Methods and means for modulating B cell-mediated immune responses

Abstract

The present invention relates to methods and means for targeted modulation of B cell-mediated immune responses by contacting B cells with a specific ratio of soluble single monovalent antigens and complex polyvalent antigens. Targeted modulation of B cell immunity can be used in mammals for the diagnosis and therapy of various conditions related to antibody-mediated immunity. Such conditions include cancer, autoimmune diseases, pathogenic infections, inflammatory diseases, allergies, and proliferative diseases such as food intolerance. The present invention is based on the observation that while complex polyvalent antigen structures induce strong IgG-type antibody B cell responses, monovalent antigen structures surprisingly have the ability to suppress such IgG responses, or, in the case of autoantigen-protective IgM responses, especially protective oligomeric anti-insulin antibodies. In this regard, the present invention provides methods, compositions, therapeutic agents, diagnostic agents, and food additives.

Description

[Technical field]

[0001] The present invention relates to methods and means for targeted modulation of B cell-mediated immune responses by contacting B cells with a specific ratio of soluble single monovalent antigens and complex polyvalent antigens. Targeted modulation of B cell immunity can be used in mammals for the diagnosis and therapy of various conditions related to antibody-mediated immunity. Such conditions include cancer, autoimmune diseases, pathogenic infections, inflammatory diseases, allergies, and proliferative diseases such as food intolerance. The present invention is based on the observation that while complex polyvalent antigen structures induce strong IgG-type antibody B cell responses, monovalent antigen structures surprisingly have the ability to suppress such IgG responses, or, in the case of autoantigen-protective IgM responses, especially protective oligomeric anti-insulin antibodies. In this regard, the present invention provides methods, compositions, therapeutic agents, diagnostic agents, and food additives. [Background technology]

[0002] 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 mellitus (T1D)

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

[22] . Moreover, anti-insulin antibodies play an important role in insulin activity, the development of diabetes, and insulin treatment [57–59]. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need to develop approaches for the controllable modulation of immune responses in order to detect or treat or avoid conditions induced or characterized by the presence or activity of an immune response in a subject, particularly an immune response against insulin. [Means for solving the problem]

[0007] BRIEF DESCRIPTION OF THE DRAWINGS Generally, as a brief description, the main embodiments of the present invention can be described as follows.

[0008] 1. Oligomeric anti-insulin antibodies, (i) Kd<5×10, preferably as measured by surface plasmon resonance -7 and / or has an affinity for insulin and / or proinsulin of (ii) Oligomeric anti-insulin antibodies that are monospecific for insulin and / or proinsulin.

[0009] 2. The oligomeric anti-insulin antibody of embodiment 1, wherein the oligomeric anti-insulin antibody is an anti-insulin antibody of the IgM isotype.

[0010] 3. The oligomeric anti-insulin antibody of embodiment 1 or 2, wherein the oligomeric anti-insulin antibody is chimeric, humanized, or human.

[0011] 4. Immunoglobulins: 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 a CDR1 defined in SEQ ID NO: 9, a CDR2 defined in SEQ ID NO: 10 and a CDR3 defined in SEQ ID NO: 11, and a variable light chain (VL) comprising a CDR1 defined in SEQ ID NO: 13, a CDR2 defined in sequence GAS and a CDR3 defined in SEQ ID NO: 14, or c) An oligomeric anti-insulin antibody of embodiments 1 to 3, comprising 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.

[0012] 5. Oligomeric anti-insulin antibodies a) comprising a variable heavy chain (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 with SEQ ID NO: 1, and a variable light chain (VL) sequence comprising the 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, b) comprising a variable heavy chain (VH) sequence comprising the 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 the 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 c) An oligomeric anti-insulin antibody of embodiment 4, comprising a variable heavy chain (VH) sequence comprising the 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 a variable light chain (VL) sequence comprising the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 19.

[0013] 6. A polynucleotide encoding the oligomeric anti-insulin antibody of any one of Examples 1-5.

[0014] 7. A host cell comprising the polynucleotide of embodiment 6.

[0015] 8. A method for producing oligomeric anti-insulin antibodies, comprising culturing the host cell of embodiment 7.

[0016] 9. A pharmaceutical composition comprising an oligomeric anti-insulin antibody of any one of embodiments 1 to 5, a polynucleotide of embodiment 6, a host cell of embodiment 7, and a pharma- ceutically acceptable carrier.

[0017] 10. The pharmaceutical composition of embodiment 9, comprising a further therapeutic agent.

[0018] 11. The oligomeric anti-insulin antibody of any one of embodiments 1-5, the polynucleotide of embodiment 6, the host cell of embodiment 7, or the pharmaceutical composition of embodiments 9-10 for use in therapy.

[0019] 12. The oligomeric anti-insulin antibody of any one of embodiments 1-5, the polynucleotide of embodiment 6, the host cell of embodiment 7, or the pharmaceutical composition of embodiment 9-10 for use in treating an insulin-related disease or disorder.

[0020] 13. A method for diagnosing and / or predicting an insulin-related disease or disorder, comprising: (i) determining the affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample, the sample is obtained from a subject, the subject being diagnosed with or at risk for having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; (iii) diagnosing and / or predicting an insulin-related disease or disorder in said subject based on the comparison made in step (ii), preferably wherein a lower affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin indicates a higher risk of an insulin-related disease or disorder.

[0021] 14. A method for determining whether a subject is susceptible to treatment of an insulin-related disease or disorder, comprising: (i) determining the affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample, the sample is obtained from a subject, the subject being diagnosed with or at risk for having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; (iii) determining whether the subject is susceptible to said treatment of an insulin-related disease or disorder, preferably wherein a lower affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin indicates a higher susceptibility to treatment of an insulin-related disease or disorder.

[0022] 15. The oligomeric anti-insulin antibody for use in embodiment 12, the polynucleotide for use in embodiment 12, or the host cell for use in embodiment 12, or the pharmaceutical composition for use in embodiment 12, the method of embodiment 13 or 14, wherein the insulin-related disease or disorder is selected from the group of pancreatic damage, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, gestational diabetes, and dysglycemia.

[0023] 16. The oligomeric anti-insulin antibody for use of embodiment 15, the polynucleotide for use of embodiment 15, or the host cell for use of embodiment 15, the pharmaceutical composition for use of embodiment 15, or the method of embodiment 15, wherein the glycemic abnormality is glycemic abnormality in a patient with an insulin-related disease or disorder selected from the group consisting of pancreatic injury, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, and gestational diabetes.

[0024] 17. A method for producing oligomeric anti-insulin and / or anti-proinsulin antibodies, preferably of the IgM isotype, comprising immunizing a mammal with a mixture of at least one monovalent insulin particle and at least one multivalent insulin particle.

[0025] 18. A method for the treatment and / or prevention of an insulin-related disease or disorder, comprising the step of administering a therapeutically effective amount of an oligomeric anti-insulin antibody of any one of embodiments 1-5, a polynucleotide of embodiment 6, a host cell of embodiment 7, or a pharmaceutical composition of embodiments 9-10. [Brief description of the drawings]

[0026] The figure shows:

[0027] [Figure 1] Figure 2: Soluble haptens inhibit antibody immune responses 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] Figure 1 shows that a very high ratio of soluble to complexed NP suppresses 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 NP 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 2: Induction of autoantibodies is dependent on autoantigen titers and regulated by their 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. 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. Boost on d42 was performed without CpG (e). Points represent mice, mean ± SD. f: ELISpot assay showing CP-specific immunoglobulin-producing spleen-derived cells at d14. Top lane shows representative images of wells. 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: 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. Three independent experiments are represented. 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). The right panel shows the 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 pancreases of InsNat immunized (red) and CI mice (grey) on day 27. The left panel shows pancreatic macrophages (CD11b+Ly6G-), neutrophils (Ly6G+CD11b+) and B cells (CD19+) pre-gated on live cells. The right panel shows histograms of insulin binding (top) and streptavidin (SAV) binding (bottom). Two independent experiments with n=5 / group are represented. 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 ratio on the x-axis refers 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), as well as 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 (gray, n=6), memory PR-IgM (black, protective insulin-IgM d85, n=5), or primary insulin-IgM (red, d7, n=4) after insulin pull-down. Mean ± SD. Statistical analysis compares the time points of the red line with the time points of the black line. [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) Quantitative data for affinity measurements. Interferometric assay of direct insulin:IgM interactions showing differences in affinity of primary IgM compared to PR-IgM. [Figure 21]Figure 1: Autoantibodies are required to balance homeostasis in mice. A: Insulin-specific IgG concentration of different IgG pull-downs measured by ELISA (coating: native insulin). Total: total IgG pull-down via protein G (n=5), insulin-specific: IgG pull-down via insulin bait column (n=5), control IgG (n=3). B: Coomassie stained SDS page showing total IgG (pull-down 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 times. 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 22]Figure 1: 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 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 23]Figure 1: 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, red 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 intraperitoneally injected with PBS (control injection; CI, n=5), cInsulin (n=5), Insulin:SAV (n=5) on day 0. Means, ±SD, statistical significance was 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 ctrl 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. [Figure 24]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. The 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 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 ctrl on day 22. F: 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 ctrl 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 anti-Insulin IgM (high affinity) or 100 μg IgM ctrl on day 22. [Diagram 25]No antibody secreting cells in mb1 deficient mice. A: Flow cytometric analysis of blood of wild type and B cell deficient mice. Left panel shows forward and side scatter of cells. Middle and right panels show cells pre-gated on lymphocytes. B: IgG secreting splenocytes of 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 of wild type and B cell deficient mice measured by ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In the following, various embodiments of the present invention are described. 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 the context.

