framework regions being of a human immunoglobulin consensus sequence. A humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region, typically of a human immunoglobulin, which (e.g., human) immunoglobulin constant region may be modified (e.g., by mutation or glycoengineering) to optimize one or more properties of such region and / or to improve the function of the (e.g., therapeutic) antibody, e.g., to increase or decrease Fc effector function or to increase serum half-life. Exemplary such Fc modifications (e.g., Fc engineering or Fc enhancement) are described elsewhere herein.

[0203] The human constant region will most likely be derived from the Mu chain sequence, but any variant thereof, such as a gamma chain constant sequence, for example with attenuated Fc region binding, may be used as an IgM variant according to the present invention.

[0204] The term "chimeric antibody" according to the present invention refers to an antibody whose light and / or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions that are identical or homologous to the corresponding sequences of different species, such as mouse and human. Alternatively, the variable region genes are from a particular antibody class or subclass, while the rest of the chains are from another antibody class or subclass of the same or different species. Also encompassed are fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen binding domains from a mouse antibody and the constant or effector domains from a human antibody, although other mammalian species may be used.

[0205] Among such embodiments, the monospecific IgM type antibody or variant thereof of the invention comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain is that of a human antibody. Preferably, the monospecific oligomeric antibody or variant thereof comprises an antigen-binding domain of an antibody or antigen-binding fragment thereof that is a human antigen-binding domain, (ii) the antibody is a monoclonal antibody or the antigen-binding fragment is a fragment of a monoclonal antibody, (iii) the antibody is a human antibody or a humanized antibody or the antigen-binding fragment is a fragment of a human antibody, a humanized antibody or a chimeric human antibody.

[0206] The light chains of human antibodies are generally classified as kappa and lambda light chains, each of which contains one variable region and one constant domain. The heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively, as described above. Human IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. Human IgM subtypes include IgM. Human IgA subtypes include IgA1 and IgA2. In humans, the IgA isotype contains four heavy chains and four light chains, the IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype contains 10 or 12 heavy chains and 10 or 12 light chains. The antibody according to the invention may be an IgG, IgE, IgD, IgA, or IgM immunoglobulin.

[0207] In some embodiments, the monospecific oligomeric antibody or variant thereof of the invention is an IgM antibody or a fragment thereof. Preferably, the antibody of the invention is, comprises or is derived from a human, an IgM immunoglobulin of human origin, or an IgG immunoglobulin such as an IgM of rabbit origin or of rat origin, or a fragment thereof.

[0208] Monospecific oligomeric antibodies or variants thereof of the invention that comprise at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin) may have such (e.g. human) immunoglobulin constant region modified, for example by glycoengineering or mutation, to optimize one or more properties of such region, such as to increase or decrease Fc effector function, or to increase serum half-life, and / or to improve the function of the (e.g. therapeutic) antibody.

[0209] Thus, different antibody isotypes or mutant isotypes can be used to generate any of the above ABPs of the present invention to control the degree of binding to different Fc-gamma receptors. Antibodies lacking an Fc region (e.g., Fab fragments) lack binding to different Fc-gamma receptors. The choice of isotype also influences binding to different Fc-gamma receptors. The respective affinities of various human IgG isotypes for three different Fc-gamma receptors, Fc-gamma-RI, Fc-gamma-RII, and Fc-gamma-RIII, have been determined (see Ravetch & Kinet, Annu. Rev. Immunol. 9, 457 (1991)). Fc-gamma-RI is a high affinity receptor that binds IgG in monomeric form, while the latter two are low affinity receptors that bind IgG only in multimeric form. In general, both IgG1 and IgG3 have significant binding activity to all three receptors, IgG4 to Fc-gamma-RI, and IgG2 to only one type of Fc-gamma-RII, called IIaLR (see Parren et al., J. Immunol. 148, 695 (1992)). Thus, human isotype IgG1 is usually selected for stronger binding to Fc-gamma receptors, and IgG2 or IgG4 are usually selected for weaker binding. Preferred embodiments of the present invention provide such antibodies in which Fc receptor binding is reduced or eliminated.

[0210] A correlation between increased Fc-gamma-R binding and mutated Fc has been shown using targeted cytotoxicity cell-based assays (Shields et al., 2001, J. Biol. Chem. 276:6591-6604; Presta et al., 2002, Biochem Soc. Trans. 30:487-490). Methods for increasing ADCC activity through specific Fc region mutations include Fc variants containing at least one amino acid substitution at a position selected from the group consisting of 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330 and 332, where the numbering of the residues in the Fc region is that of the EU index in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)).

[0211] In certain embodiments, the Fc variants are L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D, S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T, V240M, F241W, F241L, F241Y, F241L ... 1E, F241R, F243W, F243L, F243Y, F243R, F243Q, P244H, P245A, P247V, P2 47G, V262I, V262A, V262T, V262E, V263I, V263A, V263T, V263M, V264L, V 264I, V264W, V264T, V264R, V264F, V264M, V264Y, V264E, D265G, D265N, D265Q, D265Y, D265F, D265V, D265I, D265L, D265H, D265T, V266I, V266A , V266T, V266M, S267Q, S267L, E269H, E269Y, E269F, E269R, Y296E, Y296 Q, Y296D, Y296N, Y296S, Y296T, Y296L, Y296I, Y296H, N297S, N297D, N29 7E, A298H, T299I, T299L, T299A, T299S, T299V, T299H, T299F, T299E, W3 13F, N325Q, N325L, N325I, N325D, N325E, N325A, N325T, N325V, N325H, A and at least one substitution selected from the group consisting of 327N, A327L, L328M, L328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T, L328H, L328A, P329F, A330L, A330Y, A330V, A330I, A330F, A330R, A330H, I332D, I332E, I332N, I332Q, I332T, I332H, I332Y and I332A, wherein the numbering of the residues in the Fc region is that of the EU index in Kabat.

[0212] <h2 style=";text-align:left;direction:ltr">Fcバリアントはまた、V264L、V264I、F241W、F241L、F243W、F243L、F241L / F243L / V26 2I / V264I、F241W / F243W、F241W / F243W / V262A / V264A、F241L / V262I、F243L / V264I、F243L / V262I / V264W、F241Y / F243Y / V262T / V264T、F241E / F243R / V2 62E / V264R、F241E / F243Q / V262T / V264E、F241R / F243Q / V262T / V264R、F241E / F243Y / V262T / V264R、L328M、L328E、L328F、I332E、L3238M / I332E、P244H、 P245A、P247V、W313F、P244H / P245A / P247V、P247G、V264I / I332E、F241E / F24 3R / V262E / V264R / I332E, F241E / F243Q / V262T / 264E / I332E, F241R / F243Q / V262T / V264R / I332E, F241E / F243Y / V262T / V264R / I332E, S298A / I332E, S23 9E / I332E, S239Q / I332E, S239E, D265G, D265N, S239E / D265G, S239E / D265N, S239E / D265Q, Y296E, Y296Q, T299I, A327N, S267Q / A327S, S267L / A327S, A3 27L, P329F, A330L, A330Y, I332D, N297S, N297D, N297S / I332E, N297D / I332E, N297E / I332E, D265Y / N297D / I332E, D265Y / N297D / T299L / I332E, D265F / N 297E / I332E, L328I / I332E, L328Q / I332E, I332N, I332Q, V264T, V264F, V240I, V263I, V266I, T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N S239F, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239N / I332N<h2 style=";text-align:left;direction:ltr">S239N / I332Q, S239Q / I332D, S239Q / I332N, S239Q / I332Q, Y296D, Y296N, F241Y / F243Y, V262T / V264T, N297D / I332E, A330Y / I332E, V264I / A330Y / I332 E, A330L / I332E, V264I / A330L / I332E, L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L23 5H, L235Y, L235I, L235V, L235F, S239T, S239H, S239Y, V240A, V240T, V240M, V263A, V263T, V263M, V264M, V264Y, V266A, V266T, V266M, E269H, E269Y, E 269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330I, A330F, A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H, L328D / I332E L328E / I332E, L328N / I332E, L328Q / I332E, L328V / I332E, L328T / I332E, L328H / I332E, L328I / I332E, L328A, I332T, I332H, I332Y, I332A, S239E / V264 I / I332E, S239Q / V264I / I332E, S239E / V264I, A330Y / I332E, S239E / V264I, S298A / A330Y / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D26 5V / N297D / I332E,S239D / D265I / N297D / I332E,S239D / D265L / N297D / I332E,S239D / D265F / N297D / I332E,S239D / D265Y / N297D / I332E,S239D / D265H / N 297D / I332E, S239D / D265T / N297D / I332E, V264E / N297D / I332E, Y296D / N297D / I332E, Y296E / N297D / I332E, Y296N / N297D / I332E, Y296Q / N297D / I332EY296H / N297D / I332E, Y296T / N297D / I332E, N297D / T299V / I332E, N297D / T299I / I332E, N297D / T299L / I332E, N297D / T299F / I332E , N297D / T299H / I332E, N297D / T299E / I332E, N297D / A330Y / I332E, N297D / S298A / A330Y / I332E, S239D / A330Y / I332E, S239N / A330 Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, and S239D / 264I / A330L / I332E, where the numbering of residues in the Fc region is that of the EU index in Kabat. See also WO2004 / 029207, which is incorporated herein by reference.

[0213] In certain embodiments, mutations on, adjacent to, or close to sites in the hinge link region (e.g., replacing residues 234, 235, 236, and / or 237 with another residue) can be made to reduce affinity for Fc-gamma receptors, particularly Fc-gamma-RI receptors, in all isotypes (see, e.g., US6624821). Optionally, positions 234, 236, and / or 237 are substituted with alanine and position 235 is substituted with glutamate. (See, e.g., US5624821.) Position 236 is absent in human IgG2 isotypes. Exemplary segments of amino acids at positions 234, 235, and 237 of human IgG2 are Ala Ala Gly, Val Ala Ala, Ala Ala Ala, Val Glu Ala, and Ala Glu Ala. A preferred combination of mutations is L234A, L235E and G237A, or for human isotype IgG1, L234A, L235A and G237A. A particular preferred variant of the monospecific IgM type antibody of the invention is an antibody with human isotype IgG1 and one of these three mutations in the Fc region. Other substitutions that reduce binding to Fc-gamma receptors are the E233P mutation (particularly in mouse IgG1) and D265A (particularly in mouse IgG2a). Other examples of mutations and combinations of mutations that reduce Fc and / or C1q binding are E318A / K320A / R322A (particularly in mouse IgG1), L235A / E318A / K320A / K322A (particularly in mouse IgG2a). Similarly, residue 241 (Ser) in human IgG4 can be replaced, for example, with proline to disrupt Fc binding.

[0214] Additional mutations can be made to the constant region to modulate effector activity. For example, mutations can be made in the IgG1 or IgG2 constant region at A330S, P331S, or both. For IgG4, mutations can be made at E233P, F234V, and L235A, with G236 deleted, or any combination thereof. IgG4 can also have one or both of the following mutations S228P and L235E. The use of disrupted constant region sequences to modulate effector function is further described, for example, in WO2006 / 118,959 and WO2006 / 036291.

[0215] Additional mutations can be made in the constant region of human IgG to modulate effector activity (see, for example, WO2006 / 03291). These include substitutions relative to human IgG1: (i) A327G, A330S, P331S; (ii) E233P, L234V, L235A, G236 deletion; (iii) E233P, L234V, L235A; (iv) E233P, L234V, L235A, G236 deletion, A327G, A330S, P331S; and (v) E233P, L234V, L235A, A327G, A330S, P331S; or particularly (vi) L234A, L235E, G237A, A330S and P331S (e.g. relative to human IgG1), where the numbering of residues in the Fc region is that of the EU index in Kabat. See also WO2004 / 029207, which is incorporated herein by reference.

[0216] The affinity of an antibody to Fc-gamma-R can be altered by mutating certain residues in the heavy chain constant region. For example, disruption of the glycosylation site of human IgG1 can reduce the Fc-gamma-R binding and thus the effector function of the antibody (see, for example, WO2006 / 036291). The tripeptide sequences NXS and NXT (X is any amino acid except proline) are enzyme recognition sites for glycosylation of N residues. Disruption of any of the tripeptide amino acids, especially in the CH2 region of IgG, prevents glycosylation at that site. For example, mutation of N297 of human IgG1 prevents glycosylation and reduces Fc-gamma-R binding to the antibody.

[0217] Although activation of ADCC and CDC is often desirable for therapeutic antibodies, there are circumstances in which it is preferential for the monospecific IgM antibodies of the invention or variants thereof to be unable to activate effector functions (e.g., the antibodies of the invention that are agnostic regulators). For these purposes, IgG4 has been commonly used, but it has fallen out of favor in recent years due to the unique ability of this subclass to undergo Fab arm exchange, where heavy chains can be swapped between IgG4 and residual ADCC activity in vivo. Thus, an Fc engineering approach can be used to determine the key interaction sites of the Fc domain with Fc-gamma receptors and C1q, and then mutate these positions, such as the Fc of the monospecific IgM antibodies of the invention or variants thereof, to reduce or abolish binding. Through alanine scanning, Duncan and Winter (1998; Nature 332:738) first isolated the binding site of C1q to the region covering the hinge and upper CH2 of the Fc domain. Genmab researchers identified the mutations K322A, L234A, and L235A, which in combination were sufficient to almost completely abolish Fc-gamma-R and C1q binding (Hezareh et al, 2001; J Virol 75:12161). Similarly, MedImmune later identified a set of three mutations, L234F / L235E / P331S (termed TM), with very similar effects (Oganesyan et al, 2008; Acta Crystallographica 64:700). An alternative approach is modification of the glycosylation on asparagine 297 of the Fc domain, known to be required for optimal FcR interaction.Loss of binding to Fc-gamma-R has been observed in N297 point mutations (Tao et al, 1989; J Immunol 143:2595), enzymatically deglycosylated Fc domains (Mimura et al, 2001; J Biol Chem 276:45539), recombinantly expressed antibodies in the presence of glycosylation inhibitors (Walker et al, 1989; Biochem J 259:347), and expression of the Fc domain in bacteria (Mazor et al 2007; Nat Biotechnol 25:563). Thus, the present invention also includes embodiments of monospecific oligomeric antibodies or variants thereof in which such techniques or mutations have been used to reduce effector function.

[0218] IgG naturally persists for long periods in (e.g., human) serum due to FcRn-mediated recycling, resulting in a typical half-life of approximately 21 days. Despite this, several efforts have been made to manipulate the pH-dependent interactions of the Fc domain with FcRn to increase affinity at pH 6.0 while maintaining minimal binding at pH 7.4. Researchers at PDL BioPharma identified the T250Q / M428L mutation that resulted in an approximately two-fold increase in IgG half-life in rhesus monkeys (Hinto et al, 2004; J Biol Chem 279:6213), and researchers at MedImmune identified the M252Y / S254T / T256E (referred to as YTE) mutation that resulted in an approximately four-fold increase in IgG half-life in cynomolgus monkeys (Dall'Acqua, et al 2006; J Biol Chem 281:23514). The combination of M252Y / S254T / T256E mutations with the point mutations H433K / N434F results in a similar effect (Vaccaro et al., 2005, Nat Biotechnol. Oct; 23(10): 1283-8). The ABP of the present invention may also be PEGylated. PEGylation, i.e. chemical conjugation with the synthetic polymer polyethylene glycol (PEG), has emerged as a recognized technique for the development of long-acting biologics, with approximately 10 clinically approved protein and peptide drugs to date (Jevsevar et al., 2010; Biotechnol J 5: 113). The monospecific oligomeric antibodies of the present invention or variants thereof may also be subjected to PASylation, a biological alternative to PEGylation to extend the plasma half-life of pharma- ceutical active proteins (Schlapschy et al., 2013; Protein Eng Des Sel 26: 489; XL-protein GmbH, Germany). Similarly, Amunix's XTEN half-life extension technology offers another biological alternative to PEGylation (Schellenberger, 2009, Nat Biotechnol.;27(12):1186-90. doi:10.1038 / nbt.1588).Thus, the invention also includes embodiments of antibodies in which such techniques or mutations have been used to extend serum half-life, particularly in human serum.

[0219] Antibody fragments include "Fab fragments," which consist of one constant domain and one variable domain of each of the heavy and light chains, held together by the adjacent constant region of the light chain and the first constant domain (CH1) of the heavy chain. These may be formed from conventional antibodies by protease digestion, for example with papain, although similar Fab fragments may also be produced by genetic engineering. Fab fragments include Fab', Fab, and "Fab-SH," which are Fab fragments that contain at least one free sulfhydryl group.

[0220] Fab' fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the first constant domain of the heavy chain including one or more cysteines from the antibody hinge region. Fab' fragments include "Fab'-SH", which are Fab' fragments containing at least one free sulfhydryl group.

[0221] Further, antibody fragments include F(ab')2 fragments that contain two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains ("hinge region"), resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, F(ab')2 fragments are composed of two Fab' fragments that are held together by disulfide bonds between the two heavy chains. F(ab')2 fragments can be prepared from conventional antibodies by proteolytic cleavage with an enzyme that cleaves below the hinge region, for example, by pepsin, or by genetic engineering.

[0222] The "Fv region" comprises the variable regions of both the heavy and light chains, but lacks the constant regions. A "single-chain antibody" or "scFv" is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain, which forms the antigen-binding region.

[0223] The "Fc region" comprises two heavy chain fragments containing the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0224] Thus, in some embodiments, the antibody of the invention is an antibody fragment selected from the list consisting of Fab', Fab, Fab'-SH, Fab-SH, Fv, scFv and F(ab')2.

[0225] In a preferred embodiment, an antibody of the invention is an antibody in which at least a portion of the framework sequences of the antibody or fragment thereof are human consensus framework sequences, e.g., an antibody comprising human germline-encoded framework sequences.

[0226] In certain other embodiments, the monospecific oligomeric antibodies of the invention or variants thereof are modified to increase their serum half-life, particularly in human serum. For example, the antibodies of the invention may be PEGylated and / or PASylated or have an Fc region with T250Q / M428L, H433K / N434F / Y436, or M252Y / S254T / T256E / H433K / N434F modifications.

[0227] In a preferred embodiment, an antibody of the invention may comprise at least one antibody constant domain, in particular, the at least one antibody constant domain is a CH1, CH2, or CH3 domain, or a combination thereof.

[0228] In further such embodiments, antibodies of the invention having an antibody constant domain include a mutated Fc region, e.g., to reduce interaction of the Fc region with an Fc receptor (an Fc receptor on an immune effector cell) (e.g., Saxena & Wu, 2016; Front Immunol 7:580). Examples and embodiments thereof are described elsewhere herein.

[0229] In other embodiments, the monospecific oligomeric antibodies or variants thereof of the invention may comprise an effector group and / or a label group. The term "effector group" refers to any group that acts as a cytotoxic agent, particularly a group attached to another molecule such as an antigen binding protein. Examples of suitable effector groups are radioisotopes or radionuclides. Other suitable effector groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable effector groups include calicheamicin, auristatin, geldanamycin, alpha-amanitin, pyrrolobenzodiazepines, and maytansine.

[0230] The term "label" or "label group" refers to any detectable label. Generally, labels are divided into various classes depending on the assay in which they are detected: a) isotopic labels, which may be radioactive or heavy isotopes, b) magnetic labels (e.g., magnetic particles), c) redox-active moieties, d) optical dyes, enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), e) biotinylation groups, and f) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).

