WO2024054596A1 - Méthodes et compositions pour le diagnostic et le traitement d'une néphropathie iga - Google Patents

Méthodes et compositions pour le diagnostic et le traitement d'une néphropathie iga Download PDF

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WO2024054596A1
WO2024054596A1 PCT/US2023/032240 US2023032240W WO2024054596A1 WO 2024054596 A1 WO2024054596 A1 WO 2024054596A1 US 2023032240 W US2023032240 W US 2023032240W WO 2024054596 A1 WO2024054596 A1 WO 2024054596A1
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Prior art keywords
antigen
glycomimetic
igal
igm
antibody
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PCT/US2023/032240
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English (en)
Inventor
Richard D. Cummings
Elliot Chaikof
Yasuyuki Matsumoto
Sylvain LEHOUX
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Beth Israel Deaconess Medical Center, Inc.
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Publication of WO2024054596A1 publication Critical patent/WO2024054596A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Definitions

  • IgA nephropathy also known as Berger’s Disease
  • IgAN IgA nephropathy
  • ICs immune complexes
  • IgAN demonstrates significant clinical variability arising from the underlying genetic and environmental complexity contributing to the disease pathology.
  • the cause of the majority of primary IgAN cases worldwide are unknown, as the condition is largely sporadic, and only a minority of cases have been reported within clusters of families.
  • aspects of the present disclosure relate to a method of detecting levels of an antibody in a sample derived from a subject, comprising: contacting the sample with a antigen, wherein the antibody, if present, binds to the antigen; and measuring the amount of antibody bound to the antigen.
  • aspects of the present disclosure relate to a method of detecting levels of IgM antibody in a sample derived from a subject, comprising: contacting the sample containing an IgA molecule comprising a GalNAc-al-Ser/Thr antigen (Tn antigen, also known as CD175), wherein the IgM antibody, if present, binds to the GalNAc-al- Ser/Thr antigen; and measuring the amount of IgM antibody bound to the IgA molecule.
  • the antibody is an IgM antibody that binds a GalNAc-al- Ser/Thr antigen.
  • the antigen is an IgAl comprising the GalNAc- al-Ser/Thr antigen.
  • the step of contacting further comprises incubating the sample with the antigen.
  • the incubating is for a sufficient time for the antigen to bind to the antibody.
  • the antigen is adhered to a solid substrate.
  • the solid substrate is a microbead.
  • the sample is blood, blood serum, blood plasma, blood fraction, saliva, mucous, urine, or a combination thereof. In some embodiments, the sample is blood serum.
  • the step of measuring comprises the use of flow cytometry to detect the amount of antibody bound to the antigen.
  • the method further comprises separately contacting a second sample with a control antigen and comparing the amount of antibody bound to the antigen to the amount of antibody bound to the control antigen.
  • the amount of antibody bound to the antigen being higher than the amount of antibody bound to the control antigen is indicative of a positive result.
  • the positive result is indicative of a diagnosis of IgA nephropathy or Berger’s disease.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has, is suspected of having, or is at risk of developing IgA nephropathy or Berger’s disease.
  • aspects of the present disclosure relate to a method, comprising: administering to a subject a glycomimetic, wherein the glycomimetic is based on the structure of N- acetylgalactosamine (GalNAc); wherein: the subject has, is suspected of having, or is at risk of developing IgA nephropathy or Berger’s disease.
  • GalNAc N- acetylgalactosamine
  • aspects of the present disclosure relate to a method, comprising: administering to a subject a glycomimetic, wherein the glycomimetic is a-methylGalNAc or DiaGalNAc; wherein: the subject has, is suspecting of having, or is at risk of developing IgA nephropathy or Berger’s disease.
  • the glycomimetic binds an antibody that is elevated in subjects with IgA nephropathy or Berger’s disease.
  • the glycomimetic is an aGalNAc monosaccharide or disaccharide.
  • the glycomimetic is a modified aGalNAc, wherein the aGalNAc is modified to change the - OH group(s) and/or N-acetyl group.
  • the glycomimetic is an aGalNAc disaccharide.
  • the aGalNAc disaccharide further comprises linkers linking the two aGalNAc molecules.
  • the aGalNAc disaccharide comprises a carbohydrate or non-carbohydrate linker.
  • the linker is a flexible or non-flexible linker.
  • the linker is a cleavable or stable linker.
  • the glycomimetic comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers.
  • the glycomimetic further comprises one or more constituents on the aGalNAc.
  • the constituent is a carbohydrate or a non-carbohydrate constituent.