[0029] Thus, the present invention relates to oligomeric anti-insulin antibodies, the antibodies having (i) a Kd<5×10 -7 has an affinity for insulin and / or proinsulin of

[0030] In some embodiments, the invention relates to oligomeric anti-insulin antibodies, the antibodies having (i) a Kd<5×10, preferably as measured by surface plasmon resonance -7and / or (ii) is monospecific for insulin and / or proinsulin.

[0031] In some embodiments, the invention relates to oligomeric anti-insulin antibodies, the antibodies having (i) a Kd<5×10 -7 and / or (ii) is monospecific for insulin and / or proinsulin.

[0032] The term "monospecific" in the context of antibodies, as used herein, refers to an antibody having 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, such as insulin, 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 D Such monoclonal IgM binds to antigens associated with autoimmune disorders, such as insulin, at 100° C. Thus, such monoclonal IgM does not bind to unrelated antigens other than the antigens associated with autoimmune disease, 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 disease. 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, Figure 16C, Figure 16D, and Materials and Methods).

[0033] "K DThe term "Kd" as used herein is the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The KD value of an antibody 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 broadly refers 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, i.e., 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, hi some embodiments, Kd is measured by surface plasmon resonance.

[0034] The insulin described herein can have any source. In some embodiments, the insulin described herein is a mammalian insulin, a partially or fully synthetic insulin, preferably human insulin. In some embodiments, the insulin described herein is an insulin variant or an insulin analogue, such as an insulin analogue selected from the group of aspart, lispro, glulisine, glargine, detemir, deglutec.

[0035] The anti-insulin antibodies described herein may be anti-proinsulin antibodies or anti-proinsulin and anti-insulin antibodies.

[0036] The term "proinsulin" as used herein refers to an insulin polypeptide that includes the connecting peptide or "C-peptide" that links the B and A insulin polypeptide chains.

[0037] The inventors demonstrate that insulin activity is regulated by different anti-insulin antibodies in healthy and diabetic subjects (see, for example, Examples 6, 7). Herein, we provide means and methods to use and / or influence this regulatory system. In particular, we demonstrate that oligomeric anti-insulin antibodies binding with monospecificity and / or high affinity binding to insulin have a protective effect on insulin function. Without being bound by theory, the oligomeric anti-insulin antibodies described herein protect insulin from degradation upon binding of less selective and / or specific antibodies (Example 7).

[0038] Thus, the present invention is based, at least in part, on the protective / modulatory effect of oligomeric anti-insulin antibodies on insulin activity.

[0039] 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 composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; and contacting the mixture with a combination comprising:

[0040] In some embodiments alternative to the first aspect, the invention relates to a combination for use in eliciting and / or modulating a cell-mediated target antigen-specific immune response in a subject, the combination 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 composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; and contacting the mixture with a combination comprising The combinations are used by contacting one or more immune cells of the subject with the combination.

[0041] In a further alternative of the first and second aspects, the combination is 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 combination or at least (i) or (ii) of the combination. A therapeutically effective amount in the context of this invention is an amount which induces or suppresses a specific B cell mediated immune response, such as an IgG type or IgM type (or IgA) immune response.

[0042] 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 polyvalent 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. Thus, the present invention proposes to modulate the ratio of soluble to complex immune responses in order to control the focus of B cell immunity. This approach can be used for novel controlled vaccination treatments or to tackle autoimmune diseases such as diabetes.

[0043] The methods described herein are, in some embodiments, non-therapeutic and non-surgical methods. In this embodiment, the method of the present 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 embodiment, the subject is preferably a non-human vertebrate.

[0044] A "cell-mediated antigen-specific immune response" in the context of the present invention refers to an immune response that includes one or more B lymphocytes (B cells), preferably a B cell-mediated immune response. The term "B lymphocyte" or "B cell", as used herein, refers to a lymphocyte that plays a role in the 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 ) are included.

[0045] 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.

[0046] 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 reacts specifically 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 no more than one copy 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.

[0047] 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 with a specific paratope can bind 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. 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, which allows the binding of two antibodies to the multivalent antigen particle at the same time. Preferably, two or more of the antigenic structures comprised in the antigenic moiety of the multivalent antigen particle comprise a plurality of identical antigenic structures.

[0048] 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.

[0049] 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.

[0050] In another embodiment 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.

[0051] 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.

[0052] Preferably, neither the carrier moiety, nor the linker, optionally, is capable of eliciting a cell-mediated immune response against a target antigen, such as an antigen associated with an autoimmune disease.

[0053] A "linker" in the context of the present invention is preferably a peptide linker that can have any size and length appropriate 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.

[0054] In a preferred embodiment of the present invention, contacting one or more immune cells of a subject or patient with a combination 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, where in (i), (ii) and (iii) the immune cells of the subject are monovalent and multivalent antigen particles as a result of the administration in contact with the combination. In general, the term "contacting" should be understood as presenting such antigen particles to the immune system of the subject, preferably to induce a B cell-mediated immune response. Preferably, in (i), the subject is characterized by the presence of multivalent antigen particles before administration of monovalent antigen particles, and in (ii), the subject is characterized by the presence of monovalent antigen particles before administration of multivalent antigen particles.

[0055] In the context of the present invention, it has been found that a specific ratio of monovalent and polyvalent antigens can modulate the antibody immune response mediated by B cells. Therefore, it is a preferred embodiment of the present invention that the combination 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 to 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 combination comprising more than 1, preferably 10 specific antigen ratios constitutes the control of an IgG-type (or IgM) target antigen-specific B cell response in a subject. In another embodiment of the invention, contacting one or more of the subject's B cells with a combination 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.

[0056] 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 with or without direct combination with administering multivalent antigen particles to the subject.

[0057] 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 It constitutes an increase in an IgG-type target antigen-specific B cell response in a subject by contacting the subject with a combination comprising a specific antigen ratio below 1, preferably below 10. Preferably, contacting one or more of the subject's B cells with a combination comprises a specific antigen ratio below 1, preferably below 10. -1 , 10 -2 , 10 -3 , 10 -4 It involves administering to a subject an amount of multivalent antigen particles effective to produce a specified antigen ratio below:

[0058] Contacting one or more of the subject's B cells with the amount of multivalent antigen particles is preferably administered with or without direct combination with administering monovalent antigen particles to the subject.

[0059] The term "antigen" may refer to any, preferably disease-associated, molecule or structure that contains an antigenic structure. Preferably, the antigen of the present invention is an autoantigen, a cancer-associated antigen, or a pathogen-associated antigen. 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 contains 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.

[0060] A pathogen-associated antigen of the present invention may be any antigen expressed in, on or by a pathogen, e.g., a pathogenic virus or microorganism, preferably the pathogen is selected from a parasite, a unicellular eukaryote, a bacterium, a virus or a virion.

[0061] The 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 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.

[0062] In the context of the present invention, the disease or condition is selected from those diseases or conditions characterized in that an increase or decrease in a cell-mediated immune response is beneficial for treatment. Thus, the present invention provides 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.

[0063] 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 picture. 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.

[0064] 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, B cells expressing immunoglobulin (Ig)M, IgD, IgA or IgG type antibodies and / or B cell receptors are involved in the cell-mediated target antigen-specific immune response.

[0065] 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.

[0066] Exemplary antibody Ig variants discussed in the context of the present invention include IgG, IgM, IgE, IgA, or IgD antibodies.

[0067] 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.

[0068] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody of the present invention, wherein the oligomeric anti-insulin antibody is an anti-insulin antibody of the IgM isotype.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The term "immune-mediated inflammatory disease" or "IMID" as used herein refers to any of a group of conditions or diseases that lack a definitive etiology but are characterized by a common inflammatory pathway that leads to inflammation and may result from or be triggered by dysregulation of a normal immune response. Because inflammation mediates and is a major cause of many medical and autoimmune disorders, in the context of the present invention, the term immune-mediated inflammatory disease is also intended to encompass autoimmune disorders and inflammatory diseases.

[0075] The term "autoimmune disorder" or "autoimmune disease" refers to a condition in a subject characterized by cell, tissue, and / or organ damage caused by an immunological response of the subject to its own cells, tissues, and / or organs. Illustrative, non-limiting examples of autoimmune diseases that can be treated with the methods or pharmaceutical compositions of the present invention include patchy alopecia, rheumatoid arthritis (RA), ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigus, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune deficiency, and chronic inflammatory bowel disease. CFIDS, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, patchy pemphigoid syndrome, CREST syndrome, cryoglobulinemia, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease Disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatoid arthritis, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, sclerosis, progressive systemic sclerosis, Sjögren's syndrome, Goodpa These include: Sture syndrome, generalized ankylosing syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporary aneurysm / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, antiglomerular bronchial disease, antiphospholipid syndrome, autoimmune diseases of the nervous system, familial Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyendocrinopathy, and psoriasis.

[0076] The term "inflammatory disease" refers to a condition in a subject characterized by inflammation, for example, chronic inflammation. Illustrative non-limiting examples of inflammatory disorders include, but are not limited to, acute inflammation such as celiac disease, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, Crohn's disease, ulcerative colitis, allergic disorders, septic shock, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), inflammatory vasculitis (e.g., polyarteritis, Wegener's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatous granulomatosis), post-traumatic angiogenesis (e.g., post-angiogenesis remodeling disease), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, chronic inflammation resulting from chronic viral or bacterial infection, and sepsis.

[0077] The term "treat" or "treatment" or "treating" as used herein, when used directly in relation to a patient or subject, should be construed to mean administering a therapy to a patient subject in need of said treatment for the amelioration of one or more symptoms associated with a disease or disorder. Those in need of treatment include those already suffering from a condition or disorder, as well as those prone to having a condition or disorder, or those in whom a condition or disorder is to be prevented. The term "treat" or "treatment" or "treating", when used directly in relation to damaged tissue, should be construed to mean both direct mechanisms, such as regeneration of damaged tissue, repair or replacement of damaged tissue (e.g., by scar tissue), as well as indirect mechanisms, such as amelioration of such damage by reducing inflammation, thereby allowing tissue formation.