[0231] Binding of a protective regulatory antibody, variant or fragment of the invention can restore, protect, maintain and / or prolong the biological function of a molecule comprising a target antigen in that binding of the protective regulatory antibody, variant or fragment of the invention competes with a function-limiting binding partner and / or prevents degradation of the molecule comprising the target antigen (see, e.g., Figure 16). In some embodiments, the protective regulatory antibody, variant or fragment of the invention binds reversibly to the target antigen.

[0232] Thus, the present invention is based, at least in part, on the surprising discovery that the protective regulatory antibodies, variants or fragments of the invention protect and / or regulate the function of an antigen by competing with the binding of an antigen-function-limiting antigen binding agent.

[0233] In certain embodiments, the present invention relates to a protective regulatory antibody, variant or fragment of the present invention, wherein the protective regulatory antibody, variant or fragment comprises: a) a variable light chain (VL) comprising a CDR3 defined in SEQ ID NO: 4 and a CDR3 defined in SEQ ID NO: 7; b) a variable heavy chain (VH) comprising a CDR3 defined in SEQ ID NO: 11 and a variable light chain (VL) comprising a CDR3 defined in SEQ ID NO: 14; or c) a variable heavy chain (VH) comprising a CDR3 defined in SEQ ID NO: 18 and a variable light chain (VL) comprising a CDR3 defined in SEQ ID NO: 21.

[0234] In certain embodiments, the invention provides a protective regulatory antibody, variant or fragment comprising: a) a variable heavy chain (VH) comprising CDR1 defined in SEQ ID NO:2, CDR2 defined in SEQ ID NO:3, and CDR3 defined in SEQ ID NO:4, and a variable light chain (VL) comprising CDR1 defined in SEQ ID NO:6, CDR2 defined in sequence DAS, and CDR3 defined in SEQ ID NO:7; b) a variable heavy chain (VH) comprising CDR1 defined in SEQ ID NO:9, CDR2 defined in SEQ ID NO:10, and CDR3 defined in SEQ ID NO:11, and or b) a variable heavy chain (VH) comprising a CDR1 defined in SEQ ID NO: 16, a CDR2 defined in SEQ ID NO: 17 and a CDR3 defined in SEQ ID NO: 18, and a variable light chain (VL) defined by CDR1 defined in SEQ ID NO: 20, CDR2 defined in sequence DAS and CDR3 defined in SEQ ID NO: 21.

[0235] In certain embodiments, the invention relates to a protective regulatory antibody, variant or fragment comprising: a) a variable heavy (VH) sequence comprising the amino acid sequence of SEQ ID NO:1, or a sequence having at least 90%, preferably at least 95%, sequence identity to SEQ ID NO:1; and an amino acid sequence of SEQ ID NO:4, or a sequence having at least 90%, preferably at least 95%, sequence identity to SEQ ID NO:4. In one embodiment of the present invention, the protective regulatory antibody, variant or fragment of the present invention comprises a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 8, or a sequence having at least 90%, preferably at least 95%, sequence identity to SEQ ID NO: 8, and a variable light chain (VL) sequence comprising an amino acid sequence of SEQ ID NO: 12, or a sequence having at least 90%, preferably at least 95% sequence identity to SEQ ID NO: 12, or c) a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 15, or a sequence having at least 90%, preferably at least 95% sequence identity to SEQ ID NO: 15, and a variable light chain (VL) sequence comprising an amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90%, preferably at least 95% sequence identity to SEQ ID NO: 19.

[0236] In one embodiment, the invention relates to a pharmaceutical composition of the invention, a pharmaceutical composition for use of the invention, or a method of treatment of the invention, wherein said IgM antibody comprises a variable heavy chain (VH) comprising a CDR1 sequence encoded by SEQ ID NO: 60, a CDR2 sequence encoded by SEQ ID NO: 61, and a CDR3 sequence encoded by SEQ ID NO: 62, and a variable heavy chain (VL) comprising a CDR1 sequence encoded by SEQ ID NO: 57, a CDR2 sequence encoded by GGTGCATCC, and a CDR3 sequence encoded by SEQ ID NO: 58.

[0237] In one embodiment, the invention relates to a pharmaceutical composition of the invention, a pharmaceutical composition for use of the invention, or a method of treatment of the invention, wherein the IgM antibody comprises: a variable heavy chain (VH) sequence comprising an amino acid sequence encoded by a sequence defined by SEQ ID NO:59 or by a sequence having at least 90% sequence identity to SEQ ID NO:59, preferably at least 95% sequence identity to SEQ ID NO:59; and a variable light chain (VL) comprising an amino acid sequence encoded by a sequence defined by SEQ ID NO:56 or by a sequence having at least 90% sequence identity to SEQ ID NO:56, preferably at least 95% sequence identity to SEQ ID NO:56.

[0238] In one embodiment, the invention relates to a host cell comprising a polynucleotide having: a) a sequence defined by SEQ ID NO: 59, or a sequence having at least 90% sequence identity to SEQ ID NO: 59, preferably at least 95% sequence identity to SEQ ID NO: 59; and / or b) a sequence defined by SEQ ID NO: 56, or a sequence having at least 90% sequence identity to SEQ ID NO: 56, preferably at least 95% sequence identity to SEQ ID NO: 56, wherein said polynucleotide further encodes an IgM constant region, and / or said host cell comprises a further polynucleotide encoding an IgM constant region.

[0239] In one embodiment, the present invention relates to a method of providing an IgM antibody, said method comprising the steps of a) culturing the host cell of embodiment 22; and b) isolating the IgM antibody.

[0240] With respect to a reference polypeptide sequence, "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0241] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity, specificity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, so long as the final construct has the desired properties, e.g., antigen binding.

[0242] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. The amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved target antigen binding, reduced immunogenicity, or altered ADCC or CDC.

[0243] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a certain biological property (e.g., increased affinity, increased specificity, increased protective properties, decreased immunogenicity) compared to the parent antibody and / or will substantially retain a certain biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity or specificity).

[0244] For example, modifications (e.g., substitutions) may be made in the CDRs to improve antibody affinity. Such modifications may be made in CDR "hot spots", i.e., residues encoded by codons that undergo high frequency of mutation during the somatic maturation process (see, e.g., Chowdhury, 2008, Methods Mol. Biol. 207:179-196), and / or in the SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is described, for example, in Hoogenboom et al., 2002 in Methods in Molecular Biology 178:1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-directed approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted. In another embodiment, look-through mutagenesis is used to optimize antibody affinity by a multidimensional mutagenesis method that simultaneously evaluates and optimizes combinatorial mutations of selected amino acids (Rajpal, Arvind et al., 2005, Proceedings of the National Academy of Sciences of the United States of America vol. 102, 24: 8466-71).

[0245] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions) that do not substantially reduce binding affinity and / or specificity may be made in the CDRs. Such modifications may be outside the CDR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0246] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells, 1989, Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0247] In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0248] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., 1997, TIBTECH 15:26-32. The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stalk" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to form antibody variants with specific improved properties.

[0249] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fe region residues), although Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to a small number of sequence variations in antibodies. Such fucosylation variants may have altered effects on inflammation (Irvine, Edward B, and Galit Alter., 2020, Glycobiology vol. 30, 4: 241-253). See, e.g., US2003 / 0157108, US2004 / 0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 01321 40, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. 2004 J. Mol. Biol. 336: 1239-1249, Yamane-Ohnuki et al., 2004, Biotech. Bioeng. 87: 614.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., 1986, Arch. Biochem. Biophys. 249:533-545, US2003 / 0157108, and WO2004 / 056312, especially Example 11), and alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al., 2004, Biotech. Bioeng. 87:614, Kanda, Y. et al., 2006, Biotechnol. Bioeng., 94(4):680-688, and WO2003 / 085107).

[0250] The antibody variant further comprises a bisected oligosaccharide, for example, where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such an antibody variant may have altered fucosylation and / or altered effects on inflammation (Irvine, Edward B, and Galit Alter., 2020, Glycobiology vol. 30, 4: 241-253). Examples of such antibody variants are described, for example, in WO2003 / 011878, U.S. Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0251] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0252] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), involved in the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol. 117:587 and Kirn et al., 1994 J. Immunol. 24:249) have been described in US2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, such as a substitution at Fc region residue 434 (US 2006 / 0194291).

[0253] In certain embodiments, it may be desirable to form cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, e.g., drug moieties or linker-drug moieties, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in US7521541.

[0254] In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0255] In certain embodiments, the present invention relates to an antibody, or an antigen-binding fragment thereof, comprising at least one of the above sequences, wherein the antigen-binding fragment is a Fab fragment, a F(ab') fragment, or an Fv fragment.

[0256] Binding of a protective regulatory antibody comprising a sequence described herein can restore, protect, maintain and / or prolong the biological function of insulin or a variant or fragment thereof in that binding of a protective regulatory antibody, variant or fragment of the invention competes with a functionally restricted binding partner and / or prevents degradation of insulin or a variant or fragment thereof. In some embodiments, the protective regulatory antibody, variant or fragment of the invention binds reversibly to insulin or a variant or fragment thereof.

[0257] Thus, the present invention is based, at least in part, on the surprising discovery that the protective and / or modulating antibodies, variants or fragments of the invention comprising the sequences described herein protect and / or modulate the function of antigens, in particular insulin, by competing with the binding of antigen-function-limiting antigen binding agents.

[0258] In certain embodiments, the present invention relates to polynucleotides encoding the protective regulatory antibodies, variants or fragments of the present invention.

[0259] The term "polynucleotide" as used herein refers to a nucleic acid sequence. The nucleic acid sequence may be a DNA or RNA sequence, preferably the nucleic acid sequence is a DNA sequence. The polynucleotides of the present invention either consist essentially of or comprise the aforementioned nucleic acid sequences. Thus, they may also comprise additional nucleic acid sequences. The polynucleotides of the present invention are preferably provided either as isolated polynucleotides (i.e., isolated from their natural context) or in genetically modified form. The isolated polynucleotides referred to herein also encompass polynucleotides present in their natural cellular context, i.e., a cellular context other than a heterologous polynucleotide. The term polynucleotide encompasses single-stranded and double-stranded polynucleotides. Furthermore, chemically modified polynucleotides, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides such as biotinylated polynucleotides, are also included.

[0260] In one embodiment, a polynucleotide of the invention encodes at least one of the variable heavy (VH) and / or variable light (VL) sequences of a protective regulatory antibody according to the invention.

[0261] In certain embodiments, the present invention relates to polynucleotide sequences encoding variable heavy (VH) sequences comprising the nucleotide sequence of SEQ ID NO:22 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:22, preferably comprising the sequences of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.

[0262] In certain embodiments, the present invention relates to a polynucleotide sequence encoding a variable light chain (VL) sequence comprising the nucleotide sequence of SEQ ID NO:26 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:26, preferably comprising the sequences of SEQ ID NO:27, GATGCATCC and SEQ ID NO:28.

[0263] In certain embodiments, the present invention relates to polynucleotide sequences encoding a) variable heavy chain (VH) sequences comprising the nucleotide sequence of SEQ ID NO:22 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:22, preferably comprising the sequences of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, and b) variable light chain (VL) sequences comprising the nucleotide sequence of SEQ ID NO:26 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:26, preferably comprising the sequences of SEQ ID NO:27, GATGCATCC and SEQ ID NO:28.

[0264] In certain embodiments, the present invention relates to polynucleotide sequences encoding variable heavy (VH) sequences comprising the nucleotide sequence of SEQ ID NO:29 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:29, preferably comprising the sequences of SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0265] In certain embodiments, the present invention relates to a polynucleotide sequence encoding a variable light chain (VL) sequence comprising the nucleotide sequence of SEQ ID NO: 33 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33, preferably comprising the sequences of SEQ ID NO: 34, GGTGCATCC and SEQ ID NO: 35.

[0266] In certain embodiments, the present invention relates to polynucleotide sequences encoding a) a variable heavy chain (VH) sequence comprising the nucleotide sequence of SEQ ID NO:29 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:29, preferably comprising the sequences of SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, and b) a variable light chain (VL) sequence comprising the nucleotide sequence of SEQ ID NO:33 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:33, preferably comprising the sequences of SEQ ID NO:34, GGTGCATCC and SEQ ID NO:35.

[0267] In certain embodiments, the present invention relates to polynucleotide sequences encoding variable heavy (VH) sequences comprising the nucleotide sequence of SEQ ID NO:36 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:36, preferably comprising the sequences of SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39.

[0268] In certain embodiments, the present invention relates to a polynucleotide sequence encoding a variable light chain (VL) sequence comprising the nucleotide sequence of SEQ ID NO: 40 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 40, preferably comprising the sequences of SEQ ID NO: 41, GATGCATCC and SEQ ID NO: 42.

[0269] In certain embodiments, the present invention relates to polynucleotide sequences encoding a) a variable heavy chain (VH) sequence comprising the nucleotide sequence of SEQ ID NO: 36 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 36, preferably comprising the sequences of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, and b) a variable light chain (VL) sequence comprising the nucleotide sequence of SEQ ID NO: 40 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 40, preferably comprising the sequences of SEQ ID NO: 41, GATGCATCC and SEQ ID NO: 42.

[0270] In some embodiments, a polynucleotide of the present invention is operably linked to another nucleic acid sequence, for example, a transcriptional regulatory sequence is operably linked to a polynucleotide of the present invention.

[0271] In certain embodiments, the present invention relates to a vector comprising a polynucleotide of the present invention. do.

[0272] The term "vector" as used herein refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally linked to, i.e., inserted into, the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.

[0273] In some embodiments, the vectors of the present invention are transfected with the support of a transfection enhancer, for example, a transfection enhancer selected from the group of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides.

[0274] The present invention is therefore based at least in part on the surprising discovery that vectors of the invention allow expression of antibodies, variants or fragments that protect and / or modulate the function of a target antigen, in particular insulin, by competing with the binding of an antigen-function-limiting antigen binding agent.

[0275] In certain embodiments, the present invention relates to host cells comprising the polynucleotides of the present invention.

[0276] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0277] In certain embodiments, the host cells are used directly or indirectly in therapy (e.g., cell therapy). In certain embodiments, the method for cell therapy includes (i) obtaining cells from a subject, (ii) transforming the cells using a tool (e.g., a vector) comprising a polynucleotide of the invention and / or transforming the cells to produce an antibody of the invention, and (iii) administering the transformed cells to the subject. In certain embodiments, the subject in step (i) and step (iii) of the cell therapy method is the same subject. In certain embodiments, the subject in step (i) and step (iii) of the cell therapy method is different subjects. In certain embodiments, the subject in step (i) and step (iii) of the cell therapy method is different subjects belonging to different species. In certain embodiments, the subject in step (i) of the cell therapy method is a subject of the genus Sus and the subject in step (iii) of the cell therapy method is a subject of the species Homo sapiens.

[0278] In certain embodiments, the host cell is a stem cell. In other embodiments, the host cell is a differentiated cell.

[0279] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Val. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli.)

[0280] Thus, the present invention is based at least in part on the surprising discovery that the host cells of the invention allow the production of antibodies, variants or fragments that protect and / or modulate the function of a target antigen, in particular insulin, by competing with the binding of an antigen-function-limiting antigen binding agent.

[0281] In certain embodiments, the invention relates to methods for producing antibodies comprising culturing a host cell of the invention.

[0282] In certain embodiments, the invention relates to a method for producing an antibody comprising culturing a host cell of the invention, wherein the host cell comprises a polynucleotide of the invention.

[0283] In certain embodiments, the method for producing an antibody comprises culturing a host cell of the invention under suitable conditions to allow efficient production of an antibody of the invention.

[0284] In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody of the invention and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody of the invention and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody of the invention. In one embodiment, the host cell is a eukaryote, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment is a method of making an antibody, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0285] For recombinant production of an antibody (e.g., a protective regulatory antibody) according to the invention, nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0286] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US5648237, US5789199, and US5840523; Charlton, 2003, Methods in Molecular Biology, Vol. 248; BKC Lo, 2003, Humana Press, pp. 245-254. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0287] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215.

[0288] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0289] Plant cell cultures can also be used as hosts, see, e.g., US5959177, US6040498, US6420548, US7125978, and US6417429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0290] Vertebrate cells may also be used as hosts, for example mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are macaque kidney CV1 lines transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells described in Graham et al., 1997, J. Gen Viral. 36:59); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, 1980, Biol. Reprod. 23:243-251); macaque kidney cells (CV1); African green macaque kidney cells (VER0-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (e.g., as described in Mather et al., 1982, Annals NY Aead. Sei. 383:44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., 1980, Proc. Natl. Acad. Sc. USA 77:4216); and myeloma cell lines such as YO, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 BKC Lo, 2003., Humana Press, pp. 255-268.

[0291] The amount of specific antibody obtained can be quantified using ELISA, which is also described below. Further methods for the production of antibodies are well known in the art, see, for example, Harlow and Lane, 1988, CSH Press, Cold Spring Harbor.

[0292] The present invention is therefore based at least in part on the surprising discovery that the production methods of the invention allow for the generation of antibodies, variants or fragments that protect and / or modulate the function of a target antigen, in particular insulin, by competing with the binding of an antigen-function-limiting antigen binding agent.

[0293] In certain embodiments, the present invention relates to a composition of the invention further comprising a protective modulatory antibody of the invention, a variant or fragment of the invention, and / or a vector of the invention.

[0294] The addition of a protective regulatory antibody, variant or fragment of the invention and / or a vector of the invention may enhance the effect of a composition of the invention, shorten the time to onset of the effect of a composition of the invention, and / or reduce the dependency of the effect of a composition of the invention on endogenous protective regulatory antibody production.

[0295] Thus, the present invention is based, at least in part, on the surprising discovery that protective regulatory antibodies, variants or fragments of the invention can support the effect of compositions of the invention.

[0296] In certain embodiments, the present invention relates to a pharmaceutical product comprising a therapeutic agent and a) a composition of the present invention, b) a protective regulatory antibody, variant or fragment of the present invention, c) a vector of the present invention, and / or d) a monovalent antigen particle, where the monovalent antigen particle is composed of an antigenic moiety comprising no more than one antigenic structure capable of inducing an antibody-mediated immune response against a target antigen, and the therapeutic agent is the target antigen.

[0297] In certain embodiments, the present invention relates to a pharmaceutical product comprising a therapeutic agent and a) a composition of the present invention, b) a protective regulatory antibody, variant or fragment of the present invention, and / or c) a vector of the present invention, wherein the therapeutic agent is a target antigen.

[0298] The term "therapeutic agent," as used herein, refers to a compound that provides a therapeutic benefit to a subject when administered to a subject in a therapeutically effective dose. A therapeutic agent can be any type of drug, pharmaceutical, pharmaceutical, hormone, antibiotic, protein, gene, growth factor, bioactive substance used to treat, control, or prevent a disease or medical condition. One of skill in the art will appreciate that the term "therapeutic agent" is not limited to drugs that have received regulatory approval.

[0299] In some embodiments, the therapeutic agent may be selected from the group of small molecule drugs, proteins / polypeptides, antibodies, molecular drugs with antibiotic activity, phage-based therapies, nucleic acid molecules, and siRNAs.