  • the glycomimetic is administered orally, subcutaneously, or intravenously. In some embodiments, the glycomimetic is administered as an oral capsule. In some embodiments, the glycomimetic, after administration, is absorbed through the gut.
  • the glycomimetic inhibits formation of an immune complex comprising an antibody and an antigen, wherein the antibody is Anti-Tn and the antigen is an IgA comprising a GalNAc-al-Ser/Thr antigen.
  • the glycomimetic is capable of dissociating the immune complex.
  • the glycomimetic inhibits proliferation of mesangial cells.
  • the subject is human.
  • administration of the glycomimetic is a treatment for IgA nephropathy or Berger’s disease.
  • the glycomimetic reduces symptoms of IgA nephropathy or Berger’s disease by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • the glycomimetic reduces the risk of developing IgA nephropathy or Berger’s disease by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • the method further comprises administering to the subject the glycomimetic and a pharmaceutically acceptable excipient.
  • FIG. 1 shows basic donor information.
  • FIG. 2 shows structures on Tn glycopeptide array.
  • FIGs. 3A-3H show Anti-Tn antibodies in human sera: identification and characterization.
  • FIG. 3A shows Coomassie- stained SDS-PAGE analysis of affinity-purified anti-Tn antibodies from serum using Tn(+)matrix (Elution) and control (beads alone); affinity- purified materials (top) immunoblotted as indicated (bottom).
  • FIG. 3B shows a similar analysis to FIG. 3A (healthy donors, FIG. 1), with purified control antibodies and BS A (4 mg); VVA detects Tn(+)IgAl.
  • FIG. 3A shows Coomassie- stained SDS-PAGE analysis of affinity-purified anti-Tn antibodies from serum using Tn(+)matrix (Elution) and control (beads alone); affinity- purified materials (top) immunoblotted as indicated (bottom).
  • FIG. 3B shows a similar analysis to FIG. 3A (healthy donors, FIG. 1), with purified control antibodies and BS A (4 mg); VVA detects
  • FIG. 3D shows a Tn glycopeptide array probed with purified anti-Tn antibodies from 10 healthy control sera (C3, representative), stained with Alexa Fluor 488-labeled anti-human IgM antibody (see FIGs. 9A-9D).
  • Chart ID corresponds to FIG. 2.
  • FIG. 3E and FIG. 3F show data analysis using GLAD, comparing binding preferences between Tn on IgAl and non-IgAl peptides, and correlation of binding patterns in C1-C10.
  • VVA binds Tn glycopeptides (positive control), and anti-Tn ( murine anti-Tn IgM) differs from C1-C10 binding.
  • FIG. 3H shows a Tn glycopeptide array probed with purified anti- Tn antibody (Cl) after preincubation with purified Tn(+)IgAl or Tn(-)IgAl, stained with Alexa Fluor 647- labeled anti-human IgM.
  • error bars represent ⁇ 1 SD of four replicates.
  • RFU relative fluorescence units.
  • FIGs. 4A-4F show elevated level of anti-Tn antibodies and total IgAl in IgAN patients.
  • FIG. 4A shows a depiction of anti- Tn IgM detection directly from human sera using Asialo-BSM microbeads, similar to Tn(+)matrix beads in FIGs. 7A-7B, flow cytometry utilizing Alexa Fluor 488 -labeled anti-human IgM for signal detection.
  • FIG. 4B depicts a plot of standard curve of purified anti-Tn IgM from human sera, and isotype human IgM serves as a control.
  • FIG. 4A shows a depiction of anti- Tn IgM detection directly from human sera using Asialo-BSM microbeads, similar to Tn(+)matrix beads in FIGs. 7A-7B, flow cytometry utilizing Alexa Fluor 488 -labeled anti-human IgM for signal detection.
  • FIG. 4B depicts a plot of standard curve
  • FIG. 4E shows a depiction of Tn(+)IgAl detection from human serum using HPA microbeads by flow cytometry utilizing FITC-labeled anti-human IgAl for signal detection.
  • FIG. 4F shows Sera with IgAN (P1-P20) or control (C1-C20) were analyzed by flow cytometry using HPA microbeads.
  • Tn(+)IgAl and Tn(-)IgAl were used as 100% and 0% respectively.
  • FIGs. 5A-5G shows the identification of anti-Tn CICs in IgAN patient and healthy control sera and complexes dissociate with glycomimetic.
  • FIG. 5A shows BN-APAGE analysis of Tn(+)matrix affinity purified anti-Tn antibodies and total purified IgM. Left, native molecular weight markers for approximation of molecular weight. MDa, megadaltons; CICs, circulating immune complexes; Pl, P3, P4 represent three patients, control IgM.