[0078] 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. It should be understood that in most cases the antigen structure comprises or consists of an epitope that elicits an antibody immune response, whereas it is the binding site of the 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.

[0079] In some embodiments, the present invention relates to a method for treating or preventing a disease in a subject characterized by the presence of immunoglobulin G (IgG) type antibodies specific for a disease-associated antigen, the method comprising administering to the subject a therapeutically effective amount of monovalent antigen particles, the monovalent antigen particles being composed of an antigenic moiety comprising one or less of an antigenic structure capable of inducing an antibody-mediated immune response against the disease-associated antigen.

[0080] 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 amount 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.

[0081] 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 cases where there is a host immune response to foreign antigens (e.g., major histocompatibility alloantigens or small histocompatibility alloantigens) of another individual, such as when there is host-versus-graft rejection, or alternatively when there is graft-versus-host disease, in which transplanted immune cells mediate harmful effects on the host receiving the graft.

[0082] 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.

[0083] In this embodiment, the specific embodiments disclosed above apply equally here.

[0084] 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, which is composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; The method comprises administering an effective amount of a vaccination composition comprising:

[0085] 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.

[0086] 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: (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 composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; Includes.

[0087] In some embodiments, the present invention provides a method for the preparation of a (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 the antigen; (ii) a multivalent antigen particle, which is composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against the antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; The present invention relates to an immunogenic composition comprising:

[0088] The terms "of the invention," "according to the invention," "according to the present invention," and the like are intended to refer to all aspects and embodiments of the present invention as described and / or claimed herein.

[0089] The methods of the various aspects of the 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 invention include a prime / boost immunization scheme for the subject.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In some embodiments of the invention, the boosting step may be performed using a combination of monovalent and multivalent antigen particles, as described herein.

[0094] In some embodiments, the present invention relates to monospecific IgM type antibodies or variants thereof for use in the treatment of autoimmune diseases, where the monoclonal IgM type antibodies are specific and have high affinity for antigens associated with the autoimmune disease.

[0095] In another embodiment, the monospecific IgM antibody or variant thereof of the invention is not a polyclonal antibody or the antigen-binding fragment is not a fragment of a polyclonal antibody. In a more specific embodiment, the monospecific IgM antibody or variant thereof of the invention is not a primary (multispecific) IgM antibody.

[0096] In an alternative and preferred embodiment of all monospecific IgM type antibodies or variants thereof of the invention, the monospecific IgM type antibodies or variants thereof are antibodies or antigen-binding fragments thereof, which antibodies are monoclonal antibodies or which antigen-binding fragments are fragments of monoclonal antibodies.

[0097] 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 include 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. In certain embodiments, the present invention relates to oligomeric anti-insulin antibodies of the present invention, which are chimeric, humanized, or human.

[0098] 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 particular 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 for in vitro production of monoclonal recombinant antibodies, which are further characterized based on their specificity and affinity for the immunizing antigen.

[0099] The monospecific IgM type antibodies or variants thereof according to the present invention can 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.

[0100] In a further preferred embodiment, the monospecific IgM 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.

[0101] 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).

[0102] 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.

[0103] 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 by 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 said antibody or fragment thereof may be human consensus framework sequences. In some cases, Fv framework residues of the human immunoglobulin need to be replaced by corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies 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.

[0104] 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.

[0105] 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 remainder 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.

[0106] 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 IgM type 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, and (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.

[0107] 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 lgG1, lgG2, lgG3, and lgG4. Human IgM subtypes include IgM. Human IgA subtypes include lgA1 and lgA2. 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.

[0108] In some embodiments, the monospecific IgM type 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.

[0109] Monospecific IgM type antibodies or variants thereof of the invention which comprise at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin) may have such a (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.

[0110] 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.

[0111] 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)).

[0112] 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.

[0113] <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.

[0114] 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 isotype. 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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 IgM type antibodies or variants thereof in which such techniques or mutations have been used to reduce effector function.

[0119] 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 the M252Y / S254T / T256E mutations and 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 IgM antibody or variants thereof of the present invention 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In a preferred embodiment, the antibody of the invention is an antibody in which at least a portion of the framework sequences of said antibody or fragment thereof are human consensus framework sequences, e.g., an antibody comprising human germline-encoded framework sequences.

[0127] In certain other embodiments, the monospecific IgM type antibodies or variants thereof of the invention 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 may have an Fc region with T250Q / M428L, H433K / N434F / Y436, or M252Y / S254T / T256E / H433K / N434F modifications.

[0128] 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.

[0129] 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.

[0130] In other embodiments, the monospecific IgM antibody or variant thereof of the present 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.

[0131] 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.).

[0132] In certain embodiments, the invention relates to an oligomeric anti-insulin antibody of the invention, wherein the immunoglobulin comprises 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) defined by CDR1 defined in SEQ ID NO: 20, CDR2 defined in sequence DAS, and CDR3 defined in SEQ ID NO: 21.

[0133] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody of the invention, comprising: a) a variable heavy chain (VH) sequence comprising the amino acid sequence of SEQ ID NO:1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 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 80% sequence identity to SEQ ID NO:4. 8, or b) a variable light chain (VL) sequence comprising an amino acid sequence of SEQ ID NO: 8 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably at least 95%, sequence identity to SEQ ID NO: 8. 12, or c) a variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 12 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 12; or a variable heavy chain (VH) sequence comprising a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 19, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%,Preferably, the VL sequence comprises a sequence having at least 95% sequence identity.

[0134] 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.

[0135] In some embodiments, oligomeric anti-insulin antibodies of the invention comprise a variable light chain (VL) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:21. In some embodiments, oligomeric anti-insulin antibodies of the invention comprise a variable light chain (VL) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:21, and contain substitutions, insertions, or deletions compared to the reference sequence, but retain the ability to bind insulin and / or proinsulin with high affinity and / or monospecificity. Optionally, an oligomeric anti-insulin antibody of the invention comprises the VL sequence of SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:21 (including post-translational modifications of that sequence).

[0136] In certain embodiments, oligomeric anti-insulin antibodies of the invention comprise a variable heavy chain (VH) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:15. In certain embodiments, oligomeric anti-insulin antibodies of the invention comprise a variable heavy chain (VH) sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:15 and contain substitutions, insertions, or deletions compared to the reference sequence, but retain the ability to bind insulin and / or proinsulin with high affinity and / or monospecificity. Optionally, an oligomeric anti-insulin antibody of the invention comprises the VH sequence of SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:15 (including post-translational modifications of that sequence).

[0137] In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, and / or SEQ ID NO:21. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, and / or SEQ ID NO:21.

[0138] In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). In a preferred embodiment, a total of six amino acids of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, and / or SEQ ID NO:21 are substituted to optimize expression in mammalian cells.

[0139] Amino acid sequence variants of an 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 arrive at the final construct, so long as the final construct possesses the desired properties, e.g., antigen binding.

[0140] 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 antigen binding, reduced immunogenicity, or altered ADCC or CDC.

[0141] 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 certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0142] 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, antibody affinity is optimized using look-through mutagenesis, 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).

[0143] 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 as provided herein) that do not substantially reduce binding affinity and / or monospecificity 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.

[0144] 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.

[0145] 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.

[0146] 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, modification of the oligosaccharides in the antibodies of the invention may be performed to form antibody variants with specific improved properties.

[0147] 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).

[0148] 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.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] 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.

[0153] In certain embodiments, the present invention relates to polynucleotides encoding the oligomeric anti-insulin antibodies of the invention.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] In certain embodiments, a polynucleotide encoding an antibody according to the invention is suitable for use as a vector.

[0165] In certain embodiments, the present invention relates to host cells comprising the polynucleotides of the present invention.

[0166] 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.

[0167] 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.

[0168] In certain embodiments, the host cell is a stem cell. In other embodiments, the host cell is a differentiated cell.

[0169] 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.

[0170] In certain embodiments, the invention relates to methods for producing oligomeric anti-insulin antibodies comprising culturing a host cell of the invention.

[0171] 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.

[0172] 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).

[0173] 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).

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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).

[0178] 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.

[0179] 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.

[0180] In certain embodiments, the invention relates to a pharmaceutical composition comprising an oligomeric anti-insulin antibody of the invention and a pharma- ceutically acceptable carrier. In certain embodiments, the invention relates to a pharmaceutical composition comprising a polynucleotide of the invention and a pharma- ceutically acceptable carrier. In certain embodiments, the invention relates to a pharmaceutical composition comprising a host cell of the invention and a pharma- ceutically acceptable carrier.

[0181] 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.

[0182] 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. Proteins such as albumin, 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 pharmaceutically acceptable carriers herein further 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.

[0183] Pharmaceutically acceptable carriers and / or excipients may enhance the stability, delivery and / or pharmacokinetic / pharmacodynamic properties of the means of the present invention.

[0184] In certain embodiments, the present invention relates to a pharmaceutical composition of the present invention comprising an additional therapeutic agent.

[0185] 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 amount. 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.

[0186] 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 siRNA. In some embodiments, the therapeutic agent described herein is a peptide. In some embodiments, the therapeutic agent described herein is a hormone. In some embodiments, the therapeutic agent described herein is insulin.

[0187] The inventors demonstrate that the means and methods described herein are useful for regulating endogenous insulin (see, e.g., Examples 6 and 7). The same mechanism can be used to enhance or protect the effects of therapeutic agents, such as those that affect glucose homeostasis (e.g., insulin).

[0188] Thus, the present invention is based, at least in part, on the discovery that the means and methods described herein can improve the effectiveness of other therapeutic agents.

[0189] In certain embodiments, the present invention relates to the oligomeric anti-insulin antibodies of the invention for use in therapy.