[0300] The protective modulatory effect of the binding of an antibody, fragment or variant contained in a pharmaceutical product, or induced by a component of the pharmaceutical product, can improve the pharmacokinetic and pharmacodynamic properties of a therapeutic agent. In some embodiments, the pharmaceutical product is insulin or a variant or fragment thereof and a protective regulatory antibody comprising: a) a variable heavy chain (VH) comprising CDR1 defined in SEQ ID NO:2, CDR2 defined in SEQ ID NO:3, and CDR3 defined in SEQ ID NO:4, and a variable light chain (VL) comprising CDR1 defined in SEQ ID NO:6, CDR2 defined in sequence DAS, and CDR3 defined in SEQ ID NO:7; b) a variable heavy chain (VH) comprising CDR1 defined in SEQ ID NO:9, CDR2 defined in SEQ ID NO:10, and CDR3 defined in SEQ ID NO:11, and a variable light chain (VL) comprising CDR1 defined in SEQ ID NO:13, CDR2 defined in sequence GAS, and CDR3 defined in SEQ ID NO:14; or c) a variable heavy chain (VH) comprising CDR1 defined in SEQ ID NO:16, CDR2 defined in SEQ ID NO:17, and CDR3 defined in SEQ ID NO:18, and a variable light chain (VL) comprising CDR1 defined in SEQ ID NO:20, CDR2 defined in sequence DAS, and CDR3 defined in SEQ ID NO:21. and a variable light chain (VL) having an amino acid sequence of SEQ ID NO: 1 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 1; more preferably d) a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 1 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 1, and a variable light chain (VL) sequence comprising an amino acid sequence of SEQ ID NO: 4 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 4; e) a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 8, and a variable light chain (VL) sequence comprising an amino acid sequence of SEQ ID NO: 12 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 12; or f) a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90%, preferably at least 95% sequence identity with SEQ ID NO: 15, and an amino acid sequence of SEQ ID NO: 19 or a sequence having at least 90%,The present invention comprises an insulin or a variant or fragment thereof and a protective regulatory antibody, comprising a variable light chain (VL) sequence, preferably comprising a sequence having at least 95% sequence identity, or a variant or fragment of (a), b), c), d), e), and / or f), or a host cell or vector for expression of (a), b), c), d), e), and / or f), thereof. When administered to a subject, binding of the protective regulatory antibody can protect the insulin, insulin variant or insulin fragment against the subject's immune response.

[0301] Thus, the present invention is based, at least in part, on the surprising discovery that therapeutic agents can be protected and / or modulated as described herein.

[0302] In certain embodiments, the invention relates to a pharmaceutical product according to the invention, wherein the therapeutic agent is a therapeutic antibody.

[0303] The term "therapeutic antibody," as used herein, refers to a therapeutic agent described herein that is an antibody.

[0304] In some embodiments, the therapeutic antibody is selected from the group consisting of abagovomab, abciximab, avituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab avtansine, anifrolumab, ansuvimab, anrukinzumab, apolizumab, aprilumab ixadotin, arcitumomab, asculinba. Cumab, Acelizumab, Atezolizumab, Atidortoxumab, Atinumab, Atortivimab, Atorlimumab, Avelumab, Azintuxizumab vedotin, Bamlanivimab, Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bectumomab, Begelomab, Belantamab mafodotin, Belimumab, Bemarituzumab, Benralizumab, Berlimatoxumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Bicilomab, Bimagrumab, Bimekizumab, Virutamimab, Vivatuzumab Mab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontixutuzumab, burosumab, cabilalizumab, camidanlumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, casirivimab, capromab, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, sergituzumab amna Leukin, certolizumab pegol, cetrelimab, cetuximab, civisatamab, cimutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab peridotin, cortuximab ravtansine, conatumumab, concizumab, cosfrobiximab, crenezumab, crizanlizumab, clotezumab, CR6261, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab,Denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, dellotuximab biotin, detumomab, desamizumab, dinutuximab, dinutuximab beta, diridabumab, domagurozumab, dorlimov aritox, dostarlimab, drozitumab, DS-8201, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elci Rimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, encituximab, epcolitamab, epitumomab situxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, faretuz Mab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frobocimab, fulnevetomab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab ozogamicin, gevokizumab, gilvetomab, gimsilumab, Direntuximab, Glenbatumumab vedotin, Golimumab, Gomiliximab, Goslanemab, Guselkumab, Ianalumab, Ibalizumab, Sintilimab, Ibritumomab tiucetan, Icrucumab, Idarucizumab, Ifavotuzumab, Igovomab, Iradatuzumab vedotin, Imalumab, Iprelimab, Imusiromab, Imdevimab, Imgatuzumab, Inclamab, Indatuximab vedotin, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab,Iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacinotuzumab, ladiratuzumab vedotin, lampalizumab, lanadelumab, landgrozumab, laprituximab emtansine, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lenbervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifatuzumab vedotin, regelizumab, roncatuximab tesiri , rosatuximab vedotin, rilotomab satetraxetan, lintuzumab, lirilumab, roderucizumab, loxivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, rupartumab, rupartumab amadotin, rutikizumab, maftivimab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuab, milatuzumab, minretumomab, mirikizumab, mirvetuximab soravtansine, mitsumomab, modutuximab, mo Gamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab, Moxetumomab passudotox, Muromonab-CD3, Nacolomab butafenatox, Namilumab, Naputumomab estafenatox, Naratuximab emtansine, Narutumab, Natalizumab, Nabicixizumab, Nabivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001, Nelerimomab, Nesvacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomab merpentane, Oviltoxiximab, Obinutuzumab, Ocaratuzumab, ocrelizumab, odesivimab, odurimomab, ofatumumab, olaratumab, oleculumab, orendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, ombatilimab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab, palsatuzumab,Pascolizumab, pasotuximab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, plakmab, prezalumab, prozalizumab, pogalizumab, polatuzumab vedotin, ponezumab, polgabiximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ralpanizumab, ramucirumab, la Nevetomab, ranibizumab, raxibacumab, ravagalimab, ravatumumab, refanezumab, regavirumab, regdanvimab, relatilimab, lemtolumab, reslizumab, rilotumumab, rinukumab, risankizumab, rituximab, rivavazumab pegol, lobatumumab, Rmab, loredumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirin, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samolizumab, samlotamab vedotin, sarilumab, satra Lisumab, satumomab pendetide, secukinumab, cericlerumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, SGN-CD19A, SHP647, sifalimumab, siltuximab, simtuzumab, siplizumab, siltratumab vedotin, sirukumab, sofituzumab vedotin, solanezumab, solitomab, soneptizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, subizumab, sublatoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab , tafasitamab, talaxuzumab, talizumab, talquetamab, tamtubetomab, tanezumab, taplitumab paptox, talexuzumab, tabolimab, teclistamab, tefibazumab, terimomab alitox, terisotuzumab, terisotuzumab vedotin, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tiburizumab, tildrakizumab, tigatuzumab, timigtuzumab, timolumab, tiragolumab, tilagotumab, tislelizumab,Tisotumab vedotin, TNX-650, tocilizumab, tomzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, badastaximab butarilin, banalimab, bundletuzumab At least one antibody selected from the group consisting of vedotin, vanticumab, vanucizumab, bapaliximab, valisakumab, valiliumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumabmafodotin, votumumab, bunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenoctuzumab, diralimumab, zolbetuximab, and zolimomab.

[0305] Therapeutic antibodies are capable of inducing an immune response in a subject. The protective regulatory effect of the binding of an antibody, fragment or variant contained in a pharmaceutical product, or induced by a component of the pharmaceutical product, can improve the pharmacokinetic and pharmacodynamic properties of the therapeutic antibody by protecting against the immune response.

[0306] Thus, the present invention is based, at least in part, on the surprising discovery that therapeutic antibodies can be protected and / or modulated as described herein.

[0307] In certain embodiments, the invention relates to a composition of the invention, a protective modulatory antibody, variant or fragment of the invention, a vector of the invention, or a pharmaceutical product of the invention, further comprising a pharma- ceutically acceptable carrier.

[0308] Compositions or pharmaceutical products comprising the antibodies, variants or fragments thereof, vectors, host cells described herein can be prepared, in certain instances, by mixing such antibodies / variants / fragments / polynucleotides / host cells having the desired purity in the form of a lyophilized formulation or aqueous solution, with one or more optional pharma- ceutically acceptable carriers (Osol et al., 1980 Remington's Pharmaceutical Sciences 16th edition).

[0309] Exemplary lyophilized antibody compositions are described in US 6,267,958. Aqueous antibody compositions include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations including histidine-acetate buffers.

[0310] The active ingredients of the compositions / pharmaceutical products described herein and / or the antibodies / variants / fragments / vectors / host cells described herein may be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Osol et al., 1980, Remington's Pharmaceutical Sciences 16th edition.

[0311] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the compositions, pharmaceutical products, antibodies, variants, fragments, vectors, host cells and / or polynucleotides of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0312] In some embodiments, at least one component of the composition of the invention or pharmaceutical product of the invention is a modified release formulation that differs from another component, e.g., the multivalent antigen particles but not the monovalent antigen particles are bound to a release extending agent or vice versa.

[0313] In some embodiments, the present invention relates to a composition for use in inducing and / or modulating a cell-mediated target antigen-specific immune response in a subject, wherein the composition is used by contacting one or more immune cells of the subject with the composition.

[0314] In some embodiments, the compositions described herein are used to treat or prevent (vaccinate) a disease in a subject or patient, comprising administering to the subject or patient a therapeutically or prophylactically effective amount of the composition or at least one monovalent antigen particle or one multivalent antigen particle of the composition.

[0315] A therapeutically effective dose in the context of the present invention is an amount that induces or suppresses a specific B cell-mediated immune response, such as an IgG-type or IgM-type (or IgA) immune response.

[0316] In some embodiments, the present invention relates to a method for treating or preventing a disease in a subject by vaccination, the method comprising: (i) a monovalent antigen particle composed of an antigenic moiety comprising one or less of an antigenic structure capable of inducing an antibody-mediated immune response against a disease-associated antigen; (ii) a multivalent antigen particle that is comprised of an antigenic moiety that comprises two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, wherein two or more of the antigenic structures are covalently or non-covalently crosslinked.

[0317] In this embodiment, it may be preferable to administer the treatment to the subject in a vaccination scheme, including the prime / boost schemes disclosed elsewhere herein.

[0318] In some embodiments, the present invention relates to a vaccination composition for use in the treatment or prevention of a disease in a subject, the vaccination composition comprising: (iii) a monovalent antigen particle composed of an antigenic moiety that includes one or less of an antigenic structure capable of inducing an antibody-mediated immune response against a disease-associated antigen; (iv) a multivalent antigen particle comprised of an antigenic moiety comprising two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, wherein two or more of the antigenic structures are covalently or non-covalently cross-linked.

[0319] In some embodiments, the present invention provides a method for the preparation of a (v) a monovalent antigen particle composed of an antigenic moiety that includes one or less of an antigenic structure capable of inducing an antibody-mediated immune response against the antigen; (vi) a multivalent antigen particle composed of an antigenic moiety comprising two or more of antigenic structures capable of inducing an antibody-mediated immune response against the antigen, wherein two or more of the antigenic structures are covalently or non-covalently crosslinked.

[0320] In further embodiments, the compositions described herein are for use in the treatment or prevention (vaccination) of a disease in a subject or patient, comprising administering to the subject or patient a therapeutically or prophylactically effective amount of the composition or at least (i) or (ii) of the composition, in some embodiments, a therapeutically effective dose is an amount that induces or suppresses a specific B cell-mediated immune response, such as an IgG or IgM-type (or IgA) immune response.

[0321] In certain embodiments, the invention relates to a composition of the invention, a protective modulatory antibody, variant or fragment of the invention, a vector of the invention, or a pharmaceutical product of the invention for use as a medicament.

[0322] In some embodiments, the disease or condition treated by the medicament is selected from diseases or conditions characterized in that an increase or decrease in a cell-mediated immune response is beneficial for treatment.Thus, the invention provides for the herein described modulation of the immune system by the methods described herein as a treatment for a disease, such as a disease or condition selected from an inflammatory disorder, an autoimmune disease, a proliferative disorder, or an infectious disease.

[0323] In some embodiments, the compositions of the invention, the protective regulatory antibodies, variants or fragments of the invention, the vectors of the invention, or the pharmaceutical products of the invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the physician. The compositions of the invention, the protective regulatory antibodies, variants or fragments of the invention, the vectors of the invention, or the pharmaceutical products of the invention are optionally formulated with, but not necessarily, one or more additional therapeutic agents currently used to prevent or treat the disorder in question. The effective dose of such other agents depends on the amount of the compositions of the invention, the protective regulatory antibodies, variants or fragments of the invention, the vectors of the invention, or the pharmaceutical products of the invention, the type of disorder or treatment, and other factors related to the above considerations. These are generally used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined experimentally / clinically as appropriate.

[0324] The appropriate dosage of a composition of the invention, a protective modulatory antibody, variant or fragment of the invention, a vector of the invention, or a pharmaceutical product of the invention for the prevention or treatment of disease will depend (when used alone or in combination with one or more other additional therapeutic agents) on the type of disease being treated, the type of composition / antibody / variant / fragment / vector / pharmaceutical product, the severity and course of the disease, whether the administration is for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition / antibody / variant / fragment / vector / pharmaceutical product, and the judgment of the attending physician.

[0325] The protective regulatory antibodies, variants or fragments of the present invention, and / or antibodies used as additional therapeutic agents, are suitably administered to the patient once or over a series of treatments. In some embodiments, depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody variants or fragments may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors considered above. For repeated administration over several days or more, depending on the disease state, treatment will generally be continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody or antigen-binding fragment would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20, or such as about 6 doses of the antibody variant or fragment). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0326] In some embodiments, the present invention relates to monospecific oligomeric antibodies or variants thereof for use in the treatment of autoimmune diseases, wherein the monoclonal IgM type antibodies are specific and have high affinity for antigens associated with the autoimmune disease.

[0327] In some embodiments, the present invention relates to a composition, pharmaceutical product or vector of the present invention for use in therapy, wherein the vector is administered at a concentration of at least 10 per kilogram of subject body weight to achieve a therapeutic effect. 6 , 10 7 , 10 8 , 10 9 , 1010 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 The vector may be administered at a dose ranging from 100 mg / kg to 100 mg / kg of vector genome (vg / kg) or more.

[0328] In certain embodiments, the present invention relates to a composition, pharmaceutical product or host cell of the invention for use in therapy, comprising a clinically relevant number or population of host cells, e.g., at least 10 per dose. 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , typically 10 9 More than 10 cells or at least 10 10 of cells are administered. The number of cells depends on the type of cells as well as the intended use of the composition, pharmaceutical product or host cell of the invention. For the uses provided herein, the cells are typically in a volume of a liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically 10 6 More than 10 cells / ml, typically 7 cells / ml. A clinically relevant number of host cells is greater than 10 9 , 10 10 , or 10 11 The number of cells may be allocated to multiple infusions equal to or exceeding that number.

[0329] The total dose of the host cells of the invention for one treatment cycle is typically about 1×10 4 cells / kg~1×10 10 cells / kg host cells or more.

[0330] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the invention, as described herein, can be used to therapeutically modulate the immune response to a target antigen.

[0331] In certain embodiments, the present invention relates to a composition for use of the invention, a pharmaceutical product for use of the invention, a vector for use of the invention, or a protective regulatory antibody, variant or fragment for use of the invention for use in the treatment and / or prevention of a humoral target antigen-specific disease or disorder and / or a B cell-mediated target antigen-specific disease or disorder.

[0332] The term "B cell-mediated inflammatory disease", as used herein, refers to a disease or disorder whose pathogenesis and / or progression is primarily dependent on the activity of B cells and / or macromolecules of the immune system, such as antibodies and complement proteins.

[0333] The present invention provides means and methods for modulating humoral and / or B cell mediated immune responses, for example, by protective regulatory binding.

[0334] The present invention is therefore based, at least in part, on the surprising discovery that the means and methods of the invention can be used to therapeutically modulate a B cell-mediated immune response to a target antigen and / or to therapeutically modulate a humoral antigen, as described herein.

[0335] In certain embodiments, the present invention relates to a composition for use in the present invention, a pharmaceutical product for use in the present invention, a vector for use in the present invention, or a protective regulatory antibody, variant or fragment for use in the present invention, wherein the humoral target antigen-specific disease or / and B cell mediated target antigen-specific disease or disorder is an autoimmune disease or disorder or an alloimmune disease or disorder, preferably the target antigen is an autoantigen.

[0336] The term "autoantigen," as used herein, refers to an antigen or epitope that is native to a subject and is immunogenic in an autoimmune disease or disorder or an alloimmune disease or disorder.

[0337] The term "autoimmune disease" refers to a disease or disorder resulting from an immune response directed against an individual's own tissues, organs, or symptoms or conditions resulting therefrom. In some embodiments, the autoimmune disease or disorder described herein is a condition resulting from or exacerbated by the production of autoantibodies by B cells of antibodies reactive with normal body tissues and / or antigens. In other embodiments, the autoimmune disease is a disease involving the secretion of autoantibodies specific for epitopes from autoantigens.

[0338] In some embodiments, the autoimmune disease is selected from the group consisting of myocarditis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, subacute bacterial endocarditis, anti-glomerular basement membrane nephritis, lupus nephritis, interstitial cystitis, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, anti-synthetase syndrome, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, bullous dermalysis, erythema nodosum, pemphigus gestationis, hidradenitis suppurativa, lichen planus, lichen sclerosus, linear IgA disease, morphea. pemphigoid, pemphigus vulgaris, pityriasis lichenoides, Muscha-Habermann disease, psoriasis, systemic sclerosis, vitiligo, Addison's disease, autoimmune polysecretory syndrome (type 1, 2 or 3), autoimmune pancreatitis, diabetes mellitus, autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, celiac disease, Crohn's disease, esophageal achalasia, microscopic colitis, ulcerative colitis, antiphospholipid syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmune enteropathy, Epidemic neutropenia, Autoimmune microcytopenic purpura, Cold agglutinin disease, Essential mixed cryoglobulinemia, Evans syndrome, Pernicious anemia, Pure erythrocytosis, Thrombocytopenia, Painful steatosis, Adult Still's disease, Ankylosing spondylitis, Striated body syndrome, Drug-induced lupus, Periodontitis-associated arthritis, Eosinophilic fasciitis, Felty's syndrome, IgG4-related disease, Juvenile arthritis, Lyme disease (chronic), Mixed connective tissue disease, Relapsing rheumatoid arthritis, Parry-Romberg syndrome, Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis, Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, systemic lupus erythematosus, undifferentiated connective tissue disease, dermatomyositis, fibromyalgia, inclusion body myositis, myositis, myasthenia gravis, neuromyopathy, accessory cerebellar degeneration, polymyositis, acute disseminated encephalomyositis, acute motor axonal neuropathy, anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis, Baroconcentric sclerosis, Bickerstaff encephalitis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto's encephalopathy, idiopathic inflammatory demyelinating disease, Lambert-Eaton myasthenic syndrome, multiple sclerosis , Oshtrun's syndrome, Streptococcus-associated pediatric autoimmune neuropsychiatric disease, progressive inflammatory neuropathy, restless legs syndrome, stiff person syndrome, Sydenham's chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan's syndrome, Graves' eye disease, intermediate uveitis, lipoconjunctivitis, Mooren's ulcer, optic neuromyositis, opsoclonus-myoclonus syndrome, optic neuritis, sclerosis syndrome, Susac's syndrome, sympathetic ophthalmopathy, It refers to at least one disease or disorder selected from the group consisting of Tolosa-Hunt syndrome, autoimmune inner ear disease, Meniere's disease, Behcet's disease, eosinophilic granulomatosis with polyangiitis, giant cell arteritis, granulomatosis with polyangiitis, IgA vasculitis, Kawasaki disease, leukocytoclastic vasculitis, lupus vasculitis, rheumatic vasculitis, microscopic polyangiitis, polyarteritis nodosa, polymyalgia rheumatica, urticarial vasculitis, vasculitis, and primary immunodeficiency.

[0339] The term "alloimmune disease or disorder" as used herein refers to an immune response to a non-self antigen from a member of the same species. In some embodiments, the alloimmune disease or disorder is a disease or disorder selected from the group of transfusion reactions, hemolytic disease of the fetus, and / or neonatal and transplant rejection.

[0340] The present invention provides means and methods for modulating autoimmune or alloimmune responses, for example, through protective regulatory binding.

[0341] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the invention can be used to therapeutically modulate autoimmune and / or alloimmune responses against target antigens.

[0342] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the humoral target antigen-specific disease or disorder and / or a B cell mediated target antigen-specific disease or disorder, or an autoimmune disease or disorder, or an alloimmune disease or disorder is an antibody mediated disease or disorder.

[0343] The term "antibody-mediated disease or disorder" refers to an autoimmune disease or disorder or an alloimmune disease or disorder characterized by the presence of antibodies. In some embodiments, the antibodies present in the antibody-mediated disease or disorder are disease-specific antibodies.

[0344] In some embodiments, the antibody mediated disease or disorder described herein is at least one disease or disorder selected from the group consisting of Addison's disease, ankylosing spondylitis, Behcet's syndrome, celiac disease, congenital adrenal hyperplasia, dermatitis herpetiformis, Goodpasture's syndrome, Graves' disease, Hashimoto's disease, hereditary hemochromatosis, insulin-dependent diabetes mellitus, idiopathic glomerulonephritis, multiple sclerosis, myasthenia gravis, narcolepsy, psoriasis vulgaris, pemphigus vulgaris, rheumatoid arthritis, systemic lupus erythematosus, and sarcoidosis.

[0345] The present invention provides means and methods for modulating antibody expression and / or binding of antibodies of the immune system, for example by protective regulatory binding.

[0346] The present invention is thus based, at least in part, on the surprising discovery that the means and methods of the invention can be used to therapeutically modulate antibody-mediated autoimmune responses and / or antibody-mediated alloimmune responses against target antigens.

[0347] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the target antigen is insulin for use in the treatment of an insulin-related disease or disorder.

[0348] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the target antigen is insulin and the antibody-mediated disease or disorder is an insulin-related disease or disorder.

[0349] The term "insulin-related disease or disorder," as used herein, refers to any disease or disorder in which insulin production, insulin effect, insulin signaling, insulin distribution, insulin metabolism and / or insulin clearance are dysregulated.

[0350] In some embodiments, the insulin-related disease or disorder is at least one disease or disorder selected from the group of polycystic ovary syndrome, metabolic syndrome, and diabetes.

[0351] In some embodiments, the insulin-related disease or disorder is at least one disease or disorder associated with increased levels of at least one agent selected from the group: adrenaline, glucagon, cortisol, somatostatin.

[0352] In some embodiments, the insulin-related disease or disorder is at least one side effect of treatment with an insulin regulating agent, hi some embodiments, the insulin regulating agent is selected from the group of adrenaline, glucagon, steroids, and somatostatin.

[0353] The means and methods provided by the present invention allow the modulation of immune responses to insulin. Immune responses to insulin can occur in healthy subjects and / or patients and / or during insulin treatment. The inventors show that a wide range of insulin-related conditions can be affected by the means and methods of the present invention (see, for example, Figures 11, 12, 16, 20B, 20D, 20F, 22). Thus, the means and methods can improve the effect of administered and / or endogenous insulin and reduce any insulin-related disease or disorder.

[0354] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the present invention can be used to protect and / or regulate insulin function.

[0355] In certain embodiments, the present invention relates to a composition for use according to the invention, a pharmaceutical product for use according to the invention, or a protective regulatory antibody, variant or fragment for use according to the invention, -7 Less than 10 -8 Less than 10, more preferably -9 less than about 10 -10 ~about 10 -12 K in the range d binds to insulin.

[0356] The high affinity of the antibodies, variants or fragments of the invention for insulin allows them to bind efficiently in competition with other antibodies (eg, multispecific IgG antibodies of the immune system).

[0357] Thus, the present invention is based, at least in part, on the surprising discovery that the high affinity binding enabled by the means and methods of the present invention protects and / or regulates insulin function by competing with function-limited insulin binders.

[0358] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the insulin-related disease or disorder is diabetes or a symptom thereof.

[0359] The term "diabetes" as used herein refers to a disease or disorder characterized by hyperglycemia. In some embodiments, diabetes is diagnosed by a glucose level greater than 140 mg / dl, 150 mg / dl, 160 mg / dl, 170 mg / dl, 180 mg / dl, 190 mg / dl, 200 mg / dl, 210 mg / dl, or 220 mg / dl 2 hours after glucose ingestion (typically 75 g glucose) during an oral glucose tolerance test. In some embodiments, diabetes is diagnosed by a fasting glucose level greater than 100 mg / dl or 110 mg / dl.

[0360] Symptoms of diabetes include, but are not limited to, hyperglycemia, hypoinsulinemia, insulin resistance, polyuria, polydipsia, weight loss, ketoacidosis, glycosuria, fatigue, irritability, blurred vision, slow-healing sores, frequent infections (e.g., gum or skin infections and vaginal infections), and increased inflammation (e.g., chronic low-grade inflammation).

[0361] In certain embodiments, the present invention relates to a composition of the present invention, a pharmaceutical product of the present invention, a vector of the present invention, or a protective regulatory antibody, variant or fragment of the present invention, where the target antigen is insulin for use in enhancing insulin effect. Insulin effect can also be enhanced in patients or healthy subjects, where insulin effect is regulated by the antibody without necessarily inducing a disease or disorder. For example, the composition of the present invention, the pharmaceutical product of the present invention, the vector of the present invention, or the protective regulatory antibody, variant or fragment of the present invention, where the target antigen is insulin, can be used to increase weight gain, such as muscle gain. In some embodiments, enhancing insulin effect includes, but is not limited to, increased glucose uptake, increased DNA replication, increased protein synthesis, increased liposynthesis, increased fatty acid esterification, decreased lipolysis, induction of glycogen synthesis, decreased gluconeogenesis and glycogenolysis, decreased protein degradation, decreased autophagy, increased amino acid uptake, increased blood flow, increased hydrochloric acid secretion in the stomach, increased potassium uptake, and decreased renal sodium excretion.

[0362] The means and methods provided by the present invention allow for the modulation of immune responses to insulin, which may occur in any form of diabetes and in any form of insulin treatment. Thus, the means and methods can improve the effect of administered and / or endogenous insulin, for example, alleviating any insulin deficiency-related symptoms in diabetes.

[0363] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the present invention protect against and / or regulate dysregulated insulin function in diabetes.

[0364] In certain embodiments, the present invention relates to a composition for use in the present invention, a pharmaceutical product for use in the present invention, a vector for use in the present invention, or a protective regulatory antibody, variant or fragment for use in the present invention, wherein diabetes is selected from the group of type 1 diabetes, type 2 diabetes, and gestational diabetes.

[0365] The term "type 1 diabetes" as used herein refers to diabetes characterized primarily by decreased insulin production. Typically, type 1 diabetes is characterized by an autoimmune response that leads to damage to insulin-producing beta cells in the pancreas.

[0366] The term "type 2 diabetes" as used herein refers to diabetes that is primarily characterized by increased insulin resistance. Type 2 diabetes often occurs when insulin levels are normal or elevated, and appears to be due to the inability of tissues to respond appropriately to insulin. Most people with type 2 diabetes are obese.

[0367] The term "gestational diabetes" as used herein refers to diabetes during pregnancy. Gestational diabetes. Gestational diabetes conditions further include pregnancy-related conditions such as pre-eclampsia and conditions in children of mothers with gestational diabetes, including, but not limited to, growth abnormalities (e.g., macrosomia), impaired glucose homeostasis, jaundice, polycythemia, hypocalcemia, and hypomagnesemia. In some embodiments, gestational diabetes is diagnosed during pregnancy. In some embodiments, gestational diabetes is diagnosed before pregnancy.

[0368] The means and methods provided by the present invention allow for the modulation of the immune response to insulin. Since antibody types differ in their placental transfer capacity, the means and methods of the present invention allow for selective and / or simultaneous treatment of the mother and the fetus.

[0369] Immune responses to insulin occur especially during chronic insulin treatment. Thus, the means and methods can improve the therapeutic effects of type 1 diabetes, type 2 diabetes, and gestational diabetes.

[0370] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the present invention protect against and / or regulate dysregulated insulin function in type 1 diabetes, type 2 diabetes and gestational diabetes.

[0371] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the diabetes is type 1 diabetes.

[0372] The means and method provided by the present invention can regulate immune response to insulin.Immune response is considered to be an important factor in the pathology of type 1 diabetes, and the treatment of type 1 diabetes is particularly dependent on insulin.Therefore, the means and method can improve the therapeutic effect of type 1 diabetes.

[0373] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the present invention protect against and / or regulate dysregulated insulin function in type 1 diabetes.

[0374] In certain embodiments, the invention relates to a composition for use in the invention, a pharmaceutical product for use in the invention, a vector for use in the invention, or a protective regulatory antibody, variant or fragment for use in the invention, wherein the target antigen is a cancer-associated antigen, or a pathogen-associated antigen.

[0375] The term "cancer-associated antigen" as used herein refers to a protein or polypeptide antigen expressed by a cancer cell. In some embodiments, the cancer-associated antigen described herein is at least one selected from the group of surface proteins or polypeptides, nuclear proteins or glycoproteins of a cancer cell, or fragments thereof.

[0376] The term "pathogen-associated antigen" as used herein refers to a protein or polypeptide antigen expressed by a pathogen. A pathogen-associated antigen of the present invention can be any antigen expressed in, on or by a pathogen, such as a pathogenic virus or microorganism, preferably the pathogen is selected from a parasite, a unicellular eukaryote, a bacterium, a virus or a virion.

[0377] Thus, the means and methods are capable of modulating the immune response to pathology.

[0378] Thus, the present invention is based, at least in part, on the surprising discovery that means and methods can be used to increase and / or modulate the immune response to cancer or pathogens.

[0379] In certain embodiments, the present invention relates to a composition for use according to the invention, a pharmaceutical product for use according to the invention, a vector for use according to the invention or a protective regulatory antibody, variant or fragment for use according to the invention, wherein the humoral target antigen-specific disease or disorder and / or the B cell mediated target antigen-specific disease or disorder is an infectious disease, the target antigen is a pathogen-associated antigen, preferably the pathogen is at least one pathogen selected from the group of parasites, unicellular eukaryotes, bacteria, viruses and virions.

[0380] The term "parasite" as used herein refers to an organism that lives in or on a second organism. In some embodiments, the parasite described herein is a parasite selected from the group of ectoparasites, protozoan organisms, and helminths.

[0381] The term "virus" as used herein refers to an infectious agent that replicates only in living cells of an organism.In some embodiments, the virus described herein is a virus selected from the group of Adenoviridae, Anelloviridae, Arenaviridae, Astroviridae, Bunyaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Pneumoviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Rhabdovirus and Togaviridae.

[0382] In some embodiments, the unicellular eukaryote described herein is selected from the group of Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Giardia duodenalis, and Leishmania species.

[0383] The term "virion" as used herein refers to a viral nucleic acid core with a protein coat and optionally an outer envelope.

[0384] In some embodiments, the bacteria described herein are from a genus selected from Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia.

[0385] Thus, the means and methods of the present invention can be used to induce an immune response against an infectious agent (see, for example, FIG. 18).

[0386] Thus, the present invention is based, at least in part, on the surprising discovery that means and methods can be used to increase and / or modulate the immune response to infection.

[0387] In certain embodiments, the invention relates to a composition for use according to the invention or a pharmaceutical product for use according to the invention, wherein the treatment comprises administering monovalent antigen particles before multivalent antigen particles.

[0388] The means and methods of the various embodiments of the present invention in certain embodiments can be viewed as immunization methods for the generation of a particular desired antibody response in a vertebrate. In this context, preferred embodiments of the methods of the present invention include a prime / boost immunization scheme for a subject.

[0389] The term "priming" an immune response to an antigen refers to the administration to a subject of an immunogenic composition that induces a higher level of immune response to the antigen upon subsequent administration of the same or a second composition than the immune response obtained by administration of a single immunogenic composition.

[0390] The term "boosting" an immune response to an antigen refers to administering a second, boosting immunogenic composition to a subject following administration of a priming immunogenic composition. In one embodiment, the boosting administration of the immunogenic composition occurs about 2-27 weeks, preferably 1-10 weeks, more preferably 1-5 weeks, and most preferably about 3 weeks after administration of the priming dose.

[0391] In a preferred embodiment of the invention, the priming step is carried out with monovalent antigen particles composed of antigenic moieties comprising one or less of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, whereas the boosting step involves the administration of multivalent antigen particles composed of antigenic moieties comprising two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, where two or more of the antigenic structures are covalently or non-covalently cross-linked. In such a prime / boost embodiment of the invention, the antigenic structures used to induce the immune response in the prime and boost steps are the same antigenic structure.

[0392] In some embodiments of the invention, the boosting step may be carried out using a composition of monovalent and multivalent antigen particles as described herein in the first aspect of the invention.

[0393] Thus, the present invention is based, at least in part, on the surprising discovery that priming with monovalent antigen particles increases the immune response to multivalent antigen particles.

[0394] In certain embodiments, the invention relates to a composition for use according to the invention or a pharmaceutical product for use according to the invention, wherein the treatment and / or prevention comprises at least two administration times.

[0395] Thus, the components of the compositions / pharmaceutical products of the present invention can be administered at different time points to achieve a certain immune modulation (see, e.g., FIG. 19) or can be administered repeatedly to boost and achieve an enhanced effect (see, e.g., FIG. 16a, FIG. 3c).

[0396] Thus, the present invention is based, at least in part, on the surprising discovery that priming and / or boosting modulate the altered immune response elicited by the means and methods of the present invention.

[0397] In certain embodiments, the invention relates to a monovalent antigen particle, comprising an antigenic moiety that comprises not more than one antigenic structure capable of inducing an antibody-mediated immune response against a target antigen, a composition for use in the treatment and / or prevention of a disease characterized by (i) the presence of an immunoglobulin G (IgG) type antibody that binds to a target antigen, where the binding of the IgG type antibody reduces a function of the target antigen, and / or (ii) the presence of an intrinsic factor multivalent antigen particle, comprising an antigenic moiety that comprises two or more antigenic structures capable of inducing an antibody-mediated immune response against a target antigen, where two or more antigenic structures are cross-linked, the monovalent antigen particle comprising an antigenic moiety that comprises not more than one antigenic structure capable of inducing an antibody-mediated immune response against a target antigen, a composition for use in the invention, a vector for use in the invention, a protective regulatory antibody, variant or fragment for use in the invention, or a pharmaceutical product for use in the invention.

[0398] In an alternative aspect of the invention, there is provided a method for treating or preventing a disease in a subject characterized by the presence of antibodies other than IgG specific for a disease-associated antigen, the method comprising administering to the subject a therapeutically effective dose of monovalent antigen particles, the monovalent antigen particles being composed of an antigenic moiety comprising one or less of the antigenic structures capable of inducing an antibody-mediated immune response against the disease-associated antigen. Such a disorder in an alternative third aspect may, for example, be an IgE-mediated allergy.

[0399] The disease characterized by the presence of immunoglobulin G (IgG) type antibodies specific for disease-associated antigens is preferably a disease characterized by the presence of pathological IgG molecules, such as autoimmune and alloimmune IgG antibodies, in the serum of a subject. Thus, the term "IgG-mediated disease" includes autoimmune and alloimmune diseases. As used herein, the term "alloimmune disease" refers to a case where there is a host immune response to a foreign antigen (e.g., major histocompatibility alloantigen or small histocompatibility alloantigen) of another individual, such as a host-versus-graft rejection reaction, or alternatively, a graft-versus-host disease, in which transplanted immune cells mediate harmful effects on the host receiving the transplant.

[0400] In some embodiments, the present invention relates to a monovalent antigen particle for use in the treatment or prevention of a disease characterized by the presence of immunoglobulin G (IgG) type antibodies specific for a disease-associated antigen in a subject, the monovalent antigen particle being composed of an antigenic moiety comprising one or less of antigenic structures capable of inducing an antibody-mediated immune response against the disease-associated antigen.

[0401] In some embodiments, the disease characterized by the presence of immunoglobulin G (IgG) type antibodies is selected from the group consisting of Mikulicz's disease, chronic sclerosing sialadenitis, Kutner's tumor, IgG4-related eye disease, IgG4-related pharyngitis, IgG4-related thyroid disease, IgG4-related hypophysitis, IgG4-related panhypophysitis, IgG4-related adenohypophysitis, IgG4-related posterior infundibulohypophysitis, IgG4-related pachymeningitis, IgG4-related leptomeningitis, IgG4-related pancreatitis, IgG4-related pulmonary disease, IgG4-related pleuritis, IgG4-related liver disease, IgG4-related sclerosing cholangitis, IgG4-related ... The disease is selected from the group consisting of IgG4-related ductitis, IgG4-related cholecystitis, IgG4-related aortitis, IgG4-related periaortitis, IgG4-related periarteritis, IgG4-related pericardiitis, IgG4-related mediastinitis, IgG4-related retroperitoneal fibrosis, IgG4-related mesenteritis, IgG4-related mastitis, IgG4-related kidney disease, IgG4-related prostatitis, IgG4-related perivascular fibrosis, IgG4-related paratesticular pseudotumor, IgG4-related epididymitis, IgG4-related lymphadenopathy, IgG4-related skin disease and IgG4-related perineural disease.

[0402] Thus, components of the means and methods described herein can be administered in diseases where some of the compositions of the invention are already present (e.g. based on disease pathology) to modulate the immune response according to Figure 18. Thus, in diseases where IgG and / or multivalent antigen particles are present, administration of monovalent particles and / or protective regulatory antibodies, variants or fragments and / or vectors as described herein may be sufficient. Alternatively, a composition or pharmaceutical product of the invention comprising a corresponding dose of monovalent particles and / or protective regulatory antibodies, variants or fragments and / or vectors as described herein may be sufficient.

[0403] Thus, the present invention is based, at least in part, on the surprising discovery that monovalent antigen particles can support protective regulatory antibody expression.

[0404] In certain embodiments, the present invention relates to monovalent antigen particles for use according to the invention, to a pharmaceutical product for use according to the invention, or to a composition for use according to the invention, wherein the treatment and / or prevention is carried out in a (tissue) content ratio of monovalent antigen particles:multivalent antigen particles of more than 1, preferably more than 10 1 More than 10, more preferably 2 More than 10, more preferably 3 More than 10, more preferably 4 This includes the use of monovalent antigen particles at doses exceeding

[0405] Thus, the (tissue) content of the multivalent antigen particles is determined by any method known to the skilled artisan. The monovalent antigen particles (or compositions / pharmaceutical products comprising the monovalent antigen particles) are administered at an appropriate dose to achieve the desired (tissue) content. The pharmacological and / or pharmacokinetic properties of the monovalent particles, as well as multivalent antigen particle-related parameters, subject-related parameters, and / or disease-related parameters may be taken into account.

[0406] Thus, the present invention is based, at least in part, on the surprising discovery that high monovalent:polyvalent antigen particle ratios can support protective regulatory antibody expression.