[0190] In certain embodiments, the present invention relates to the polynucleotides of the present invention for use in therapy.

[0191] In certain embodiments, the present invention relates to a host cell of the present invention for use in therapy.

[0192] In certain embodiments, the present invention relates to a pharmaceutical composition of the present invention for use in therapy.

[0193] In certain embodiments, the invention relates to an oligomeric anti-insulin antibody of the invention, a polynucleotide of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use in treating an insulin-related disease or disorder.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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 (see, for example, Examples 6 and 7). 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, Figure 11, Figure 12, Figure 16, Examples 6 and 7). Thus, the means and methods can improve the effect of administered and / or endogenous insulin and reduce any insulin-related disease or disorder.

[0199] 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.

[0200] In certain embodiments, the present invention relates to a method for diagnosing and / or predicting an insulin-related disease or disorder, the method comprising:

[0201] (i) determining the binding affinity of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample, the sample being obtained from a subject, and the subject being diagnosed with or at risk of having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; and (iii) diagnosing and / or predicting an insulin-related disease or disorder in said subject based on the comparison made in step (ii), preferably wherein a lower binding affinity of anti-insulin IgM antibodies to proinsulin and / or insulin indicates a higher risk of an insulin-related disease or disorder.

[0202] The step of determining the binding affinity of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample may also be accomplished by obtaining corresponding information from a measurement instrument or a database.

[0203] In certain embodiments, the present invention relates to a method for determining whether a subject is susceptible to treatment of an insulin-related disease or disorder, the method comprising the steps of: (i) determining the binding affinity of an anti-insulin IgM antibody to proinsulin and / or insulin from a sample, the sample being obtained from a subject, the subject being diagnosed with or at risk of having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; and (iii) determining whether said subject is susceptible to treatment of an insulin-related disease or disorder, preferably wherein a lower binding affinity of an anti-insulin IgM antibody to proinsulin and / or insulin indicates a higher susceptibility to treatment of an insulin-related disease or disorder.

[0204] The present inventors have found that IgM antibody affinity predicts disease onset, progression and outcome in insulin-related diseases or disorders (Example 10).

[0205] Thus, the present invention is based, at least in part, on the predictive information contained in a subject's IgM antibody affinity status.

[0206] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody for use according to the invention, a polynucleotide for use according to the invention or a host cell for use according to the invention, or a pharmaceutical composition for use according to the invention, a method according to the invention, wherein the insulin-related disease or disorder is selected from the group of pancreatic injury, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, gestational diabetes, and dysglycemia.

[0207] The term "pancreatic injury" as used herein refers to any form of pancreatic abnormality that deregulates hormones that regulate insulin production, insulin activity, and / or insulin effect, such as adrenaline, glucagon, steroids, and somatostatin. In some embodiments, the pancreatic injury as used herein is selected from the group of drug-induced pancreatic injury, obesity-induced pancreatic injury, and cancer-induced pancreatic injury.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] The term "exogenous insulin antibody syndrome," as used herein, refers to hypersensitivity to exogenous insulin and / or insulin resistance associated with circulating insulin antibodies in insulin-treated patients.

[0212] The term "glycemic abnormality" as used herein refers to abnormalities in blood glucose stability. In some embodiments, the glycemic abnormality described herein is hypoglycemia. In some embodiments, the glycemic abnormality described herein is hyperglycemia. In some embodiments, diabetes is diagnosed by a blood 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, the glycemic abnormality is a fasting blood glucose level greater than 100 mg / dl or 110 mg / dl.

[0213] The means and methods described herein can be used to restore deregulated homeostasis insulin and hormones affected by insulin action and / or an immune response to insulin.

[0214] Thus, the present invention is based, at least in part, on the discovery that the means and methods provided herein can restore dysregulated homeostasis in a variety of insulin-related diseases or disorders.

[0215] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody for use according to the invention, a polynucleotide for use according to the invention, or a host cell for use according to the invention, a pharmaceutical composition for use according to the invention, or a method according to the invention, wherein the glycemic abnormality is glycemic abnormality in a patient with an insulin-related disease or disorder selected from the group of pancreatic injury, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, and gestational diabetes.

[0216] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody of the present invention for use in enhancing insulin effect. Insulin effect may 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, may 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.

[0217] 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.

[0218] 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.

[0219] In certain embodiments, the present invention relates to an oligomeric anti-insulin antibody for use in the present invention, a polynucleotide for use in the present invention or a host cell for use in the present invention, or a pharmaceutical composition for use in the present invention, a method of the present invention, wherein the insulin-related disease or disorder is diabetes or a symptom thereof.

[0220] 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.

[0221] 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).

[0222] In one particular embodiment, the present invention relates to a method for producing oligomeric anti-insulin antibodies, preferably of the IgM isotype, comprising immunizing a mammal with a mixture of at least one monovalent insulin particle and at least one multivalent insulin particle.

[0223] The term "insulin particle" as used herein refers to an antigen particle (e.g., a multivalent or monovalent antigen particle) in which the antigen is at least partially contained in insulin and / or proinsulin. In some embodiments, the insulin particle comprises an antigen that includes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or all amino acids of insulin and / or proinsulin.

[0224] In certain embodiments, the present invention relates to a method for treating and / or preventing an insulin-related disease or disorder, the method comprising the step of administering a therapeutically effective amount of any one of the oligomeric anti-insulin antibodies of the present invention, the polynucleotide of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0225] In addition to the above, the present invention further relates to the following specific itemized embodiments. Item 1. A method for inducing and / or modulating a humoral and / or cell-mediated target antigen-specific immune response in a subject, comprising inducing one or more immune cells of the subject with (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 composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; The method of claim 1, further comprising contacting the subject with a combination comprising: Item 2. The method according to item 1, wherein 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, are involved in a cell mediated target antigen specific immune response. Item 3. The method of item 1 or 2, wherein B cells expressing immunoglobulin (Ig)M, IgD, IgA or IgG type antibodies and / or B cell receptors are involved in a cell-mediated target antigen-specific immune response. Item 4. The method of any one of items 1 to 3, wherein two or more of the antigen structures contained in the antigenic portion of the multivalent antigen particle include a plurality of identical antigen structures. Item 5. The method of any one of items 1 to 4, wherein the monovalent antigen particle further comprises a carrier moiety attached to the antigenic moiety, optionally via a linker, wherein the carrier, and optionally the linker, do not comprise another copy of the antigen structure, and wherein the carrier moiety, and optionally the linker, are not capable of eliciting a cell-mediated immune response against the target antigen. Item 6. The method of any one of items 1 to 5, wherein the multivalent antigen particle further comprises a carrier moiety optionally linked to the antigen moiety via a linker. Item 7. The method of item 6, wherein the carrier moiety, and optionally the linker, is incapable of eliciting a cell-mediated immune response against the target antigen. Item 8. The method of any one of items 5 to 7, wherein the carrier moiety is 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), 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. Item 9. The method of any one of items 1 to 8, wherein contacting one or more immune cells of the subject with a combination comprising monovalent antigen particles and multivalent antigen particles involves (i) administration of monovalent antigen particles to the subject, (ii) administration of multivalent antigen particles to the subject, or (iii) administration of monovalent antigen particles and multivalent antigen particles to the subject, wherein in (i), (ii) and (iii), the immune cells of the subject are monovalent antigen particles and multivalent antigen particles as a result of the administration in contact with the combination. Item 10. The method of item 9, wherein 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. Item 11. The method of any one of items 1 to 10, wherein the combination comprising monovalent antigen particles and multivalent antigen particles comprises a specific antigen ratio of monovalent antigen particles:multivalent antigen particles. Item 12. Modulation of a cell-mediated target antigen-specific immune response in a subject comprises modulating one or more of the subject's B cells by more than 1, preferably 10 1 Super, 10 2 Super, 10 3Super, 10 4 The method of claim 11, comprising reducing the IgG-type target antigen-specific B cell response in a subject by contacting the subject with a combination comprising a specific antigen ratio of greater than or equal to 100%. Item 13. Contacting one or more of the subject's B cells with the combination is performed in a subject in a dose of more than 1, preferably 10 1 Super, 10 2 Super, 10 3 Super, 10 4 13. The method of claim 12, comprising administering to the subject an amount of monovalent antigen particles effective to produce a specific antigen ratio that is greater than or equal to the specific antigen ratio. Item 14. The method of item 12 or 13, wherein contacting one or more of the subject's B cells with the amount of monovalent antigen particles is administered with or without direct combination of administering to the subject multivalent antigen particles. Item 15. Modulation of a cell-mediated target antigen-specific immune response in a subject comprises modulating one or more of the subject's B cells by less than 1, preferably less than 10 -1 , 10 -2 , 10 -3 , 10 -4 The method of claim 11, comprising increasing an IgG-type target antigen-specific B cell response in a subject by contacting the subject with a combination comprising a specific antigen ratio below: Item 16. Contacting one or more of the subject's B cells with the combination is performed in a subject in a period of less than 1, preferably less than 10 -1 , 10 -2 , 10 -3 , 10 -4 16. The method of item 15, comprising administering to a subject an amount of multivalent antigen particles effective to produce a specific antigen ratio below: Item 17. The method of items 15 or 16, wherein contacting one or more of the subject's B cells with an amount of multivalent antigen particles is administered with or without direct combination with administering monovalent antigen particles to the subject. Item 18. The method of any one of items 1 to 17, wherein the multivalent antigen particle comprises at least two copies of the antigen structure in spatial proximity to each other, preferably within nanometer range. Item 19. The method of any one of items 1 to 18, wherein the antigen is an autoantigen, a cancer-associated antigen, or a pathogen-associated antigen. Item 20. The method of item 19, wherein the pathogen is selected from a parasite, a unicellular eukaryote, a bacterium, a virus, or a virion. Item 21. The method of any one of items 1 to 20, wherein the antigen is an antigen associated with a disease or condition, preferably a disease or condition that the subject is suffering from or suspected of suffering from. Item 22. The method of any one of items 1 to 21, wherein the antigen is a natural or synthetic immunogenic substance, such as a complete, fragmented or partial immunogenic substance, and the immunogenic substance may be selected from a nucleic acid, a carbohydrate, a peptide, a hapten, or any combination thereof. Item 23. The method of any one of the preceding items, which is a method for treating a disease or condition in a subject. Item 24. The method of item 23, wherein the disease or condition is selected from diseases or conditions characterized in that an increase or decrease in a cell-mediated immune response is beneficial for treatment. Item 25. The method of items 23 or 24, wherein the disease or condition is selected from an inflammatory disorder, an autoimmune disease, a proliferative disorder, or an infectious disease. Item 26. A method for treating or preventing a disease characterized by the presence of immunoglobulin G (IgG) type antibodies specific for a disease-associated antigen in a subject, comprising administering to the subject a therapeutically effective amount 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. Item 27. The method of item 26, wherein the disease is an autoimmune disease. Item 28. The method of items 26 or 27, wherein the disease-associated antigen is an autoantigen. Item 29. The method of any one of items 26 to 28, wherein the disease is characterized by the presence of endogenous multivalent antigen particles that are composed of antigenic moieties that include two or more of the antigenic structures that are capable of inducing an antibody-mediated immune response against disease-associated antigens, and two or more of the antigenic structures are covalently or non-covalently cross-linked to form a complex disease-associated antigenic structure. Item 30. The method of item 29, wherein the therapeutically effective amount of the monovalent antigen particles is an amount that, when administered to a subject, results in a (serum / tissue) ratio of administered monovalent antigen particles to endogenous polyvalent antigen particles of greater than 1. Item 31. A method for treating or preventing a disease by vaccination in a subject, 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 composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against a disease-associated antigen, and in which two or more of the antigenic structures are covalently or non-covalently crosslinked; Administering an effective amount of a vaccination composition comprising: Item 32. The method of item 31, wherein the disease-associated antigen is a foreign antigen. Item 33. The method of item 31 or 32, wherein the vaccination composition has a ratio of (i) to (ii) of less than 1. Item 34. An immunogenic composition 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 the antigen; (ii) A multivalent antigen particle that is composed of an antigenic moiety that includes two or more of the antigenic structures capable of inducing an antibody-mediated immune response against the antigen, and two or more of the antigenic structures are covalently or non-covalently cross-linked. and an immunogenic composition comprising: Item 35. The immunogenic composition of item 30, wherein the antigen structures capable of inducing an antibody-mediated immune response against the antigens of (i) and (ii) are identical. Item 36. The immunogenic composition of item 34 or 35, further comprising a pharma- ceutically acceptable carrier and / or excipient. Item 37. A monospecific IgM antibody or variant thereof for use in the treatment of an autoimmune disease, wherein the monoclonal IgM antibody is specific and has high affinity for an antigen associated with the autoimmune disease. Item 38. Antibodies, 10 -7 Less than 10 -8 Less than 10, more preferably -9 less than about 10 -10 K D 38. The use of item 37, wherein the monospecific IgM antibody or a variant thereof binds to an antigen associated with an autoimmune disease. Item 39. A monospecific IgM type antibody or a variant thereof for use according to item 37 or 38, wherein the monoclonal IgM does not bind to unrelated antigens other than the antigen associated with the autoimmune disease. Item 40. A monospecific IgM type antibody or variant thereof for use according to any one of items 37 to 39, wherein the treatment does not include the use of a multispecific antibody specific for an unrelated antigen other than the antigen associated with the autoimmune disease. Item 41. The monospecific IgM type antibody or variant thereof for use according to any one of items 37 to 40, wherein the variant is a monospecific IgG type antibody or variant thereof, having an attenuated Fc and preferably defective in interaction with Fcγ receptors or C1q for use in the treatment of an autoimmune disorder or an alloimmune disorder.

[0226] 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 (i) A, (ii) B, and (iii) each of 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] Unless otherwise specified, the general methods and techniques described herein are 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).

[0231] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. EXAMPLES

[0232] 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.

[0233] An example is as follows.

[0234] 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 (Figures 2a and 2b). 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).

[0235] 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.

[0236] 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 (Figure 1e, Figure 1f; Figure 2c).

[0237] 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.

[0238] 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).

[0239] Either biotinylated C-peptide conjugated by incubation was used with streptavidin (SAV). Alternatively, KLH was used as a carrier coupled to C-peptide to generate multivalent conjugated antigen (cCP). The unconjugated form of C-peptide (sCP) was used as 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 autoreactive antibody responses (Figure 3c). As expected, sCP did not induce detectable IgM or IgG immune responses, 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).

[0240] 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 revealed no significant differences in the different groups of mice (Suppl. Fig. 3b, Fig. 3c). Furthermore, no differences were detected in the IgG response to the carrier of C-peptide (Fig. 4d).

[0241] 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).

[0242] 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).

[0243] 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.

[0244] 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 may 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 elicits 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 when injected in soluble form, as it regulates glucose metabolism similarly to its non-biotinylated endogenous counterpart (data not shown). Nat The conjugates were injected into mice 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 (Fig. 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) (Fig. 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 immunized with 10 μg of Insulin. NatThe complex was injected 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 not related to the upregulation of the complex Ins Nat Insulin-injected wild-type mice had detectable glucose in their urine (Fig. 5d). Nat Water consumption in wild-type mice injected with α-glucose was dramatically increased (Figure 5e). Due to the unexpected severity of diabetic symptoms, the mice were sacrificed on day 27 and the pancreas and spleen were analyzed.

[0245] In contrast to control mice, the complex Ins Nat Immunized mice showed greatly increased recruitment of macrophages, neutrophils, and B cells to the pancreas (Fig. 5f). Nat IgG+ macrophages from complex-immunized mice showed binding of native insulin (Fig. 5f), thus suggesting an autoantibody-mediated acute inflammatory process in the pancreas. FACS analysis showed that IgG+ macrophages from complex-immunized mice were significantly more sensitive to native insulin than those from control mice. Nat Although ELISpot analysis showed no difference in splenic B cells between mice immunized with or without the complex Ins (Fig. 6), Nat revealed significantly increased numbers of splenic B cells secreting anti-insulin IgG in mice injected with IgG (Fig. 5g).

[0246] To test whether secreted IgG was responsible for diabetic symptoms, the complex Ins Nat We performed IgG pull-down experiments using sera from mice injected with insulin-specific 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 the anti-insulin IgG in total IgG after purification. NatUp to 40% (0.4 mg / mg) of the IgG isolated from the mice was found to be insulin-responsive, suggesting that direct serum IgG measurements fail to detect total insulin-specific IgG due to binding to endogenous insulin (compare Fig. 5a and Fig. 5h). To test the pathogenicity of the isolated anti-insulin IgG, equal amounts of IgG from control immunizations were 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 caused diabetic symptoms (Fig. 5j).

[0247] 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.

[0248] 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 significant reduction of this autoreactive IgG on day 28 (Figure 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 described above (Figure 5a, i).

[0249] 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 (Figure 7a). This was not observed in mice immunized with the viral peptide (Figure 8a, Figure 8b). 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 (Figure 7b).

[0250] 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).

[0251] 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 (Figure 7e and Figure 7f).

[0252] The presence of antigen-specific B cells at d28 after immunization was confirmed by FACS analysis (Figures 9a and 9b). 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).

[0253] Taken together, the data suggest that increasing the proportion of complex multivalent self-antigens leads to increased amounts of autoreactive IgG and subsequent self-destructive autoimmune responses in wild-type animals.

[0254] 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 11b).

[0255] To directly test whether the increased ratio of autoreactive anti-insulin IgM counteracts the negative effect 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 (Figure 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 (Figures 13a-c).

[0256] 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 of 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, 7i, and 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. It was found that IgM generated after booster InsA immunization showed higher anti-insulin affinity compared to primary IgM collected on day 7 (Figure 7j). To further investigate the protective role of pIgM, mice were immunized with cInsA or cInsA together with intravenous injection of 50 μg purified IgM containing 5 μg pIgM (Figures 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 (Figure 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 (Figure 15c).

[0257] 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.

[0258] 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 16b). 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 (Figure 16c) and indirect immunofluorescence (Figure 16d) using HEp-2 slides showed that, in contrast to primary IgM, memory PR-IgM was not polyreactive but specifically bound insulin (Figures 16c and 16d).

[0259] To show that anti-insulin IgM is specifically responsible for the observed effects, we performed insulin-specific pull-down assays using serum from InsA-immunized mice. Pull-downs yielded pure insulin-specific IgM as revealed by Western blot analysis against insulin (Figure 17). We performed anti-DNA ELISA (Figure 16e) and indirect immunofluorescence on HEp-2 slides (Figure 16f) using purified primary anti-insulin IgM or memory anti-insulin PR-IgM. The results support the observation that, in contrast to primary IgM, purified anti-insulin PR-IgM is not polyreactive and specifically binds insulin (Figures 16e and 16f). To directly test the hypothesis that primary anti-insulin IgM and not PR-IgM form large immune complexes, we incubated anti-insulin primary IgM or PR-IgM with insulin and DNA and determined immune complex formation using size-exclusion spin columns. In contrast to PR-IgM, we found that primary anti-insulin IgM mainly formed large complexes of >104 kD (Figure 16g). To show that purified primary anti-insulin IgM was responsible for the dysregulation of glucose metabolism, we intravenously injected 5 μg of purified anti-insulin primary IgM or PR-IgM and monitored blood glucose. In contrast to PR-IgM, a strong increase in blood glucose was observed after injection of purified primary anti-insulin IgM (Figure 16h). Interestingly, the increase in blood glucose appeared earlier after injection of purified anti-insulin primary IgM compared to total primary IgM (Figure 16h).