[0407] In certain embodiments, the invention relates to a multivalent antigen particle, a pharmaceutical product for use in the treatment and / or prevention of a disease, characterized by the presence of (i) an oligomeric antibody that binds to a target antigen, whereby the binding of the oligomeric antibody protects the function of the target antigen; and / or (ii) a monovalent antigen particle, whereby the monovalent antigen particle is composed of an antigenic moiety that comprises not more than one antigenic structure capable of inducing an antibody-mediated immune response against the target antigen, whereby two or more antigenic structures are cross-linked, a pharmaceutical product for use in the invention, or a composition for use in the invention.

[0408] In certain diseases or disorders, the protective regulatory effect of endogenous IgM antibodies may be undesirable (e.g., cytokine protection in inflammatory diseases). Multivalent antigen particles or means and methods of the invention comprising multivalent antigen particles can be used to modulate the immune response and suppress IgM antibody production or increase competitively binding antibodies. Immune responses in diseases or disorders characterized by the presence of monovalent antigen particles can be modulated by multivalent antigen particles or means and methods of the invention comprising multivalent antigen particles.

[0409] Thus, the present invention is based, at least in part, on the surprising discovery that monovalent antigen particles can support protective regulatory antibody expression.

[0410] In certain embodiments, the present invention relates to a vector for use of the invention, a pharmaceutical product for use of the invention, or a protective regulatory antibody, variant or fragment for use of the invention for use in the treatment and / or prevention of a disease or disorder in a subject with reduced IgD-type levels.

[0411] The term "patient" or "subject" as used herein refers to any animal classified as a mammal, suffering from a disorder or disease, including, but not limited to, domestic and farm animals, primates and humans, such as humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, a patient is a human, male or female, of any age or race.

[0412] Endogenous IgM expression and / or maturation is reduced in subjects with IgD-type antibody expression (see, e.g., Examples 8-11). Thus, the effects of the means and methods of the present invention are particularly pronounced in this subject population, especially when the means and methods include or induce the expression of protective regulatory antibodies, variants or fragments.

[0413] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the invention are capable of modulating immune responses in subjects with reduced endogenous protective regulatory antibody production / maturation.

[0414] In certain embodiments, the invention relates to a vector for use of the invention, a pharmaceutical product for use of the invention, or a protective regulatory antibody, variant or fragment for use of the invention for use in the treatment and / or prevention of a disease or disorder in a subject having an IgD-type antibody-related gene deficiency.

[0415] The term "IgD-type antibody-related gene deficiency," as used herein, refers to any disease or disorder in which the expression, production, and / or function of IgD-type antibodies is reduced.

[0416] Endogenous IgM expression and / or maturation is reduced in subjects with IgD-type antibody-related gene deficiencies (see, e.g., Examples 8-11). Thus, the effects of the means and methods of the present invention are particularly pronounced in this subject population, especially when the means and methods include or induce the expression of protective regulatory antibodies, variants or fragments.

[0417] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the invention are capable of modulating immune responses in subjects with reduced endogenous protective regulatory antibody production / maturation.

[0418] In certain embodiments, the present invention relates to a vector for use of the invention, a pharmaceutical product for use of the invention, or a protective regulatory antibody, variant or fragment for use of the invention for use in the treatment and / or prevention of a disease or disorder in a pediatric subject, preferably a pediatric subject under the age of 11.

[0419] The term "pediatric subject," as used herein, refers to a subject under the age of 18, 17, 16, 15, 14, 13, 12, 11, or 10. In some embodiments, a pediatric subject is a subject with a reduced proportion of mature B cells.

[0420] Endogenous IgM expression and / or maturation is reduced in pediatric subjects due at least in part to incomplete development and / or ratios of required B cell types. Thus, the effects of the means and methods of the invention are particularly pronounced in this subject population, especially when the means and methods include or induce the expression of protective regulatory antibodies, variants or fragments.

[0421] Thus, the present invention is based, at least in part, on the surprising discovery that the means and methods of the invention are capable of modulating immune responses in subjects with defective development of endogenous protective regulatory antibody production / maturation.

[0422] The terms "of the invention," "according to the invention," "according to the present invention," and the like, when used herein, are intended to refer to all embodiments of the invention as described and / or claimed herein.

[0423] As used herein, the term "comprising" encompasses both "including" and "consisting of," and both meanings are specifically intended and should therefore be interpreted as encompassing each individually disclosed embodiment according to the present invention. As used herein, the term "and / or" should be understood as a specific disclosure of each of the two specific features or components with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±20%, ±15%, ±10%, and for example ±5%. As will be understood by a person skilled in the art, the specific such deviation from the numerical value of a given technical effect depends on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater such deviation than one of a man-made or engineered technical effect. As will be appreciated by one of ordinary skill in the art, the particular such deviation for the numerical value of a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater such deviation than one of a man-made or engineered technical effect. When an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes the plural of that noun unless specifically stated otherwise.

[0424] It should be understood that application of the teachings of the present invention to a particular problem or environment, and modifications of the present invention or the inclusion of additional features thereto (such as further aspects and embodiments), are within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0425] In particular, each definition provided, as well as a particular embodiment described in the context of one aspect of the invention, applies equally to other aspects of the invention.

[0426] Unless otherwise indicated by context, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0427] Unless otherwise specified, the general methods and techniques described herein may 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 the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d 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).

[0428] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0429] [Figure 1]Figure 2 shows that soluble haptens inhibit the antibody immune response induced by hapten-carrier complexes. a: Schematic wild-type B cells expressing IgM (green) and IgD (yellow) B cell receptors. b: Serum anti-NP-Ig titers of immunized NP-KLH (red and green) and CI mice (grey) measured by ELISA on the indicated days. The indicated ratios refer to the molar ratio of soluble to complexed NP (sNP:cNP). Points represent mice, mean ± SD. c: Serum anti-KLH-IgG titers measured by ELISA on the indicated days. Points represent mice, mean ± SD. d: ELISpot assay showing NP-specific immunoglobulin producing cells. n=2 / group, mean ± SD. e: Schematic IgD BCR knockout B cells. f: Serum anti-NP-Ig titers of immunized NP-KLH (red and green) and CI mice measured by ELISA on the indicated days (IgD- / - mice). Points represent mice, mean ± SD. CI: control immunization. [Diagram 2] A very high ratio of soluble to complexed NPs indicates suppression of antigen-specific IgM responses. a: Scheme showing 4-hydroxy-3-nitrophenylacetyl hapten solubilized or conjugated to keyhole limpet hemocyanin (KLH). b: Scheme showing immunization schedule with soluble / complexed NPs and CpG-ODN1826. c: Antibody titers of NP-injected mice were analyzed via ELISA. Serum was plated in duplicate on NP-BSA coated plates and serially diluted 1:3. [Diagram 3]Figure 1 shows that the induction of autoantibodies depends on the autoantigen titer and is regulated by the ratio. a: Scheme of full-length CP derived from proinsulin bound to KLH carrier. b: Table comparing human and mouse CP and insulin-A chain amino acid sequences. The sequences used as peptides are underlined, conserved amino acids are in bold. c: Schematic immunization schedule. d-e: Serum anti-CP-Ig titers of immunized CP-SAV (red and green) and CI mice (grey) measured by ELISA on the indicated days. The boost on d42 was performed without CpG (e). Points represent mice, mean ± SD. f: ELISpot assay showing CP-specific immunoglobulin-producing spleen-derived cells on d14. The top lane shows a representative image of a well. n=4 mice / group, mean ± SD. g: Serum anti-CP-Ig titers of immunized CP-SAV (red and green) and CI IgD- / - mice (grey) measured by ELISA. Points represent mice, mean ± SD. CP: C-peptide, KLH: keyhole limpet hemocyanin, SAV: streptavidin, CI: control immunization. [Figure 4]Figure 2 shows that soluble antigens interfere with plasma cell differentiation. a: Flow cytometric analysis (FACS) of splenocytes from C-peptide (CP)-immunized mice. Data represent two independent experiments (n=4). The ratio on the x-axis refers to the molar ratio of monovalent (sCP) to multivalent (cCP) CP. CD138+ and B220- cells were identified as plasma cells. The top panel shows 0:1-injected mice and the bottom panel shows 20:1-injected mice. b: Statistical analysis of the presented FACS data. Mean +- SD. c: Flow cytometric analysis (FACS) of splenocytes from C-peptide (CP)-immunized mice. Data represent two independent experiments (n=4). The ratio on the x-axis refers to the molar ratio of monovalent (sCP) to multivalent (cCP) CP. The top panel shows 0:1-injected mice and the bottom panel shows 20:1-injected mice. Right panel: quantification. d: Western blot of pancreatic lysates with C-peptide (CP) mouse serum as primary antibody. Proinsulin (15 kD). e: Streptavidin (carrier)-specific IgG titers of C-peptide (CP)-immunized mice were measured by ELISA. Sera from CP:SAV-immunized mice were applied in duplicate to CP-coated ELISA plates and serially diluted 1:3. [Diagram 5]Figure 1: Conjugated natural insulin (InsNat) elicits an autoreactive IgG response that induces autoimmune diabetic symptoms in wild-type mice. a: Serum anti-insulin-Ig titers of immunized InsNat and CI mice measured by ELISA on the indicated days. Dots represent mice, mean ± SD. b: Flow cytometric analysis of blood showing B cells (CD19+Thy1.2-) and T cells (Thy1.2+CD19-) of wild-type (left) and B cell-deficient (right) mice. Cells were pre-gated on lymphocytes. Represents three independent experiments. c: Blood glucose levels of immunized InsNat (red: WT, yellow: B cell-deficient) and CI mice (grey) were assessed on the indicated days after immunization. Dots represent mice, mean ± SD. d: Urinary glucose levels of immunized InsNat (red) and CI mice (grey) were monitored on the indicated days after immunization. The left panel shows the visualization of glucose standards (top lane) and representative pictures of test animals (middle and bottom lanes). Right panel shows quantification. Points represent mice, mean ± SD. e: Water intake of CI and InsNat immunized mice monitored from d21 to d26. f: Flow cytometry analysis of pancreas of InsNat immunized (red) and CI mice (grey) on day 27. Left panel shows pancreatic macrophages (CD11b+Ly6G-), neutrophils (Ly6G+CD11b+) and B cells (CD19+) pre-gated on live cells. Right panel shows histograms of insulin binding (top) and streptavidin (SAV) binding (bottom). n=5 / group representative of two independent experiments. g: ELISpot of InsNat immunized (red) and CI mice (grey) showing insulin-specific IgG producing spleen-derived cells (d27). Representative wells are shown (top lane). n=3 / group, mean ± SD. h: Quantification of total IgG (red) and insulin-specific IgG (salmon) after serum IgG purification of InsNat-immunized mice. i: Coomassie stained SDS-page showing purified serum IgG of InsNat-immunized (red) and CI mice (grey) under reducing (β-ME) (left lane) and non-reducing conditions (right lane). HC: heavy chain, LC: light chain. Two independent experiments are represented. j: Blood glucose levels of intravenously (iv) injected WT mice.20 μg of purified serum IgG was obtained from InsNat-immunized (red) or CI (grey) mice at the indicated times post-injection. Points represent mice, mean ± SD. CI: control immunization, InsNat: conjugated native insulin, β-ME: β-mercaptoethanol. [Figure 6] Immunization with self-antigen does not alter the splenic B cell compartment. a: Flow cytometry analysis of splenocytes from immunized InsNat and CI mice. Top panel, gating strategy for lymphocytes and single cells. Middle panel shows B cells pre-gated on lymphocytes. Bottom panel shows IgM and IgD expression on B cells. Left: control immunization (CI), right: InsNat immunization (complexed native insulin). n=3 / group. [Figure 7]Figure 1: The ratio of autoantigen-specific IgM to IgG controls the deleteriousness of autoimmune responses and induces protective IgM. a: Serum anti-insulin-Ig titers of immunized InsA peptide (red and green) and CI mice (grey) measured by ELISA on the indicated days. Dots represent mice, mean ± SD. b: Blood glucose levels of immunized InsA peptide (red and green) and CI mice (grey) were assessed on the indicated days. Dots represent mice, mean ± SD. c: Urinary glucose levels of immunized InsA peptide (red and green) and CI mice (grey) were monitored on the indicated days after immunization. Dots represent mice, mean ± SD. d: IgG to IgM ratios obtained from ELISA values ​​plotted against the molar ratio of antigens. n=5 / group, mean ± SD. e: Western blot analysis of insulin-specific serum IgG obtained from InsA peptide-immunized mice. Top panel (green): 100:1 serum, bottom panel (red): 0:1 serum (sInsA:cInsA). Black filled arrows: proinsulin (12 kD), black unfilled arrows: insulin (6 kD), β-actin (42 kD, loading control). Represents two independent experiments. f: ELISpot of d14 immunized InsA peptide (red) and CI mice (grey) showing insulin-specific IgG producing spleen-derived cells. Representative wells are shown (top lane). n=4 / group, mean ± SD. g: IgG to IgM ratios derived from ELISA values ​​plotted on a 2D graph against blood glucose levels (left panel) and urine glucose levels (right panel). n=5 / group, mean ± SD. h: Serum anti-insulin-Ig titers of InsA peptide immunized mice (black) and CI mice (grey) with γ / μ ratios <0.1 measured by ELISA on the indicated days. Points represent mice, mean ± SD. i: Blood glucose levels of InsA peptide-immunized mice (γ / μ<0.1; black) and CI mice (grey) were assessed on the indicated days after immunization. Points represent mice, mean ± SD. j: Insulin-specific IgM affinity maturation of InsA-peptide-immunized mice (left panel) and virus-peptide-immunized mice (right panel) was measured by ELISA on the indicated days.k: Blood and urinary glucose levels of mice immunized with cInsA (red) and cInsA+pIgM iv (salmon). Points represent mice, mean ± SD. CI: control immunization, cInsA: conjugated insulin-A peptide. [Figure 8] Monovalent soluble virus-derived peptide antigens modulate IgG vs. IgM antibody responses induced by the corresponding complexed antigen. a: Determination of viral peptide-specific serum immunoglobulin titers. Sera of virus-peptide immunized mice were applied in duplicate to virus-peptide-bio:streptavidin (SAV) coated plates in serial dilutions of 1:3. Mean +-SD. b-c: Determination of KLH (carrier)-specific serum IgG titers. The indicated ratios on the x-axis refer to the molecular ratio of soluble vs. complexed virus-peptide. Mean +-SD. [Figure 9] Figure 1. Increase in IgMhigh / IgDlow positive compartments after immunization with self- but not foreign antigens, and pancreatic macrophage binding InsA peptide by IgG. a-b: Flow cytometric analysis of splenocytes from virus- or insulin peptide-immunized mice. Top panel (a) shows B cells (CD19+B220+) pre-gated on lymphocytes. Bottom panel (b) shows B cell subsets: mature B cells (IgDhi IgMlo), transitional / marginal zone B cells (IgDlo IgMhi). Cells were pre-gated on B cells. Left: PBS (grey), center: viral peptide (grape), right: insulin peptide (teal). Right outer panel shows quantification, mean +-SD. c: Flow cytometric analysis of pancreatic cells. Left panel shows gating strategy for cells (top) and macrophages (bottom). Right panel shows histograms of InsA-peptide and peptide control binding. [Figure 10]Figure 1: Splenic macrophages bind insulin-specific IgG in cInsA-peptide immunized mice. a: Flow cytometric analysis (FACS) of splenocytes from cInsA-peptide immunized mice. The left panel shows the gating strategy for macrophages (CD11b+CD19-). The top panel shows IgG binding histograms of control immunized (black) and cInsA immunized (red) mice. The bottom panel shows macrophage InsA-peptide binding. Representative data from three separate experiments. [Figure 11] Dysregulated glucose metabolism is prevented by increasing IgM upon repeated reloading with cInsA complex. a: Determination of insulin-specific serum immunoglobulin titers. Sera of InsA-peptide immunized mice were applied in duplicate on native insulin-coated ELISA plates in serial dilutions of 1:3. The left panel shows anti-insulin IgM on d49 and the right panel shows anti-insulin IgG in arbitrary units (AU). The indicated ratios on the X-axis refer to the molecular ratio of soluble vs. complexed InsA-peptide. Mean +- SD. b: Urinary glucose levels were monitored by test stripes. Mean +- SD. [Figure 12] Polyreactive IgM induced by InsA peptide immunization results in diabetic symptoms depending on antigen titer and day. a: Blood glucose levels were monitored by AccuCheck system (Roche). Freshly drawn blood from the tail vein was applied to the test stripe and blood glucose was measured in mmol / L. Mean +- SD. b: Urine glucose levels were monitored by Combur M stripe (Roche). Freshly drawn urine was applied to the glucose field of the test stripe and analyzed according to the manufacturer's standards. The green bar indicates 100:1 (soluble:complex) InsA peptide. Mean +- SD. The dots represent the mice used in this study. [Figure 13]Generation of autoreactive IgM by increasing ratios of monovalent antigens (100:1, sInsA:cInsA) protects against dysregulation of glucose metabolism induced by complexed antigens (0:1, sInsA:cInsA). a: Blood glucose levels were monitored by AccuCheck system (Roche). Freshly drawn blood from the tail vein was applied to the test stripe and blood glucose was measured in mmol / L. Mean +- SD. b: Urine glucose levels were monitored by Combur M stripe (Roche). Freshly obtained urine was applied to the glucose field of the test stripe and analyzed according to the manufacturer's standards. The green bar indicates 100:1 (soluble:complexed) InsA peptide. Mean +- SD. Dots represent mice. c: Determination of insulin-specific serum immunoglobulin titers. Sera of InsA-peptide immunized mice were applied in duplicate on native insulin-coated ELISA plates in serial dilutions of 1:3. (a) shows anti-insulin IgM on d59, while (b) shows anti-insulin IgG in arbitrary units (AU). The indicated ratios on the X-axis refer to the molecular ratio of soluble vs. complexed InsA-peptide. Mean +- SD. [Figure 14] Repeated rechallenge with cInsA complexes results in accumulation of insulin-specific IgM+ B cells. a: Flow cytometric analysis (FACS) of splenocytes (d79) of cInsA-immunized (d71) WT mice. Left panel shows forward and side scatter with lymphocyte gating. Middle panel pre-gated on lymphocytes shows B cells (CD19+B220+). Right panel pre-gated on B cells shows histogram of InsA-peptide binding. Red: g / μ<0.1, black: g / μ<0.1 SAV only control. [Figure 15]Figure 1 shows that intravenous administration of purified serum pIgM does not result in autoimmune dysglycemia. a: Coomassie stained SDS-page shows purified serum IgM of InsA peptide (d49) immunized (red) and CI mice (grey) under reducing (b-ME) (left lane) and non-reducing conditions (right lane). HC: heavy chain, LC: light chain. Represents two independent experiments. b-c: Blood glucose levels of mice intravenously injected with 20 μg of either CI IgM (grey) or InsA IgM (black). Points represent mice, mean ± SD. CI: control immunized, pIgM: protective IgM. d: Anti-KLH-IgM serum titers measured by ELISA. [Figure 16]Figure 1: Differences in affinity and specificity of primary versus memory IgM control autoimmune responses. a: Schematic of the immunization schedule with complexed Ins-A-peptide (cInsA) intraperitoneally and insulin-specific protective IgM (PR-IgM) in a 48-h cycle intravenously (iv). *Monitoring: Diabetic symptoms were observed only in the cInsA only group. b: Blood and urinary glucose levels of wild-type mice on day 7 immunized with complexed InsA-peptide (cInsA) (red, n=5) and cInsA plus intravenously injected (iv) pIgM (salmon, n=5). Points represent individual mice, mean ± SD. c: Serum anti-dsDNA-IgM titers of insulin-A peptide immunized mice on day 7 (n=8) and day 85 (n=4) measured by ELISA. Points represent individual mice, mean ± SD. d, f: Serum antinuclear IgM (ANA) of control immunized mice (CI, n=3), insulin A peptide immunized mice at day 7 (n=3) and day 85 (n=3), and total serum or insulin specific IgM (isotype control: n=3, day 7: n=3, day 85: n=3) analyzed via HEp-2 slides. Scale bar: 10 μm. Green fluorescence indicates that IgM is bound to nuclear structures. e: Coomassie stained SDS-page showing primary (cInsA d7) and memory (cInsA d85) insulin specific IgM after incubation with insulin / DNA and size exclusion with a cutoff at 10.000 kD (see > / <104 kD). IgM heavy chain: 69 kD, IgM light chain: 25 kD, J-segment: 15 kD. Data shown are representative of three independent experiments. g: Blood glucose levels in wild-type mice injected intravenously with either IgM isotype control (grey, n = 6), memory PR-IgM (black, protective insulin-IgM d85, n = 5), or primary insulin-IgM (red, d7, n = 4) after insulin pull-down. [Figure 17]Figure 1. Insulin-specific pull-down of sera from cInsA-immunized mice containing insulin-reactive IgM. a: Western blot analysis of insulin-specific pull-down of cInsA-immunized mice sera. CI: control immunization. The top panel (green) shows IgM heavy chain (IgM HC, 69 kD) and the bottom panel shows IgG heavy chain (IgG HC, 55 kD). b: Serum IgM of control immunized mice against DNA (left) and insulin (right) measured by ELISA. Mean +- SD. Points represent individual mice. [Figure 18] Schematic summary of the case of insulin: Insulin-specific B cell responsiveness is controlled by antigen valency resulting in inducible protective autoreactive IgM under physiological conditions. pIgM: protective IgM, sInsulin: soluble (monovalent), cInsulin: complex (multivalent). [Figure 19] Antibody responses after immunization with SARS-CoV-2 derived RBD. Mice were pretreated as indicated 2 weeks prior to immunization. Mice were then immunized on days 1 and 21. Serum was collected 28 days after immunization and used in ELISA to determine Ig concentrations. [Figure 20]Immunization of mice with cInsulin induces acute inflammatory pancreatitis. A) FACS measurements showing germinal center B cells binding native insulin. B) ELISA measurements showing serum pancreatic lipase used as a marker of pancreatic injury. Consistent with an autoimmune reaction induced by multivalent insulin, a significant increase in serum pancreatic lipase was detected as a clear sign of organ injury. C) Competition assay of insulin binding to IgM. Serum from wild-type mice immunized with cInsA was preincubated with either BSA (untreated control, UT) or 50 μg / mL calf thymus dsDNA (+DNA). The data show a relative decrease in insulin binding to primary IgM (d7) after preincubation with dsDNA, suggesting that dsDNA competes with insulin for binding to primary IgM, which is polyspecific in contrast to PR-IgM. D) Quantitative data for the affinity measurement interference assay of direct insulin:IgM interaction showing the difference in affinity of primary IgM compared to PR-IgM. E) Flow cytometry-based bead array of pancreatic supernatants from mice immunized with cInsulin (n=3) or control immunization (n=3). Representative histograms of cytokine beads (left) and cytokine detection (right). F) Quantification of FACS bead arrays for the indicated cytokines. Dots represent individual mice. [Figure 21] FIG. 1 is a schematic diagram of insulin-4mer (cInsulin) and CP-4mer (cCP). [Figure 22]CpG adjuvant is not required for the initiation of autoantibody responses against InsA peptide. a: Serum anti-insulin-IgM titers (coating: insulin) of mice injected with conjugated insulin-A peptide (cInsA, n=5) or control injections (PBS, n=3) measured by ELISA. Dots represent individual mice. Mean ± SD, statistical significance calculated using Mann-Whitney-U test. b: Blood glucose levels of mice injected with conjugated insulin-A peptide (cInsA, n=5) or control injections (PBS, n=3) were monitored with a commercially available blood glucose monitoring device. Dots represent individual mice. Mean ± SD, statistical significance calculated using Mann-Whitney-U test. [Figure 23] IgD-deficient mice mount a robust polyreactive IgM response one day after immunization. A-B: Serum immunoglobulin titers of mice injected with NP-KLH (IgD-deficient n=5, WT n=4) and CpG ODN1826 (control immunization: Cl, IgD-deficient n=4, WT n=2) measured by ELISA. NP-reactive IgM on days 1 and 3 (A) and day 7 (B). Mean, ±SD. C: Serum immunoglobulins reactive to self molecules (DNA / RNA) of NP-KLH and Cl-injected IgD-deficient and WT mice tested via HEp2 slides. Fluorescence microscopy images are representative of three independent experiments. Scale bar: 10 pm. D: Serum immunoglobulin titers of NP-KLH (IgD-deficient n=5, WT n=4) and Cl-injected (IgD-deficient n=4, WT n=2) mice measured by ELISA. dsDNA-reactive IgM 7 days after immunization. Student's t test with Welch's correction was used to compare two groups within one experiment. Mean, ±SD. [Figure 24]We show that IgD class BCRs are necessary to prevent rapid immune responses against self-antigens and induce affinity maturation. A: Schematic of insulin-A-chain derived peptide (InsA) multivalent complexes with keyhole limpet hemocyanin (KLH). The amino acid sequence of InsA is shown in the figure. B: Immunization schedule in which IgD-deficient and WT mice were injected with InsA-KLH+CpG ODN1826 on day 0 and with InsA-KLH on days 21 and 42. C: Serum immunoglobulin titers reactive to native insulin in IgD-deficient (n=4) and WT (n=10) mice immunized with InsA-KLH or Cl (n=3), measured by ELISA. Days are indicated in the figure. Mean, ±SD. D: Serum immunoglobulins reactive to self molecules (DNA / RNA) of InsA-KLH and Cl-injected IgDko (n=5 / day) and WT (n=5 / day) mice tested via HEp2 slides. Fluorescence microscopy images are representative of three independent experiments. Scale bar: 10 pm. Student's t-test with Welch's correction was used to compare two groups within one experiment. [Diagram 25]Figure 1: IgD class BCR is required for affinity maturation of insulin-IgM to be protective and prevent autoimmune pathology. A: Affinity of IgM to InsA peptide measured by peptide ratio ELISA in clnsA (InsA-KLH+CpG ODN1826) immunized IgDko (n=4), WT (n=5) and Cl (n=3) mice. Plates were coated with streptavidin with one (lnsA(1)) or four (lnsA(4)) biotin binding sites. Mean, ± SD. B: Urinary glucose values ​​(mmol / L) measured by commercial urine stripes (Roche) in IgD-deficient mice (n=4) and WT mice (n=5) immunized with InsA-KLH or Cl (n=3). Mean, ± SD. C: Blood glucose levels (mmol / L) of IgD-deficient mice (n=4) and WT mice (n=5) immunized with InsA. Injections were performed on days 0 (InsA-KLH+CpG ODN1826), 21 (InsA-KLH) and 42 (InsA-KLH). D: Coomassie stained SDS-page showing reduced (+β-ME) and non-reduced (-β-ME) IgM of clnsA and control immunized mice. Total serum IgM was isolated by HiTrap IgM column (clnsA d85 refers to PR-IgM). IgM monomer: 150 kD, IgM HC: 70 kD, IgM LC: 25 kD. E: Blood glucose levels (mmol / L) of IgD-deficient mice immunized with InsA-KLH (n=9), WT control immunized mice (n=4), and IgD-deficient mice immunized with InsA-KLH and injected with PR-IgM iv (n=5). Mean, ±SD. F: Serum immunoglobulins reactive to self molecules (DNA / RNA) of IgM isolated from InsA-KLH immunized mice (PR-IgM and primary IgM on day 7) examined via HEp2 slides. Fluorescence microscopy images are representative of three independent experiments. Scale bar: 10 pm. G: Interferometric assay to determine the affinity of IgM for insulin. Insulin-specific isolated IgM of IgD-deficient mice (upper panel) and WT mice (lower panel) immunized with clnsA. The affinity of IgM on different days is shown in pm. Graph is representative of three independent experiments. [Figure 26] IgD class BCRs are rapid responders capable of secreting autoantibodies 24 hours after immunization.