[0260] In summary, these data suggest that increased specificity for self-antigens is important for autoreactive memory PR-IgM to be protective during immune responses (Figure 18). Furthermore, the inducible generation of autoreactive PR-IgM is most likely a critical step in B cell tolerance.

[0261] Example 5: Immunization Scheme The impact of the immune concept of the present invention on vaccine design was tested using a pathogen-specific antigen derived from the SARS-CoV-2 coronavirus, which causes Covid-19. During infection, the SARS-CoV-2 coronavirus binds to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its receptor binding domain (RBD). Thus, eliciting an antibody response that blocks the RBD / ACE2 interaction is believed to be key to preventing coronavirus infection. Therefore, we used the RBD from SARS-CoV-2 to determine the role of the antigen in the immune response during immunization.

[0262] Immunization with complexed RBD (cRBD) (streptavidin and biotinylated RBD were used in a 4:1 ratio for complexation) was found to induce a stronger IgG immune response compared to soluble RBD (sRBD). For production of RBD, an expression vector encoding a hexahistidine-tagged version of RBD was transiently transfected into HEK293-6E cells (Amanat, F., et al., 2020, Nature medicine, 26(7), 1033-1036). Soluble RBD was purified from the supernatant 5 days after transfection by nickel-based immobilized metal affinity chromatography (TaKaRa). However, antibody concentrations were not sufficient to allow virus neutralization using in vitro infection experiments. Therefore, we tested whether pre-treating mice with sRBD before immunization would boost the immune response. Indeed, pretreatment of mice with soluble RBD 2 weeks prior to immunization significantly enhanced the immune response (Figure 19). Importantly, sera from pretreated mice showed a highly potent ability to prevent SARS-CoV-2 infection in vitro.

[0263] Furthermore, it has been found that different ratios of sRBD to cRBD in the composition of the immunization cocktail result in different ratios of immunoglobulin isotypes (i.e., IgG to IgM) that allow for precise control of the immune response following immunization.

[0264] Example 6: Anti-insulin IgG regulates blood glucose concentrations. We noticed that a significant amount of total IgG isolated from wild-type (WT) mice was reactive to insulin (Figure 21A and Figure 21B). To confirm these data, we performed an ELISpot assay and found that anti-insulin IgG-secreting B cells were present in the spleens of WT mice (Figure 21C). We measured blood glucose concentrations in WT and B cell-deficient mice unable to produce antibodies and detected a surprising difference. Unexpectedly, B cell-deficient mice showed abnormally reduced blood glucose levels compared to WT controls (Figure 21D).

[0265] To test whether this abnormal decrease was caused by antibody deficiency, we intravenously injected B cell-deficient mice with total IgG from WT mice, or with an anti-insulin IgG-depleted control of the same total IgG. We found that blood glucose concentrations increased with total mouse IgG, but not with anti-insulin IgG-depleted controls (Figure 21E). To test the consequences of the steady-state blood glucose reduction on fitness, we performed a wire hanging test to assess motor function and found that B cell-deficient mice significantly reduced wire hanging time compared to WT controls. Importantly, this loss in wire hanging time was restored after intravenous injection of whole mouse IgG (Figure 21F). In addition, B cell-deficient mice also showed dysregulated blood glucose levels after rotarod exercise.

[0266] Because total IgG preparations from healthy donors are often used as intravenous immunoglobulin (IVIg) injections in the treatment of immune deficiencies, we tested the presence of anti-insulin IgG in these preparations. All preparations contained significant amounts of anti-insulin IgG. However, anti-insulin IgG concentrations appeared to be increased when the United States was the country of origin. Because insulin is highly conserved between humans and mice, we injected human IVIg into B cell-deficient mice and detected reduced insulin concentrations (Figure 21G). Furthermore, injection of 50 μg human IVIg into WT mice elevated blood glucose, and this effect required anti-insulin IgG, because depletion of anti-insulin IgG from human IVIg prevented IVIg-induced blood glucose elevation (Figure 21H).

[0267] To test whether IVIg injection would show similar results in human patients suffering from antibody deficiency, we monitored blood glucose before and after IVIg injection. Similar to B cell-deficient mice, antibody-deficient patients showed reduced blood glucose concentrations compared to healthy donors. Importantly, after IVIg injection, blood glucose concentrations increased and reached normal levels (Figure 21I). Furthermore, immunodeficient patients who received IVIg showed reduced serum insulin levels.

[0268] To demonstrate that the anti-insulin IgG present in IVIg is specific for insulin, we determined the affinity via biolayer interferometry (BLI). The dissociation constants of 10–11 suggest that the anti-IgG is highly specific for insulin (Figure 21J).

[0269] These data suggest that anti-insulin IgG is present in healthy individuals and may be necessary for the regulation of blood glucose levels.

[0270] Example 7: Regulation of blood glucose by anti-insulin IgM To further confirm our findings regarding the presence of anti-insulin antibodies in healthy individuals, we evaluated anti-insulin IgG and IgM in the blood of different age groups. We found that anti-insulin IgG was similar in young and old humans, whereas anti-insulin IgM appeared to decrease with age in men and women (Figure 22A). Interestingly, human anti-insulin IgM recognizes multiple epitopes on insulin.

[0271] Consistent with the high specificity, anti-insulin IgG did not show binding to any cellular structures in an indirect immunofluorescence assay (IIFA) on HEp-2 cells, a method commonly used for the detection of antinuclear antibodies. However, anti-insulin IgM consists of two fractions that can be biochemically separated according to their affinity for insulin. Low affinity anti-insulin IgM elutes from the insulin column at a higher pH (5) compared to high affinity anti-insulin IgM, which requires acidic conditions (pH = 2.8) for elution (Figure 22B, Figure 22C). Low affinity IgM shows polyreactivity detected by binding to nuclear structures in IIFA and dsDNA binding in ELISA, whereas high affinity IgM is virtually negative in these assays (Figure 22D, Figure 22E). Furthermore, we confirmed the difference in affinity by performing a BLI assay, with 10% of each antibody binding to nuclear structures in IIFA and 10% of each antibody binding to insulin. -10 and 10 -7We found high-affinity IgM and low-affinity IgM with dissociation constants of 0.01 and 0.01 (Fig. 22F). To test the effect of the different IgM fractions on glucose metabolism, we injected WT mice with equal amounts of insulin-responsive IgMhigh and IgMlow. In mice that received IgMlow, we observed an increase in blood glucose within 2 h after injection, whereas IgMhigh did not significantly change blood glucose levels (Fig. 22G). Furthermore, we tested whether IgMhigh plays a regulatory role under conditions of abnormally increased insulin concentrations that could lead to hypoglycemia. To this end, we injected 0.1 μg of insulin in combination with IgMhigh or a nonspecific IgM isotype control. Surprisingly, the presence of anti-insulin IgMhigh, but not IgM isotype control, prevented the dramatic drop in blood glucose that occurred immediately after insulin injection (Fig. 22H). To further test the regulatory role of IgMhigh in protecting insulin from IgG-mediated degradation, we combined anti-insulin IgMhigh with anti-insulin IgG purified from IVIg preparations. The data indicate that anti-insulin IgMhigh functions as PR-IgM to prevent IgG-mediated neutralization of insulin, which leads to elevated blood glucose levels (Figure 22I). These data suggest that anti-insulin IgMhigh is important for regulating glucose metabolism by protecting insulin from IgG-mediated neutralization and binding excess insulin, thereby preventing a dramatic drop in insulin concentrations. The age-related decrease in insulin-responsive IgM (Figure 21A) prompted us to test whether anti-insulin IgMhigh or IgMlow was affected by this decrease. We determined the amount of anti-insulin IgMhigh or IgMlow in young and old healthy donors and found that the proportion of anti-insulin IgMhigh increased with age (Figure 22J).

[0272] Taken together, these data suggest that glucose metabolism is regulated by distinct classes of antibodies and that anti-insulin IgMhigh functions as a PR-IgM that regulates glucose metabolism by regulating insulin homeostasis, which may be particularly important with age.

[0273] Example 8: Induction of anti-insulin antibodies by insulin conjugates. To examine whether the conjugated autoantigen can induce an autoreactive antibody response independent of any adjuvant, we incubated insulin with 1,2-phenylene-bis-maleimide, a typical homobifunctional crosslinker, which covalently binds to free sulfhydryl groups in proteins, thereby crosslinking the protein of interest (Figure 23A). Importantly, sulfhydryl-containing drugs have been reported to induce anti-insulin autoantibodies. Furthermore, increased pancreatic activity and elevated insulin production may lead to abnormal formation of disulfide bonds between insulin peptides, which may generate abnormal insulin forms that are more susceptible to sulfhydryl-mediated crosslinking and thus more susceptible to conjugation under conditions of oxidative stress. The homobifunctional crosslinking of insulin with 1,2-phenylene-bis-maleimide was examined by SDS page, and the crosslinked insulin was purified using a size-exclusion spin column that excludes monomeric and dimeric insulin (Figure 23B). Insulin conjugates were dialyzed and injected into WT mice at 5 μg per mouse without additional adjuvant. As a control, a typical immunization was performed using CpG as an adjuvant and streptavidin as an exogenous carrier. We found that insulin conjugates led to an increase in blood glucose and anti-insulin IgM at d7 of treatment similar to immunization (Figure 23C, Figure 23D). In addition, insulin-reactive IgG was detectable by ELISA at d14 and d26. Repeated injection of insulin conjugates at d21 led to further deregulation of glucose metabolism (Figure 23E). For this reason, anti-insulin IgMhigh was injected at d22, one day after injection of insulin conjugates. We found that anti-insulin IgMhigh could prevent blood glucose deregulation induced by injection of insulin conjugates (Figure 23E).

[0274] Furthermore, we found that anti-insulin IgMhigh prevented pancreatic inflammation and damage, as shown by reduced macrophage (CD11b+ / LY6G+) and neutrophil (LY6G+) infiltration in the pancreas, as well as reduced serum pancreatic lipase in the blood (Figures 23F and 23G).

[0275] As a mechanism for the protective role of anti-insulin IgMhigh compared to anti-insulin IgMlow, we proposed that the polyreactivity of the latter, which also binds to dsDNA, induces the formation of immune complexes that can be phagocytosed by macrophages, whereas anti-insulin IgMhigh is highly specific for insulin and therefore does not form large immune complexes that are easily phagocytosed by macrophages. To test this, we incubated anti-insulin IgMhigh or anti-insulin IgMlow with insulin in the presence of genomic dsDNA (Figure 23H). We found increased binding / phagocytosis of anti-insulin IgMlow compared to anti-insulin IgMhigh (Figure 23). Furthermore, IgMhigh could protect insulin from degradation, since there was a greater decrease in insulin in the supernatants containing anti-insulin IgMlow compared to anti-insulin IgMhigh antibodies.

[0276] These data indicate that anti-insulin antibodies can be generated under conditions that activate the formation of insulin complexes, leading to deregulation of glucose metabolism that can be counteracted by anti-insulin IgMhigh acting as PR-IgM.

[0277] Example 9 Recombinant anti-insulin IgM can regulate blood glucose. The above results suggest that insulin-specific PR-IgM may be of great therapeutic interest because it may regulate insulin homeostasis and prevent pancreatic insufficiency, both of which are essential for normal physiology and prevention of diabetes. According to our data, anti-insulin IgM can function as PR-IgM if it has high affinity for insulin and does not react to self-antigens such as dsDNA or nuclear structures in IIFA. We hypothesized that human insulin-specific IgG antibodies could be converted into insulin-specific PR-IgM by exchanging the constant region.

[0278] Therefore, we cloned and expressed a published human insulin-specific antibody

[60] as IgG1 (anti-insulin IgGrec) and IgM (anti-insulin IgMrec) (Figure 24A). To test the quality of the in vitro produced antibodies, we assessed their glycosylation by PNGaseF treatment, which resulted in a reduced molecular weight compared to the untreated control, suggesting functional glycosylation. We increased the affinity of both IgG and IgM by 10 -9 We determined that the dsDNA binding was significantly greater than that of the total human serum IgM (Fig. 24B). Compared to total human serum IgM, little dsDNA binding was observed by ELISA and no nuclear staining was observed by IIFA (Fig. 24C,D). Furthermore, we tested whether monomeric anti-insulin-IgM could protect insulin from degradation. Anti-insulin IgG caused an increase in blood glucose that was abolished when monomeric anti-insulin IgM was present (Fig. 24E).

[0279] To test whether the resulting recombinant human anti-insulin IgMrec has a protective regulatory function, we injected it together with insulin and found that anti-insulin IgMrec prevented the dramatic drop in glucose concentration induced by excess insulin (Fig. 24F). Moreover, anti-insulin IgMrec protects insulin from anti-insulin IgGrec-mediated neutralization, thus preventing the increase in blood glucose induced by anti-insulin IgGrec (Fig. 24G). In addition, anti-insulin IgMrec counteracts glucose leakage into the urine (Fig. 24H).

[0280] These data suggest that expressing high-affinity insulin-specific antibodies as IgM may modulate insulin homeostasis, prevent dysregulation of blood glucose concentrations, and provide a novel strategy for the treatment of insulin-related diseases and disorders.

[0281] Example 10: Prediction of disease parameters Highly autoreactive primary IgM repertoire represents a high risk of autoreactive damage if high affinity PR-IgM cannot be generated by secondary immune responses and somatic hypermutation. Thus, memory IgM repertoire consists of the majority of PR-IgM generated during adaptive tolerance. Somatic hypermutation results in failure of PR-IgM generation and autoimmune damage induced by primary IgM. Furthermore, all forms of hyper IgM syndrome (HIGM) are associated with severe autoimmunity. HIGM patients are particularly prone to develop IgM-mediated autoimmune diseases such as immune thrombocytopenia, hemolytic anemia and nephritis. We measure the affinity of autoreactive IgM antibodies that cause autoimmune and / or insulin-related diseases and consider (i) low affinity to autoantigens as a risk factor for disease onset, disease progression and / or death, and (ii) high affinity autoreactive IgM as protective.

[0282] Materials and Methods Mice used in Examples 1 to 5 C57BL / 6 and B cell deficient mice aged 8-30 weeks were immunized intraperitoneally (ip) with a mixture of 13-50 μg of antigen containing 50 μg of CpG-ODN1826 (Biomers) in 1× PBS. Control immunized (CI) mice received PBS and CpG-ODN1826 (50 μg / mouse). Native biotinylated mouse insulin was purchased from BioEagle.

[0283] Mice used in Examples 6 to 9 Female C57BL / 6 and mb1 mice aged 8–15 weeks were intraperitoneally (i.p.) injected with a mixture of 10 μg of antigen (cInsulin or Insulin-bio:SAV) in 1x PBS. Control-injected (CI) mice received PBS in a total volume of 100 μL / mouse. Animal experiments were performed in accordance with License 1484 for animal experiments at the responsible regional committee in Tübingen, Germany. All mice used in this study were bred and housed in the animal facility of the University of Ulm under specific pathogen-free conditions or obtained from Jackson at 6 weeks of age. All animal experiments were performed in accordance with the guidelines of German legislation and approved by the Animal Care and Committee of the University of Ulm, as well as the local authorities.

[0284] peptide C-Peptide peptides (RoyoBiotech, Shanghai), insulin and virus-derived peptides (SEQ ID NO: 43; SEQ ID NO: 44) (Peptides&Elephants, Berlin) were dissolved according to their water solubility in pure water, 1% DMSO or 1% dimethylformamide (DMF). Virus-derived peptides (SEQ ID NO: 43; SEQ ID NO: 44) were conjugated to biotin or KLH, respectively. Quantities of 1 mg were purchased and dissolved in a volume of 1 ml. 10–50 μg of KLH-conjugated peptides were used for immunization of mice via intraperitoneal injection. For covalent conjugation of peptides to keyhole limpet hemocyanin (KLH), an N-terminal cysteine ​​was added. Conjugation of peptides to streptavidin (SAV, ThermoScientific) was performed by addition of biotin to the N-terminus. The C-terminus left an OH group for better handling.

[0285] Cross-linking of native insulin and InsA peptides. Native human insulin (Merck) was pre-diluted to 1 mg / mL in PBS. Chemical thiol cross-linking was performed with 1,2-phenylene-bis-maleimide (Santa Cruz, 13118-04-2) at 10 μg / mL, followed by removal using a 10 kD cut-off spin column (Abcam, ab93349). Purified insulin conjugate (cInsulin) was used for intraperitoneal injection of 10 μg per mouse in a total volume of 100 μL.

[0286] Flow cytometry Cell suspensions were blocked with polyclonal rat IgG-UNLB (2,4G2; BD) and stained according to standard protocols. Biotin-conjugated peptides / antibodies were detected using streptavidin Qdot605 (Molecular Probes; Invitrogen). Viable cells were distinguished from dead cells by the use of the fixable viability dye eFluor780 (eBioscienc). Cells were acquired on a Cato II Flow Cytometer (BD). Unless otherwise stated, numbers in plots indicate percentages within the respective gates and numbers in histogram plots indicate mean fluorescence intensity (MFI).

[0287] Enzyme-linked immunosorbent assay (ELISA) 96-well plates (Nunc, Maxisorp) were coated with 10 μg / mL of native insulin (Sigma-Aldrich, Cat. 91077C), streptavidin (ThermoScientific, Cat. 21125), or calf thymus DNA (ThermoScientific, Cat. 15633019), or anti-IgM, anti-IgG antibodies (SouthernBiotech). Biotinylated peptides (2.5 μg / mL) of SAV plates were loaded and blocking was performed in 1% BSA blocking buffer (Thermo Fisher). Serial dilutions of 1:3 IgM or IgG antibodies (SouthernBiotech) were used as standards. Relative concentrations, shown as arbitrary units (AU), were determined by detection with alkaline phosphatase (AP)-labeled anti-IgM / anti-IgG (SouthernBiotech), respectively. p-Nitrophenyl phosphate (pNPP; Genaxxon) in diethanolamine buffer was added and data were acquired at 405 nm using a Multiskan FC ELISA plate reader (Thermo Scientific). All samples were measured in duplicate.

[0288] For affinity maturation analysis, results from plates coated with either peptide (1) or peptide (4) were calculated by dividing peptide (1) by peptide (4), and therefore results are reported as relative units [RU] in the figures.

[0289] Enzyme-linked immunospot assay (ELISpot) Total splenocytes were measured in triplicate at 300.000 cells / well. ELISpot plates were pre-coated with either native insulin (Sigma-Aldrich, Cat. 91077C) or C-peptide (RoyoBiotech). Cells were incubated at 37°C for 12-24 h before antigen-specific IgM or IgG was detected by anti-IgM-bio:SAV-AP or anti-IgG-bio:SAV-AP (Mabtech). Handling of plates and antibody concentrations were performed according to the manufacturer's recommendations.

[0290] HEp-2 slides and fluorescence microscopy HEp-2 slides (EUROIMMUN, F191108VA) were used to evaluate serum IgM reactivity against nuclear antigens (ANA). Sera from insulin-A-peptide immunized mice on days 7 and 85 post-immunization were diluted to equal concentrations of IgM (approximately 300ng / mL of anti-insulin-IgM in both immunized samples) and applied onto HEp-2 slides. Anti-IgM-FITC (eBioscience, Cat. 11-5790-81) was used for detection of ANA-IgM. Stained HEp-2 slides were analyzed using a fluorescent microscope Axioskop2 (Zeiss) and DMi8 software (Leica).