[0430]

number

[0431] Regulates B cell populations. A-E: Flow cytometry analysis of IgD-deficient (n=4 / group) and WT (n=4 / group) mice immunized with InsA-KLH+CpG ODN1826 or CpG ODN1826(Cl). All panels show representative plots pre-gated on lymphocytes (FSC / SSC), single cells (SSC-H / SSC-W) and viable cells (FVD). A: Left panel shows histogram of CD19 expression used to gate B cells (CD19+) within the lymphocyte gate. Right panel shows expanded (activated) lgM+ B cells (FSChi B220+) within the B cell gate. B: Histogram showing activated B cells by CD69 expression pre-gated on lgM+ B cells. C: Representative plots showing marginal zone B cell (CD21hi CD23'°), follicular B cell (CD21'° CD23hi), and CD2T CD23" (double negative) B cell populations. D: Left panel shows histogram of CD2T CD23" B cell IgM expression. Right panel shows histogram of CD2T CD23" B cell IgD expression. E: Histogram showing CD23 expression of IgM+ splenic B cells. [Figure 27]CD21 / CD23 negative B cell populations are the major source of IgM secreting cells under the control of IgD class BCR. A-D: ELISpot analysis showing IgM secreting splenocytes of InsA-KLH+CpG ODN1826 or control (CpG ODN1826) immunized (Cl) mice 24 h after injection. (A) IgM secreting total splenocytes, (B) IgM secreting CD21 / CD23 negative sorted B cells, (C) IgM secreting CD23+ follicular B cells, (D) representative images of ELISpot wells of the indicated cells and genotypes. Two independent experiments were performed with n=3 / group for Cl and n=6 / group for InsA-KLH. Means, ±SD. Student's t-test with Welch's correction was used to compare two groups within one experiment. [Figure 28] Primary IgM is antigen-specific and polyreactive, but not cross-reactive. A, C: Blood glucose levels (mmol / L) of IgD-deficient and WT mice immunized with NP-KLH+CpG and control (CpG-ODN1826) (A) or InsA-KLH+CpG and control (C). Mean, ±SD. B, D: Serum immunoglobulin titers measured by ELISA of IgD-deficient and WT mice immunized with either NP-KLH+CpG and control (B) or InsA-KLH+CpG and dsDNA-reactive control (D). Mean, ±SD. E, F: Serum immunoglobulin titers measured by ELISA in IgD-deficient and WT mice immunized with NP-KLH+CpG or InsA-KLH+CpG and control reactive to insulin (top panels), or control immunized mice reactive to InsA-KLH+CpG and NP (bottom) (E) and control immunized mice reactive to NP or insulin (F). Means, ±SD. [Figure 29] Schematic overview: IgD is required for IgM maturation. [Diagram 30]IgD-deficient mice require multiple boosts to control autoreactivity. a) Immunization schedule of IgD- / - (n=5) and WT (n=5) mice injected with cInsA (KLH+CpG ODN1826). Days of injection and boost are indicated in the scheme. b) Blood glucose titers of IgDko and WT mice immunized with cIsA (InsA-KLH+CpG ODN1826) and control (Cl, CpG ODN1826). [Diagram 31] IgD-deficient mice require multiple boosts for affinity maturation of insulin-specific IgM. Affinity of IgM to InsA peptides in cInsA (InsA-KLH+CpG ODN1826)-immunized IgD- / - (n=4), WT (n=5) and Cl (n=3) mice measured by peptide ratio ELISA (Shimizu et al. 2004). Plates were coated with streptavidin with one (InsA(1)) or four (InsA(4)) biotin-binding sites. Mean ± SD [Diagram 32] IgD-deficient mice show activated B cells within the CD21-CD23- B cell population one day after immunization. A: General gating strategy used in this study. Top panel shows total splenocytes with gating on lymphocytes. Middle panel shows lymphocytes with gating on single cells. Bottom panel shows single cells with gating on viable cells (fixable viability dye (FVD) negative). B-C: Flow cytometry analysis of splenic B cells from InsA (InsA-KLH+CpG ODN1826) immunized WT and IgD- / - mice. Histograms showing FSC (cell size) pre-gated on CD21-CD23- B cells (C) and CD23+FO (follicular) B cells (D). Data shown are representative of two independent experiments. [Diagram 33] IgD class BCR controls plasma cell differentiation in the peritoneal cavity. A: Flow cytometric analysis of peritoneal cells from clnsA (InsA-KLH+CpG ODN1826) and control (ctrl) immunized mice. Panels show CD138+ plasmablasts and plasma cells. Data shown are representative of two independent experiments with n=3 / group. [Diagram 34] Schematic diagram of A) 1,2-phenylene-bis-maleimide RBD dimer, B) activated RBD monomer, C) reaction with cysteine, D) conjugation with IgG, E) polymerization with IgG, F) complexation with endogenous proteins. [Diagram 35] Mimicking immune complexes by chemical cross-linking of the RBD results in robust antibody responses. A. Concentrations of RBD-specific IgM (left), IgG (middle), and total Ig (right) determined by ELISA in samples used for neutralization assays. B-C. Neutralization potential measured in sera from mice immunized with cRBD*MM. Results were compared to neutralization capacity determined in mice immunized with cRBD-SAV after RBD pretreatment. IgM is not exclusively required to achieve virus neutralization, which can also be achieved by samples containing mainly IgG. Higher concentrations of RBD-specific total Ig correlate with stronger neutralization capacity. cRBD MM: RBD conjugated with maleimide (MM) [Diagram 36] Activating antigens form IgG complexes that boost immune responses. A. Schematic of SARS-CoV-2 spike protein with localization of the receptor binding domain (RBD). B. Reaction scheme of chemical cross-linking. At pH 6.5-7.5, the reactive groups of 1,2-phenylene-bis-maleimide oxidize with sulfhydryl groups on cysteine ​​residues of proteins to form stable thioether linkages. C. Coomassie staining for RBD complexed by 1,2-phenylene-bis-maleimide (Bismale). RBD shows native RBD without cross-linking. D and E. Immunization with RBD. [Figure 37]Autoantibodies are required to balance homeostasis in mice. A: Insulin-specific IgG concentration of different IgG pulldowns measured by ELISA (coating: native insulin). Total: total IgG pulldown via protein G (n=5), insulin-specific: IgG pulldown via insulin bait column (n=5), control IgG (n=3). B: Coomassie stained SDS page showing total IgG (pulldown from serum) and IgG control (total IgG depleted for anti-insulin-IgG). Images shown are representative of three independent experiments. Markers on the left are shown in kilodaltons (kD). C: Anti-insulin-IgG secreting splenocytes of native wild-type and B cell deficient (B cell def) mice measured by ELISpot (coating: native insulin). Cells were seeded at 300.000 cells / well and incubated for 48 hours. D: Blood glucose levels of native wild-type and B cell deficient mice measured using a commercial blood glucose monitor (mmol / L). E: Blood glucose levels of wild type and B cell deficient mice injected with 200 μg total IgG, IgG depleted for anti-insulin-IgG measured at the indicated times. F: Motor function of wild type (WT) and B cell deficient (B cell def) mice measured by wire hanging test (on-wire seconds). Grey: WT untreated, blue: B cell def untreated, green: B cell def injected with 200 μg total IgG. G: Insulin titers of B cell deficient (B cell def) mice injected with 100 μg commercial human IVIg measured by ELISA at the indicated time points. H: Blood glucose levels (mmol / L) of wild type mice injected with 200 μg commercial human IVIg (black) and commercial human IVIg depleted for anti-insulin IgG (grey) measured by commercial blood glucose monitor at the indicated time points. I: Serum glucose levels in immunocompromised patients (common variable immunodeficiency, CVID) receiving IVIg before (pre) and after (post) treatment (500 mg / kg) compared to healthy donor (HD) controls. J: Insulin binding affinity of human anti-insulin-IgG determined by biolayer interferometry (BLI). Kd (dissociation constant) was calculated using Ka (association constant): 1 / Ka.Data shown are representative of three independent experiments. [Figure 38]Neutralizing and PR-IgM are present in humans. A: Serum anti-insulin-IgM concentrations in young (<30 years) and older (>65 years) individuals measured by ELISA (coating: native insulin). Female (young): n=25, Female (old): n=11, Male (young): n=15, Male (old): n=12. Means, ±SD, statistical significance calculated using the Kruskal-Wallis test. B: Scheme showing column-based purification of insulin-specific IgM fractionated into low and high affinity fractions. C: Coomassie stained SDS page showing low affinity anti-insulin-IgM (red) and high affinity anti-insulin-IgM (green) after purification. Images shown are representative of three independent experiments. Markers on the left are indicated in kilodaltons (kD). HC (heavy chain): 70 kD, LC (light chain): 25 kD, J (J segment): 15 kD. D: HEp2 slide showing anti-DNA reactive IgM of insulin specific IgM pulldown. Black: monoclonal IgM control (n=6), red: low affinity anti-insulin IgM (n=6), green: high affinity anti-insulin IgM (n=6). Scale bar: 10 μm. Green fluorescence indicates HEp2 cell binding. Images are representative of three independent experiments. E: Anti-dsDNA-IgM concentration of insulin specific IgM pulldown measured by ELISA (coating: calf thymus DNA). IgM control (ctrl, n=3), IgMlow (n=3), IgMhigh (n=3). Mean, ±SD, statistical significance calculated using Kruskal-Wallis test. F: Insulin binding affinity of human anti-insulin-IgM pulldown determined by biolayer interferometry (BLI). Kd (dissociation constant) was calculated using Ka (association constant): 1 / Ka. Data shown are representative of three independent experiments. Capital letters refer to affinity fractions. G: Blood glucose levels of wild type mice injected intravenously with 100 μg human insulin-specific IgM (capital letters refer to affinity fractions) and human IgM control. H, I: Blood glucose levels of wild type mice injected intravenously with 100 μg human insulin-specific IgM (capital letters refer to affinity fractions) and human IgM control together with 500 ng native insulin (H) and together with 100 μg human anti-insulin-IgG (I).J: Proportion of insulin-specific IgM in young (<30 years) and old (>65 years) individuals as determined by ELISA. Prior to the experiment, insulin-specific IgM was isolated via an insulin bait column. [Figure 39]Endogenous insulin conjugates induce robust autoimmunity in mice. A: Schematic of insulin tetramer (cInsulin) generated by thiol-mediated disulfide cross-linking via 1,2-phenylene-bis-maleimide. Black lines: endogenous disulfide bonds, grey lines: induced disulfide bonds. B: Coomassie-stained SDS page showing insulin (left lane) and cross-linked insulin (right lane; left panel) and cInsulin conjugates after purification on a 10 kD size exclusion column (right panel). Images shown are representative of three independent experiments. Markers on the left are indicated in kilodaltons (kD). C: Blood glucose levels of wild-type mice injected intraperitoneally with PBS (control injection; CI, n=5), cInsulin (n=5), Insulin:SAV (n=5) on day 0. Means, ±SD, and statistical significance were calculated using repeated measures ANOVA test. D: Serum anti-insulin-IgM concentrations of wild-type mice injected intraperitoneally with PBS (control injection; CI, n=5) and cInsulin (n=3) on day 0 measured by ELISA on the indicated days (coating: native insulin). Means, ±SD, statistical significance calculated using the Kruskal-Wallis test. E: Blood glucose levels of wild-type mice injected intraperitoneally with PBS (control injection; CI, n=5) and cInsulin (n=5) on days 0 and 21, followed by intravenous injection of 100 μg anti-Insulin IgM (high affinity) or 100 μg IgM control on day 22. F: Flow cytometric analysis of mice injected intraperitoneally with PBS (n=5) and cInsulin (n=5 / group) together with intravenous 100 μg anti-insulin-IgM (high affinity) or 100 μg IgM control. Panels show pancreatic macrophages (CD11b+) and neutrophils (Ly6G+) pre-gated on viable cells. Images are representative of three independent experiments. G: Serum pancreatic lipase levels in wild-type mice injected intraperitoneally with PBS (n=5) and cInsulin (n=5 / group) along with 100 μg of anti-insulin-IgM (high affinity) or 100 μg of IgM control intravenously. H: Schematic of the macrophage assay used to assess phagocytic activity.I: Flow cytometry analysis of bead-based phagocytosis assays performed with high or low affinity mouse anti-insulin-IgM. The left panel shows representative FACS plots of the percentage of phagocytic macrophages in the presence of low or high affinity IgM. The right panel shows a quantitative analysis of the percentage of phagocytic macrophages. [Diagram 40] Monoclonal human insulin-IgM can protect insulin in vivo. A: Coomassie stained SDS page showing monoclonal anti-insulin-IgM and IgG after purification. The images shown are representative of three independent experiments. Markers on the left are shown in kilodaltons (kD). B: Insulin binding affinity of monoclonal human anti-insulin-Ig determined by biolayer interferometry (BLI). Kd (dissociation constant) was calculated using Ka (association constant): 1 / Ka. The data shown are representative of three independent experiments. C: Anti-dsDNA-IgM concentration of insulin-specific IgM pull-down measured by ELISA (coating: calf thymus DNA). IgM control (ctrl, n=4), IgMMY (n=4), IgGMY (n=4). D: HEp2 slide showing anti-DNA reactive monoclonal IgMMY (n=6) and IgGMY (n=6). Scale bar: 10 μm. Green fluorescence indicates HEp2 cell binding. Images are representative of three independent experiments. E: Blood glucose levels of wild type mice injected intraperitoneally with PBS (control injections; CI, n=5) and cInsulin (n=5) on days 0 and 21, followed by intravenous injection of 100 μg of anti-Insulin IgM (high affinity) or 100 μg of IgM control on day 22. F: Blood glucose levels of wild type mice injected intraperitoneally with PBS (control injections; CI, n=5) and cInsulin (n=5) on days 0 and 21, followed by intravenous injection of 100 μg of anti-Insulin IgM (high affinity) or 100 μg of IgM control on day 22. G: Urinary glucose levels in wild-type mice injected intraperitoneally with PBS (control injection; CI, n = 5) and cInsulin (n = 5) on days 0 and 21, followed by intravenous injection of 100 μg of anti-Insulin IgM (high affinity) or 100 μg of IgM control on day 22. [Diagram 41] There are no antibody secreting cells in mb1 deficient mice. A: Flow cytometric analysis of blood from wild type and B cell deficient mice. Left panel shows forward and side scatter cells. Middle and right panels show cells pre-gated on lymphocytes. B: IgG secreting splenocytes from wild type and B cell deficient mice measured by ELISpot. 