[0291] Glucose Level Monitoring Assessment of urinary glucose levels was performed using Combur 10 M test stripes (Roche Diagnostics, Mannheim). Sterile stripes were used during daily mouse handling and the color displayed after testing was compared to the manufacturer's standard for glucose levels in mmol / L. Blood glucose levels of mice were measured using an AccuCheck (Roche Diagnostics, Mannheim) blood glucose monitor. Blood was collected from the tail vein of ad libitum-fed mice and transferred to sterile test stripes. Glucose levels were measured in mmol / L for each mouse per group on the days indicated in the figure. Control immunizations were performed with littermates and measured at similar times of day.

[0292] SDS page, Coomassie and Western blot. Organs were harvested immediately after sacrifice and lysed in RIPA buffer containing protease and phosphatase inhibitors (50 mM TrisHCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA (pH 8), 1 mM sodium orthovanadate, 1 mM NaF, protease inhibitor cocktail (Sigma-Aldrich). Samples were resolved on 10–20% SDS-polyacrylamide gels and blotted onto PVDF membranes (Millipore) or incubated with Coomassie (Coomassie Brilliant Blue R-250, ThermoFisher) for 45 min followed by destaining. Membranes were subsequently blocked in 5% BSA PBS for 1 h at room temperature with constant agitation. Primary antibodies were diluted in 5% BSA PBS (BIOMOL Research Laboratories). Secondary antibodies were diluted in 5% BSA PBS. Membrane development and data recording were performed using the Fusion optical system. This was performed using SL (Vilber).

[0293] Total serum immunoglobulin pull-down Immediately after euthanasia, serum from immunized mice was collected and either IgM or IgG was purified. Removal of antigens bound to antibodies was achieved by repeated freeze-thaw cycles of serum and pH shift during elution. For IgG protein G, Sepharose beads (Thermo Fisher) were used according to the manufacturer's protocol and dialyzed overnight in 10x sample volume in 1x PBS. For IgM, HiTrap IgM columns (GE Healthcare, Sigma-Aldrich) were used according to the manufacturer's protocol and dialyzed overnight in 10x sample volume 1x PBS. Quality check of isolated immunoglobulins was addressed by SDS page and Coomassie and the amount of insulin-specific immunoglobulins was determined by ELISA. Finally, 20–50 μg (1–10 μg insulin-specific Ig) was injected intravenously.

[0294] Isolation of insulin-specific serum immunoglobulins Immediately after euthanasia, serum from InsA and control immunized mice was collected and prepared for insulin-specific immunoglobulin isolation. Streptavidin bead columns (Thermo-Scientific, Cat. 21115) were loaded with 10 μg Bio-Insulin (BioEagle). Serum was incubated at room temperature for 90 min to ensure binding of insulin-specific antibodies to the beads. Isolation of insulin antibodies was performed by pH shift using the manufacturer's elution and neutralization solutions. The quality of the isolated immunoglobulins was examined by Coomassie and Western blot analysis using anti-IgM heavy chain (Thermo-Scientific, Cat. 62-6820) and anti-IgG heavy chain (Cell Signaling Technologies, Cat. 7076) antibodies. The isolated antibodies were dialyzed for further in vivo experiments.

[0295] Biolayer Interferometry (BLI) An interference assay (BLItz device, ForteBio) was used to determine the affinity of protein-protein interactions

[61] . Here, insulin-specific IgM (see Isolation of insulin-specific immunoglobulins) and Insulin-Bio (ThermoFisher) were used as targets. The targets were loaded onto a streptavidin biosensor (ForteBio). The binding affinity of IgM to insulin was obtained in nm. The calculated affinity value (Ka) was then used to calculate the dissociation constant (K d ) was determined. d =1 / K a The following protocol was used: 30 sec baseline, 30 sec loading, 30 sec baseline, 240 sec association, 120 sec dissociation. Manufacturer's sample buffer (ForteBio) was used for buffering samples, targets, and probes.

[0296] Wire hanging test The straight wire hanging test is used to evaluate the motor strength and function of mice. Individual mice were placed on a horizontal wire at a height of 36 cm above the cage, and then the mouse tried to stay on the wire using its legs and muscle strength. The ability of each mouse to stay on the wire in seconds was recorded. The maximum duration was set at 240 seconds. Each mouse underwent three consecutive tests. The average value was calculated from the measured data. Blood glucose levels were measured before and after the test.

[0297] statistical analysis Graphs were generated and statistical analysis was performed using GraphPad Prism (version 6.0h) software. The number of individual replicates or mice (n) is stated in the figures or figure legends. P values ​​were calculated by the tests stated in the respective figure legends. Student's t-test with Welch's correction was used to compare two groups within one experiment. P values ​​>0.05 were considered statistically significant (ns=not significant; *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001).

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Claims

1. 1. An oligomeric anti-insulin antibody, (i) a Kd<5×10, preferably as measured by surface plasmon resonance －７ and / or has an affinity for insulin and / or proinsulin of (ii) Oligomeric anti-insulin antibodies that are monospecific for insulin and / or proinsulin.

2. The oligomeric anti-insulin antibody of claim 1 , wherein the oligomeric anti-insulin antibody is an anti-insulin antibody of the IgM isotype.

3. The oligomeric anti-insulin antibody of claim 1 or 2, wherein the oligomeric anti-insulin antibody is chimeric, humanized, or human.

4. The immunoglobulin, 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 a CDR1 defined in SEQ ID NO: 9, a CDR2 defined in SEQ ID NO: 10, and a CDR3 defined in SEQ ID NO: 11, and a variable light chain (VL) comprising a CDR1 defined in SEQ ID NO: 13, a CDR2 defined in sequence GAS, and a 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. The oligomeric anti-insulin antibody of claims 1 to 3, comprising:

5. The oligomeric anti-insulin antibody a) comprising a variable heavy chain (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 with SEQ ID NO: 1, and a variable light chain (VL) sequence comprising the 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; b) comprising a variable heavy chain (VH) sequence comprising the 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 the 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 c) comprising a variable heavy chain (VH) sequence comprising the 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 a variable light chain (VL) sequence comprising the amino acid sequence of SEQ ID NO: 19 or a sequence having at least 90%, preferably at least 95%, sequence identity with SEQ ID NO: 19; The oligomeric anti-insulin antibody of claim 4.

6. A polynucleotide encoding the oligomeric anti-insulin antibody of any one of claims 1 to 5.

7. A host cell comprising the polynucleotide of claim 6.

8. A method for producing oligomeric anti-insulin antibodies comprising culturing the host cell of claim 7.

9. A pharmaceutical composition comprising an oligomeric anti-insulin antibody according to any one of claims 1 to 5, a polynucleotide according to claim 6, or a host cell according to claim 7, and a pharma- ceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, further comprising an additional therapeutic agent.

11. 11. A composition or pharmaceutical composition for use in therapy according to claim 9 or 10, said composition comprising an oligomeric anti-insulin antibody according to any one of claims 1 to 5, a polynucleotide according to claim 6, or a host cell according to claim 7.

12. 11. A composition or pharmaceutical composition according to claim 9 or 10 for use in the treatment of an insulin related disease or disorder, said composition comprising an oligomeric anti-insulin antibody according to any one of claims 1 to 5, a polynucleotide according to claim 6, or a host cell according to claim 7.

13. 1. A method for measuring the affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample for use in the diagnosis and / or prognosis of an insulin-related disease or disorder, comprising: (i) determining the affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample, comprising the steps of: the sample is obtained from a subject, the subject being diagnosed with or at risk for having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; (iii) diagnosing and / or predicting an insulin-related disease or disorder in said subject based on the comparison made in step (ii), preferably wherein a lower affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin indicates a higher risk of an insulin-related disease or disorder.

14. 1. A method of measuring the affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample for use in determining whether a subject is susceptible to treatment of an insulin-related disease or disorder, comprising: (i) determining the binding affinity of anti-insulin IgM antibodies to proinsulin and / or insulin from a sample, said sample being obtained from a subject, said subject diagnosed with or at risk of having an insulin-related disease or disorder; (ii) comparing the level determined in step (i) with a reference value; (iii) determining whether the subject is susceptible to said treatment of an insulin-related disease or disorder, preferably wherein a lower affinity of binding of anti-insulin IgM antibodies to proinsulin and / or insulin indicates a higher susceptibility to treatment of an insulin-related disease or disorder.

15. The composition or pharmaceutical composition of claim 12, or the method of claim 13 or 14, wherein the insulin-related disease or disorder is selected from the group of pancreatic injury, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, gestational diabetes, and dysglycemia.

16. 16. The composition, pharmaceutical composition or method of claim 15, wherein the glycemic abnormality is in a patient having an insulin-related disease or disorder selected from the group consisting of pancreatic injury, type 1 diabetes, type 2 diabetes, exogenous insulin antibody syndrome, and gestational diabetes.

17. A method for producing oligomeric anti-insulin and / or anti-proinsulin antibodies, preferably of the IgM isotype, comprising immunizing a mammal with a mixture of at least one monovalent insulin particle and at least one multivalent insulin particle.

18. 11. A composition or pharmaceutical composition according to claims 9-10 for the treatment and / or prevention of insulin related diseases or disorders, said composition comprising an oligomeric anti-insulin antibody according to any one of claims 1-5, a polynucleotide according to claim 6, or a host cell according to claim 7, wherein a therapeutically effective amount of said oligomeric anti-insulin antibody, said polynucleotide, or said host cell is administered.

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