50.000 splenocytes were seeded per well. C, D: Serum total IgG (C) and total IgM (D) titers from wild type and B cell deficient mice measured by ELISA. [Diagram 42] Recombinant low affinity anti-insulin IgM destroys insulin in vivo. A) Schematic of recombinant anti-insulin IGHV purified in-house highlighting two mutations in CDR2 that were reverted to the germline version of the IGHV3-74*01 allele. Light grey: α-insulin IgMhigh (WT-IGHV); Medium grey: α-insulin IgMlow (gl-IGHV). B) Coomassie stained SDS-PAGE showing purified α-insulin IgMhigh and purified α-insulin IgMlow under reducing conditions (with β-mercaptoethanol). Images are representative of three independent experiments. C) Insulin binding affinity of α-insulin IgMhigh and α-insulin IgMlow measured by biolayer interferometry. KD (dissociation constant) was calculated by the software. Experiment shown is representative of three independent experiments. D) Blood glucose concentrations in WT mice injected intravenously (iv) with 100 μg of α-insulin IgMhigh (n=4) or α-insulin IgMlow (n=4) measured at the indicated time points. Mean ± SD, statistical significance calculated using two-way ANOVA with Tukey's multiple comparison test. *p<0.05 [Diagram 43]High affinity RF enhances the effect of autoreactive IgG. A) Blood glucose concentrations of WT mice injected intravenously with 100 μg anti-insulin IgG alone (black bars, n=4) or 20 μg RF concentrate from rheumatoid arthritis patients (RFhigh, green bars, n=4) or in combination with monoclonal antibody IgM control (mIgM, blue bars, n=4) were measured at the indicated time points. Mean ± SD, statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison test. **p<0.01. B) Scheme showing the procedure for isolation of total IgM from healthy donor (HD) serum. C) Coomassie stained SDS-PAGE showing total IgM isolation from n=2 healthy donors (IgMHD) under reducing conditions (with β-mercaptoethanol). Images are representative of three independent experiments. D) IgG binding affinity of IgM (dark red line), RFhigh (green line), and mIgM (blue line) isolated from healthy donors measured by biolayer interferometry. KD (dissociation constant) was calculated by software. Experiment shown is representative of three independent experiments. E) Hep-2 slide showing antinuclear structurally reactive IgM (ANA) (IgMHD, dark red squares), RFhigh (green squares), and monoclonal IgM control (blue squares) for total IgM isolation. Scale bar is 65 μm. Green fluorescence indicates IgM binding to Hep-2 cells. Images are representative of three independent experiments. F) Blood glucose concentrations measured at the indicated time points in WT mice injected intravenously (iv) with a combination of 100 μg anti-insulin IgG and 20 μg total IgM purified from healthy donors (dark red line, n=4) or monoclonal IgM control (blue line, n=4). Mean ± SD, statistical significance was calculated using two-way ANOVA with Sidak's multiple comparison test. *p<0.01 [Diagram 44]Recombinant low affinity RF is polyreactive and binds to DNA. A) Schematic of the immunoglobulin heavy and light chain variable genes (IGHV and IGLV, respectively) of recombinant purified low affinity RF compared to the respective alleles of the closest germline. Mutations are shown in bold. IGHM: immunoglobulin heavy constant μ; IGVK: immunoglobulin variable kappa. B) Coomassie stained SDS-PAGE showing recombinant monoclonal (purified in-house) low affinity RF (RFlow), commercial RF (RFhigh) from a rheumatoid arthritis patient and monoclonal control IgM (mIgM) under reducing conditions (with β-mercaptoethanol). Images are representative of three independent experiments. C) IgG binding affinity of RFlow, RFhigh and monoclonal IgM (blue line) measured by biolayer interferometry. KD (dissociation constant) was calculated by the software. Experiment shown is representative of three independent experiments. D) Anti-IgG IgM concentrations (coating: human IgG) detected in purified RFlow (n=3), RFhigh (n=3), and mIgM control (n=3) measured by ELISA (coating: human IgG). Mean ± SD. Statistical significance was calculated using ordinary one-way ANOVA with Tukey's multiple comparison test. **p<0.01. E) Anti-dsDNA-IgM concentrations in RFlow (n=3), RFhigh (n=3), and IgM control (n=3) measured by ELISA (coating: calf thymus dsDNA). Mean ± SD. Results are representative of three independent measurements. F) Hep-2 slide showing anti-nuclear structural reactive IgM (ANA). Scale bar 65 μm. Green fluorescence indicates IgM binding to Hep-2 cells. Images are representative of three independent experiments. G) Schematic of RFhigh and RFlow characterization. [Diagram 45]RFlow regulates the in vivo function of IgG by promoting its degradation. A Blood glucose concentrations were measured at the indicated time points in WT mice injected intravenously (iv) with 100 μg insulin-rich IgG alone (n=4) or in combination with 20 μg RFlow (n=4) or mIgM control (n=4). Mean ± SD, statistical significance calculated using two-way ANOVA with Tukey's multiple comparison test. **p<0.01 B Serum human IgG concentrations in WT mice on days 0 and 1 after a single iv injection of 20 μg α-CD20 human IgG (rituximab) alone (n=4) or in combination with RFhigh (n=4) or mIgM control (n=4), as measured by ELISA. Mean ± SD, statistical significance calculated using two-way ANOVA with Tukey's multiple comparison test. ****p<0.0001 C Serum human IgG concentrations in WT mice on days 0 and 1 after a single intravenous injection of 20 μg α-CD20 human IgG (rituximab) in combination with RFhigh (n=4), RFlow (n=4) or IgM control (ctrl) (n=5) as measured by ELISA. Mean ± SD, statistical significance calculated using two-way ANOVA with Tukey's multiple comparison test. *p<0.05;****p<0.0001 [Diagram 46]A Deregulated ratio of high affinity RF to low affinity RF in autoimmune diseases. A Total IgM detected in serum of young (n=20) and elderly (n=17) healthy donors (HD), rheumatoid arthritis (RA) patients (n=15) and multiple sclerosis (MS) patients (n=28) measured by ELISA. Bars represent mean ± SD, individual values ​​are represented by single points. Statistical significance was calculated using the Kruskal-Wallis test. *p<0.05; **p<0.01. Mean values ​​of IgM (μg / ml) are as follows: young HD 1517.55; elderly HD 1258.02; MS patients 2143.72; rheumatoid arthritis patients 2361.29. B Deregulated ratio of high affinity RF to low affinity RF in autoimmune diseases. A) Amount of total IgG detected in serum of young (n=20) and elderly (n=17) healthy donors (HD), rheumatoid arthritis (RA) (n=15) and multiple sclerosis (MS) (n=28) patients, measured by ELISA. Bars represent mean ± SD, individual values ​​are represented as single points. Statistical significance was calculated using the Kruskal-Wallis test. **p<0.01. Mean values ​​of IgG (μg / ml) are as follows: young HD 7733.22; elderly HD 6856.48; MS patients 10419.28; rheumatoid arthritis patients 10345.23. Total RF-IgM detected in serum of young (n=20) and elderly (n=17) healthy donors (HD), rheumatoid arthritis (RA) patients (n=15) and multiple sclerosis (MS) patients (n=28) measured by C ELISA (coating: human IgG). Bars represent mean ± SD, individual values ​​are represented by single points. To simplify visualization, values ​​of RA patients were plotted separately. Statistical significance was calculated using the Kruskal-Wallis test. *p<0.05; **p<0.01; ****p<0.0001 Mean values ​​(AU) of RF-IgM were: young HD 4.71; elderly HD 2.31; MS patients 1.72; RA patients 737.58. [Figure 47]Anti-insulin IgM concentrations detected in in-house purified recombinant anti-insulin IgMhigh (WT, n=3) and anti-insulin IgMlow (gl, n=3) as measured by ELISA (coating: human insulin). Mean ± SD is shown. Data are representative of three independent experiments. [Figure 48] A Kinetic plots showing the mean ± SD of blood glucose levels after injection of 100 μg of anti-insulin IgG (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 B IgG-bound IgM concentrations in RF-IgM elutions from healthy donors (RF-IgMHD, n=3) and RA patients (RF-IgMRA, n=3) measured by ELISA (coating: human IgG). Mean ± SD, statistical significance was calculated using an unpaired t-test. *p<0.05. C IgG-binding affinity of RF-IgM isolated from healthy donors and RA patients measured by biolayer interferometry. KD (dissociation constant) was calculated by the software. Experiments shown are representative of three independent experiments. D Comparison of IgG-bound IgM concentrations in total IgM isolated from healthy donors (n=3) with IgG-bound IgM detected by RFhigh (n=3) and monoclonal IgM (n=3) measured by ELISA (coating: human IgG). Mean ± SD, statistical significance calculated using one-way ANOVA with Tukey's multiple comparison test. ***p<0.001 [Figure 49]A Serum human IgG concentrations in WT mice after a single intravenous injection of 20 μg α-CD20 IgG alone (n=4), 20 μg RFhigh alone (n=5), or 20 μg α-CD20 IgG+RFhigh (n=4). Mean ± SD, statistical significance calculated using two-way ANOVA with Sidak's multiple comparison test. ***p<0.001, ****p<0.0001 B Serum human IgG concentrations in WT mice after a single intravenous injection (day 0) of 20 μg α-CD20 IgG (rituximab) alone (n=4) or 20 μg RFhigh (n=4) or 20 μg mIgM control (ctrl) at the indicated time points (n=4). Mean ± SD, statistical significance calculated using two-way ANOVA with Tukey's multiple comparison test. **p<0.01; ****p<0.0001 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0432] Certain aspects and embodiments of the invention will now be described, by way of example, and with reference to the description, figures, and tables set forth herein. Such examples of methods, uses, and other aspects of the invention are merely representative and should not be construed as limiting the scope of the invention to only such representative examples. EXAMPLES

[0433] An example is as follows.

[0434] Example 1: Immunization experiments and antibody responses The presence of soluble hapten suppresses IgG production: To test the concept of relative B cell responsiveness in vivo, immunization experiments were performed using NP (4-hydroxy-3-nitrophenylacetyl) as hapten coupled to KLH (keyhole limpet hemocyanin) as carrier (Figure 2a and b). For this purpose, groups of wild-type mice were injected with either NP as soluble compound (sNP) or NP-KLH, termed multivalent complex antigen (cNP), in equal molar ratio to NP (Figure 1a). Antibody responses were determined 7 days (IgM) and 14 days (IgG) after immunization (Figure 1b). Injection of soluble hapten alone (sNP:cNP, 1:0) failed to induce clear IgM or IgG antibody responses, as did control immunization (CI) lacking the tested antigen (CI), whereas injection of cNP as multivalent antigen (sNP:cNP, 0:1) was able to induce both. Addition of sNP to cNP at different molar ratios interfered with the antibody response. Interestingly, the IgG response was significantly inhibited already at a ratio of sNP to cNP of 100:1. Using higher ratios of sNP to cNP (>10.000:1) was also able to significantly suppress the IgM antibody response to the NP hapten (Figure 2c). Importantly, the IgG response to the carrier (KLH) was similar regardless of the amount of soluble hapten (Figure 1c).

[0435] To further confirm these findings, an ELISpot assay was performed to directly assess the proportion of antibody-secreting cells. Consistent with the serum immunoglobulin data, the ELISpot results showed that combining soluble hapten with hapten-conjugated carrier at a ratio of 100:1 reduced the number of IgG-secreting cells, while IgM-secreting cells were unaffected (Fig. 1d). These data are consistent with the inventor's concept that soluble monovalent antigens inhibit immune responses to complex forms of the same antigen. In contrast to IgM, the suppressive effect on IgG immune responses is observed at low concentrations of soluble monovalent antigens.

[0436] Importantly, the presence of IgD-type BCR was suggested to be important for this regulation. Therefore, the role of IgD was tested by performing NP immunization experiments in IgD knockout mice lacking IgD-type BCR. IgD knockout mice did not show an inhibitory effect when soluble NP was added to cNP immunization (Fig. 1e, f; Fig. 2c).

[0437] Taken together, these data suggest that mature B cells are able to fine-tune the immune response according to antigenic determinant density, thereby resulting in distinct IgM and IgG responses to different epitopes of the same antigen.

[0438] The presence of soluble peptides enhances IgM antibody responses: After testing the hapten-specific antibody responses, we tested whether the concept could be valid against autoantigens and thus provide a different scenario for the selection of B cells and the control of self-destructive immune responses. To avoid the use of transgenic mice artificially carrying monospecific B cells expressing a defined BCR recognizing either a transgene product or an endogenous structure, we chose the insulin-related autoantigen as a physiologically relevant system of autoimmune disease. During biosynthesis in the pancreas, proinsulin is cleaved into the well-known hormone insulin and the so-called C-peptide (CP), both of which are secreted into the bloodstream. While insulin is found in nanomolar amounts in the blood and plays a key role in regulating blood glucose levels and diabetes, C-peptide is barely detectable, present in low picomolar amounts in the blood and does not appear to have a homeostatic function

[30] . Using full-length C-peptide or insulin-derived peptides, the autoreactive antibody response against an abundant and functionally important (insulin) in comparison to a barely detectable autoantigen without physiological function (C-peptide) should be investigated (Figure 3a). Moreover, in contrast to insulin, the C-peptide is not conserved (Figure 3b).

[0439] Either biotinylated C-peptide conjugated by incubation with streptavidin (SAV) was used. Alternatively, KLH was used as a carrier coupled to C-peptide to generate a multivalent conjugated antigen (cCP). The unconjugated form of C-peptide (sCP) was used as a soluble antigen. Similar to the NP hapten, wild-type mice were injected with sCP, cCP, or a combination of these to test their potential to induce an autoreactive antibody response (Figure 3c). As expected, sCP did not induce a detectable IgM or IgG immune response, whereas the multivalent form of cCP induced both IgM and IgG when measured at d7 and 14, respectively (Figure 3d). In addition to ELISA experiments, sera from immunized mice were used to determine the specificity of the generated antibody response. Western blot analysis using mouse sera revealed that mice immunized with cCP were positive for IgG antibodies recognizing pancreatic C-peptide (Figure 4a). This is entirely consistent with hapten immunization, showing that soluble peptides, which alone cannot induce detectable immune responses, prevent the generation of IgG memory B cells. Indeed, postchallenge with the same antigen on d21 resulted in weak IgG responses in mice immunized with sCP:cCP ratio 20:1 compared to mice immunized with cCP alone, sCP:cCP ratio 0:1 (Fig. 3d, d14 and d28 IgG). To confirm the memory response to C-peptide as a self-antigen, recall immunization was performed on d42 using cCP without the adjuvant CpG, and robust IgG responses to C-peptide were detected in mice immunized only with sCP:cCP ratio 0:1 (Fig. 3e).

[0440] In contrast to IgG, C-peptide-specific IgM antibody responses were induced upon recall immunization with a 20:1 ratio of sCP:cCP (Fig. 3d, d28 IgM). FACS analysis of splenic B cells did not reveal significant differences in the different groups of mice (Suppl. Fig. 3b, c). Moreover, no differences were detected in the IgG response to the carrier of C-peptide (Fig. 4d).

[0441] These data suggest that soluble monovalent antigens regulate immune responses and determine the IgG:IgM ratio of antibody-secreting cells during immune responses. This conclusion was confirmed by performing ELISpot analysis to determine the number of IgG- or IgM-secreting cells in the different mouse groups. In full agreement with the serum Ig results, ELISpot experiments showed that the number of IgM-secreting cells was increased in mice immunized with sCP:cCP at a ratio of 20:1, while the number of IgG-secreting cells was decreased compared to mice immunized with cCP, sCP:cCP at a ratio of 0:1 (Figure 3f).

[0442] Similar to the NP immunization experiments, to test whether IgD is required for the modulation of B cell responsiveness by the sCP:cCP ratio, C-peptide immunization was performed in IgD knockout mice. IgD knockout mice generally showed reduced IgG responses, and no modulatory effect of soluble peptide on IgG antibody responses was observed in mice immunized with a 0:1 ratio of sCP:cCP (Figure 3g).

[0443] Taken together, these data indicate that antibody responses can be directed against self-antigens, suggesting that the respective autoreactive B cells are neither clonally deleted by central tolerance nor functionally silenced by anergy. Most importantly, regardless of self or non-self antigens, the results show that B cell responses are induced by polyvalent antigens and regulated by soluble counterparts, thereby regulating B cell responsiveness and the isotype of antibodies produced. This results in a dynamic and important B cell function that is quite different from current thinking.

[0444] Example 2: Autoantibody responses to insulin Multivalent native insulin induces a deleterious anti-insulin IgG response: since C-peptide is barely detectable in blood and has no known physiological relevance, it is not excluded that an autoantibody response could be viable against an autoantigen present at such a very low concentration. Therefore, the autoantibody response against insulin was tested. First, we tested the basic assumption that autoreactive B cells are naturally present in the periphery and are not deleted by central tolerance or become unresponsive by anergy as proposed in current thinking. According to this concept, the formation of an autoantigen complex induces the secretion of autoreactive antibodies from naturally occurring autoreactive peripheral B cells. To test this, the autoantigen was a complex generated by incubating biotinylated native mouse insulin with streptavidin (Ins Nat). Importantly, biotinylated mouse insulin is biologically active, since when injected in soluble form it regulates glucose metabolism similarly to its non-biotinylated endogenous counterpart (data not shown). Wild-type mice were injected with 10 μg of InsNat complexes and monitored over time for the presence of anti-insulin antibodies in serum. In parallel, we tested whether immunized mice developed diabetes-like dysregulation of glucose metabolism by monitoring blood and urinary glucose levels. Significant amounts of anti-insulin IgM were detected on day 7, whereas anti-insulin IgG was detected on d14 after injection of conjugated insulin (Figure 5a). Both isotypes were detected after a boost immunization on d28 (d21). Importantly, mice showed clear signs of diabetes, as measured by an increase in blood glucose concentrations that started on d7 (data not shown), continued until d14, and further increased after a boost on d26 (d21) (Figure 5c). To show that the increase in blood glucose levels was dependent on autoantibody production, B cell-deficient mice (mb-1 knockout mice lacking the BCR component Igα, also known as CD79A) were injected with 10 μg of the InsNat complex and blood glucose was monitored (Fig. 5b, c). Interestingly, no increase in blood glucose was observed in the B cell-deficient mice, suggesting that the presence of B cells and autoantibody secretion are important for the development of the diabetic symptoms observed in wild-type mice (Fig. 5c). Furthermore, the increase in blood glucose was accompanied by detectable glucose in the urine of wild-type mice injected with the complex InsNat (Fig. 5d). Consistent with the onset of diabetes, the water consumption of wild-type mice injected with the complex InsNat was dramatically increased (Fig. 5e). Due to the unexpected severity of the diabetic symptoms, the mice were sacrificed on day 27 and the pancreas and spleen were analyzed.

[0445] In contrast to control mice, complexed InsNat immunized mice showed a highly increased recruitment of macrophages, neutrophils and B cells to the pancreas (Fig. 5f). Moreover, IgG+ macrophages from InsNat complex immunized mice showed binding of native insulin (Fig. 5f), thus suggesting an autoantibody-mediated acute inflammatory process in the pancreas. Although FACS analysis showed no difference in splenic B cells between control and complexed InsNat immunized mice (Fig. 6), ELISpot analysis revealed a significantly increased number of splenic B cells secreting anti-insulin IgG in complexed InsNat injected mice (Fig. 5g).

[0446] To test whether the secreted IgG was responsible for the diabetic symptoms, we performed IgG pull-down experiments with serum from mice injected with conjugated InsNat and control immunizations (Fig. 5h, i). Because IgG purification is expected to result in dissociation of endogenous insulin from serum insulin-specific IgG (see Methods), we determined anti-insulin IgG in total IgG after purification. Up to 40% (0.4 mg / mg) of the IgG isolated from InsNat mice was found to be insulin reactive, suggesting that direct serum IgG measurements cannot detect the entire insulin-specific IgG due to binding to endogenous insulin (compare Fig. 5a and 5h). To test the pathogenicity of the isolated anti-insulin IgG, an equal amount of IgG from the control immunization was injected intravenously into wild-type animals or mice were injected with conjugated insulin and blood glucose was monitored. Injecting total IgG containing 2 μg of anti-insulin IgG was found to be sufficient to induce an increase in blood glucose in recipient mice, suggesting that IgG from mice injected with conjugated insulin causes diabetic symptoms (Fig. 5j).

[0447] These data indicate that autoreactive B cells recognizing important metabolic hormones are not deleted or functionally silenced, but are present in the periphery and can induce severe autoimmunity when the balance of autoantigens shifts towards multivalent forms.

[0448] Insulin-derived epitopes induce harmful anti-insulin IgG responses: To further confirm the above findings, we performed immunization experiments using an insulin A-chain derived peptide sequence, designated InsA (Fig. 3b), a frequently reported epitope in autoantibody responses to insulin

[32] . A virus-derived peptide from HIV gp12033 was included as a non-relevant foreign peptide (virus-peptide). For C-peptides, selected peptides were conjugated to the carrier KLH to generate a composite multivalent antigen (cInsA), which was then used in immunization experiments alone or in combination with a soluble peptide (sInsA). Subsequently, antibody responses against the immunogen, InsA peptide, or native insulin were measured to confirm the induction of harmful autoantibody responses. We found that InsA induced IgM and IgG autoantibody responses that recognized native insulin (Fig. 7a). One week after the day 28 boost (d21), multivalent insulin-derived peptide alone (sInsA:cInsA ratio 0:1) readily induced the production of anti-insulin IgG, whereas addition of soluble peptide (sInsA:cInsA ratio 100:1) led to a strong reduction of this autoreactive IgG on day 28 (Fig. 7a). Importantly, the amount of autoreactive anti-insulin IgG was most likely higher than that detected by direct serum ELISA, because anti-insulin IgG bound to endogenous insulin escapes detection as mentioned above (Fig. 5a,i).

[0449] Notably, the presence of soluble InsA led to robust insulin-specific IgM production at d28, which was slightly reduced in mice immunized with the multivalent peptide alone (sInsA:cInsA ratio 0:1), which showed detectable anti-insulin IgM at d28 (Fig. 7a). This was not observed in mice immunized with the viral peptide (Fig. 8a, b). In contrast to the control peptide, insulin is present in relatively high amounts in the organism, suggesting that the presence of endogenous soluble insulin may modulate its immune response of multivalent InsA, thereby resulting in an increase in the autoreactive booster IgM response. Taken together, the data show that the ratio of polyvalent to monovalent antigen is reflected by the ratio of antigen-specific IgG to IgM (γ / μ ratio) antibody responses at day 28 after booster immunization (Fig. 7b).

[0450] In contrast to serum IgG from mice immunized in the presence of soluble peptide (sInsA:cInsA ratio 100:1), serum IgG from mice immunized with multivalent peptide alone (sInsA:cInsA ratio 0:1) readily detected native insulin by Western blot analysis (Fig. 7c). Furthermore, ELISpot analysis using splenic B cells from mice immunized with cInsA confirmed the increased presence of autoreactive IgG-secreting cells in the respective mice (Fig. 7d).

[0451] To confirm that the increase in anti-insulin IgG is associated with a deleterious autoimmune response, we tested whether mice immunized with cInsA (sInsA:cInsA ratio 0:1) showed signs of diabetes. Approximately one week after the booster immunization on day 28 (d21), this group of mice was found to show increased blood glucose and water intake from d27 to d33 (Figure 7e and Figure 10). In addition, we tested whether the glucose concentration in the urine of mice immunized with multivalent insulin peptides (sInsA:cInsA, 0:1) was also elevated. In perfect agreement, increased autoreactive anti-insulin IgG resulted in increased urinary glucose concentration (Figure 7f). In contrast to autoreactive IgG, no detectable signs of autoimmune diabetes were observed in mice with increased amounts of autoreactive anti-insulin IgM in the booster immunization (Figures 7e and f).

[0452] The presence of antigen-specific B cells at d28 after immunization was confirmed by FACS analysis (Figures 9a and b). Compared to controls, mice immunized with the conjugated peptide alone (sInsA:cInsA ratio 0:1) show an increased percentage of macrophages in the pancreas that bound autoreactive IgG, as determined by increased InsA peptide binding (Figure 9c). Similar results were observed in the spleen (Figure 10).

[0453] Taken together, the data suggest that an increased proportion of complex multivalent self-antigens leads to increased amounts of autoreactive IgG and subsequent self-destructive autoimmune responses in wild-type animals.

[0454] Example 3: Protective anti-insulin-IgM expression after InsA-peptide immunization Monovalent autoantigens induce immune tolerance by protective IgM: Apart from the self-destructive role of autoreactive IgG, the aforementioned data point to a protective role of autoreactive IgM in diabetes. Indeed, the results suggest that high anti-insulin IgM compared to the corresponding anti-insulin IgG protects against deregulation of glucose metabolism and diabetes in mice immunized with InsA (Fig. 7a-f). In perfect agreement, mice showing a low ratio of insulin-reactive IgG to IgM (γ / μ<0.1) were protected from diabetes at d28 (Fig. 7g). A second InsA booster immunization at d42 led to anti-insulin IgM but not IgG when the monovalent peptide was included (sInsA:cInsA ratio 100:1), and the corresponding mice showed no signs of diabetes between d42 and d49 (Fig. 11a and b).

[0455] To directly test whether the increased ratio of autoreactive anti-insulin IgM counteracts the negative effects on glucose metabolism induced by autoreactive anti-insulin IgG, mice initially immunized in the presence of monovalent InsA peptide (sInsA:cInsA ratio 100:1) were challenged with polyvalent antigen (sInsA:cInsA, 0:1) alone on d51. Interestingly, treatment that induced autoimmune diabetes from d14 to 28 (Fig. 12, d7 vs. d14) generated only autoreactive anti-insulin IgM responses, but neither anti-insulin IgG nor deregulation of glucose metabolism from d51 to 59 (Fig. 13a-c).

[0456] These data suggest that primary immunization with the presence of monovalent InsA peptides (sInsA:cInsA ratio 100:1) induced resistance to pathogenic immunization with polyvalent InsA (sInsA:cInsA ratio 0:1). Furthermore, the findings indicate that this unique resistance mechanism generates a new class of memory response by inducing and maintaining the production of protective autoreactive IgM (pIgM). To test this further, we monitored the decline in anti-insulin IgM concentrations over time, followed by anti-insulin recall responses (Figure 7h). We show that anti-insulin IgM persists for several weeks, and booster cInsA immunization on day 71 induces only IgM, but not IgG, without signs of deregulation of glucose metabolism (Figures 7h, i and Figure 14). Since an increase in antibody affinity for an antigen is usually associated with a memory response, ELISA experiments were performed to compare the affinity of insulin-specific antibodies at different time points. We found that IgM generated after booster InsA immunization showed higher anti-insulin affinity compared to primary IgM collected on day 7 (Fig. 7j). To further investigate the protective role of pIgM, mice were immunized with cInsA or cInsA along with intravenous injection of 50 μg purified IgM containing 5 μg pIgM (Fig. 15a, b) every 48 h starting on d0. Interestingly, the presence of insulin-specific pIgM mitigated autoimmune dysglycemia and completely prevented glycosuria, as observed in mice immunized with cInsA alone (Fig. 7k). To exclude that pIgM iv injection neutralized the immunogen (cInsA, ip), anti-carrier ELISA was performed. As expected, no difference in anti-KLH-IgM levels was observed on day 7 (Fig. 15c).

[0457] Especially since insulin and InsA peptides are highly conserved between mice and humans (Figure 3b), the data not only present a novel and dynamic concept of B cell resistance but also introduce a fundamental animal model for understanding autoimmune diabetes induced by anti-insulin antibodies in humans.

[0458] Example 4: Protected memory anti-insulin-IgM is monospecific The above results point out an unexpected fundamental difference between autoreactive primary IgM and PR-IgM. Indeed, primary anti-insulin-IgM induced diabetic symptoms but was produced in much smaller amounts compared to memory PR-IgM, which had higher insulin affinity but did not induce pathology. To directly test the protective function of autoreactive memory PR-IgM against destructive autoimmunity, mice were immunized with cInsA alone or cInsA together with intravenous injection of 50 μg total IgM containing 5 μg anti-insulin memory PR-IgM every 48 h starting on d0 (Figures 16a and b). Interestingly, the presence of insulin-specific PR-IgM mitigated autoimmune dysglycemia and completely prevented glucosuria on day 7 compared to mice immunized with cInsA alone (Figure 16b). To exclude that PR-IgM injection neutralized the injected cInsA, anti-carrier (KLH) ELISA was performed and no difference in anti-KLH-IgM levels was found between the two groups on day 7 (Figure 15C). These data suggest that memory anti-insulin PR-IgM prevents insulin depletion by primary anti-insulin IgM, thereby preventing the onset of diabetes. One explanation for the difference between autoreactive primary and memory PR-IgM could be that primary IgM is polyreactive and may be produced by B1 B cells as a first line of immune protection. Presumably, this polyreactivity results in high molecular weight joint immune complexes containing multiple autoantigens, allowing their removal by phagocytes, thereby depleting bound insulin. In contrast, autoreactive memory PR-IgM may be monospecific for autoantigens and thus release autoantigens after binding without forming immune complexes. To test this, the polyreactivity of primary IgM compared to memory PR-IgM was analyzed. Anti-DNA ELISA (Fig. 16c) and indirect immunofluorescence (Fig. 16d) using HEp-2 slides showed that, in contrast to primary IgM, memory PR-IgM was not polyreactive but specifically bound insulin (Figs. 16c and d).

[0459] To show that anti-insulin IgM is specifically responsible for the observed effects, we performed insulin...

Claims

1. A pharmaceutical composition comprising an IgM antibody or a fragment thereof and a therapeutic antibody, wherein the IgM antibody specifically binds to the therapeutic antibody, pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the IgM antibody and the therapeutic antibody are included in a molar ratio of 5:1 to 1:10, preferably 2:1 to 1:

5. The pharmaceutical composition according to claim 1.

3. A pharmaceutical composition for use in the treatment of a disease or disorder, comprising a therapeutic antibody, a) an effective dose of the therapeutic antibody is administered, b) a corresponding dose of an IgM antibody or a fragment thereof is administered, characterized in that the IgM antibody specifically binds to the therapeutic antibody, the corresponding dose of the IgM antibody is between 10% and 400% of the effective dose of the therapeutic antibody, preferably between 20% and 200% of the effective dose of the therapeutic antibody, pharmaceutical composition.

4. The pharmaceutical composition according to claim 1 or 3, wherein the half-life of the therapeutic antibody is extended by the binding of the IgM antibody. The pharmaceutical composition according to claim 1 or 3.

5. The IgM antibody preferably has a K measured by biolayer interferometry Ｄ of at least 10 －８ and The pharmaceutical composition according to claim 1 or 3, which binds to the therapeutic antibody. The pharmaceutical composition according to claim 1 or 3.

6. The pharmaceutical composition according to claim 1 or 3, wherein the therapeutic antibody is an anti-rheumatoid arthritis antibody. The pharmaceutical composition according to claim 1 or 3.

7. The pharmaceutical composition according to claim 1 or 3, wherein the therapeutic antibody is an anti-CD20 antibody. The pharmaceutical composition according to claim 1 or 3.

8. The pharmaceutical composition according to claim 5, wherein the therapeutic antibody is rituximab. The pharmaceutical composition according to claim 5.

9. The pharmaceutical composition according to claim 7, wherein the therapeutic antibody is rituximab. The pharmaceutical composition according to claim 7.

10. For use in the treatment of an autoimmune disease or disorder, The pharmaceutical composition according to claim 1 or 3.

11. The pharmaceutical composition according to claim 10, wherein the autoimmune disease or disorder is multiple sclerosis or rheumatoid arthritis. The pharmaceutical composition according to claim 10.

12. A method for obtaining a protective regulatory antibody, comprising: (a) providing a blood sample from a subject who has experienced induction of IgG and oligomeric antibody responses by a target antigen; (b) concentrating the mature oligomeric antibody, wherein (i) the binding of the oligomeric antibody is more specific for the target antigen than the IgG-type antibody, preferably the oligomeric antibody is monospecific for the target antigen, and / or (ii) The binding affinity of the oligomeric antibody to the target antigen is equal to or greater than that of the IgG-type antibody, and preferably, the protective regulatory antibody has a K value in the range of 10 -7 less than, preferably 10 -8 less than, more preferably 10 -9 less than, and most preferably about 10 -10 to about 10 -12 and binds to the target antigen with a K ｄ value in the range of step; (c) isolating the concentrated mature oligomeric antibody to obtain the protective regulatory antibody that is protective and regulatory against the function of the target antigen. A method comprising the steps.

13. The subject has experienced induction of the IgG and oligomer antibody responses by the target antigen at least 7 days ago, preferably at least 14 days ago, more preferably at least 27 days ago. The method according to claim 12.

14. A method for obtaining a degradable oligomer antibody, comprising: (a) providing a blood sample from a subject who has experienced induction of an IgG and oligomer antibody response by a target antigen; (b) concentrating a primary oligomer antibody, (i) the binding of the oligomer antibody is equally or less specific for the target antigen than an IgG-type antibody, preferably the oligomer antibody is cross-specific for the target antigen and DNA, and / or (ii) The binding affinity of the oligomeric antibody to the target antigen is lower than that of the IgG-type antibody, preferably the protective regulatory antibody binds to the target antigen with a K -7 greater than 10 ｄ and binds to the target antigen. steps; (c) isolating the concentrated primary oligomer antibody to obtain the degradable antibody capable of forming an immunodegradable complex with the target antigen. A method comprising the above steps.

15. The blood sample is selected from the group consisting of whole blood, plasma and serum samples, preferably serum samples. The method according to claim 12 or 14.

16. Isolating the oligomer antibody includes mass-related and / or affinity-related isolation. The method according to claim 12 or 14.

17. Concentrating the oligomer antibody includes immunoprecipitation of the oligomer antibody. The method according to claim 12 or 14.

18. The oligomer antibody is an IgM antibody. The method according to claim 12 or 14.

19. The IgM antibody includes a variable heavy chain (VH) containing a CDR1 sequence encoded by SEQ ID NO: 60, a CDR2 sequence encoded by SEQ ID NO: 61, and a CDR3 sequence encoded by SEQ ID NO: 62; and a variable light chain (VL) containing a CDR1 sequence encoded by SEQ ID NO: 57, a CDR2 sequence encoded by GGTCATCC, and a CDR3 sequence encoded by SEQ ID NO:

58. The pharmaceutical composition according to claim 1 or 3 comprising the above.

20. The IgM antibody includes a variable heavy chain (VH) amino acid sequence encoded by a sequence defined by SEQ ID NO: 59 or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 59; and A variable light chain (VL) comprising an amino acid sequence encoded by the sequence defined by SEQ ID NO: 56, or by a sequence having at least 90% sequence identity to SEQ ID NO: 56, preferably at least 95% sequence identity to SEQ ID NO: 56, comprising, the pharmaceutical composition according to claim 19.

21. A host cell, a) the sequence defined by SEQ ID NO: 59, or a sequence having at least 90% sequence identity to SEQ ID NO: 59, preferably at least 95% sequence identity to SEQ ID NO: 59; and / or b) the sequence defined by SEQ ID NO: 56, or a sequence having at least 90% sequence identity to SEQ ID NO: 56, preferably at least 95% sequence identity to SEQ ID NO: 56, comprising a polynucleotide having, wherein the polynucleotide further encodes an IgM constant region, and / or wherein the host cell comprises a further polynucleotide encoding an IgM constant region, host cell.

22. A method for producing an IgM antibody, the method comprising: a) culturing the host cell according to claim 21; and b) isolating the IgM antibody. A method comprising.

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