  • FIG. 5B shows Tn(+)matrix affinity-purified ReBaGs6 antibody resolved by BN-APAGE system and blotted for murine IgM.
  • FIG. 5C shows a BN-APAGE analysis of affinity-purified anti-Tn antibodies and total purified IgM from three healthy controls (C2, C3, C6), control IgM immunoblotted for IgM.
  • FIG. 5D shows Coomassie-stained SDS-PAGE gel of affinity- purified anti-Tn antibodies from healthy control (C3) and patient (P3); control antibodies and BSA as indicated. Immunoblots (bottom) of same preparation probed as indicated. VVA and IgA blots performed with appropriate controls- purified Tn(+)IgAl and Tn(-)IgAl produced from CosmcKO and WT Dakiki cells, respectively.
  • FIG. 5D shows Coomassie-stained SDS-PAGE gel of affinity- purified anti-Tn antibodies from healthy control (C3) and patient (P3); control antibodies and BSA as indicated. Immunoblots (bottom) of same preparation probed as indicated. VVA and IgA blots performed with appropriate controls
  • FIG. 5E shows Anti-Tn CICs purified from serum of IgAN patients immunodepleted with anti-IgA or isotype control antibodies; depleted materials analyzed by BN-APAGE- WB probed for IgM.
  • FIG. 5F shows Anti-Tn CICs purified from IgAN patient’s serum treated with mock, a- methylGalNAc ( aGalNAc), or a-methylGlcNAc ( aGlcNAc); samples analyzed by BN-APAGE-WB and probed for IgM and IgA.
  • 5G shows Tn glycopeptide array probed with anti-Tn antibodies (C3) preincubated with 20 mM a-methylGalNAc or 20 mM a-methylGlcNAc, stained with Alexa Fluor 488-labeled anti-human IgM. Error bars represent ⁇ 1 SD of four replicates.
  • FIGs. 6A-6R show the activity of anti-Tn CICs on HRMCs.
  • FIGs. 6A-6C show HRMC staining- DAPI (nuclear), anti-Tn CICs (green), Vimentin (red); negative control (mixture of isotypes IgM, IgG, IgA).
  • DAPI nuclear
  • anti-Tn CICs green
  • Vimentin red
  • negative control mixture of isotypes IgM, IgG, IgA
  • HC healthy control
  • FIGs. 6D-6E show surface staining by anti-Tn CICs.
  • FIGs. 6I-6L show the proliferation of anti-Tn CICs on starved HRMCs.
  • FIG. 61 shows the unstimulated (0.5% FBS), stimulated (10% FBS); HRMCs stimulated using IgAN serum (FIG.
  • FIG. 6J shows the quantification of FIGs. 61- 6K, error bars, triplicates measure (3 areas/well); n.s., not significant.
  • FIGs. 6M-6N show the exogenous addition of purified anti-Tn CICs stimulates HRMCs and glycomimetic inhibits proliferation.
  • FIG. 6O-6P show the exogenous addition of anti-Tn CICs (50 ng/100 microliters/well) purified from IgAN patient or HC to Tn(+)matrix depleted sera from IgAN patient, with/without prior treatment of anti-Tn CICs with a-methylGalNAc or a - methylGlcNAc. Scale bars, 20mm.
  • FIG. 6Q shows the quantification of FIG. 6M-6P, fold change of Ki-67 positive cells normalized to unstimulated. Error bars, triplicate measure (3 areas/well). Representative of 3 biological replicates, IgAN and HC.
  • FIG. 7A-7C shows the characterization of Tn(+)matrix beads.
  • FIG. 8 shows the mass spectrometry analysis of the components of the purified anti- Tn antibodies.
  • FIGs. 9A-9D show the purified anti-Tn antibodies bind to Tn glycoform containing IgAl.
  • FIGs. 10A-10E shows the establishment of CosmcKO cell line to generate galactose- deficient IgAl glycoform, Tn(+)IgAl.
  • FIGs. 11A-11C show immune complexes of the total serum, from both healthy and IgAN patients.
  • FIGs. 12A-12C show how Glycomimetics -Di aGalNAc specifically inhibits the binding of anti-Tn CICs to IgAl glycopeptide.
  • FIGs. 13A-13C show the cell proliferation assay on primary mesangial cells using serum from IgAN patients and healthy controls.
  • FIGs. 14A-14C show that purified Anti-Tn CICs from both IgAN patient’s and healthy control serum do not stimulate HEK293T cells.
  • FIGs. 15A-15B show the synthesis of GalNAc dimer (Di aGalNAc) and GlcNAc dimer (Di aGlcNAc).
  • FIG. 16 shows a diagnostic assay for IgA nephropathy.
  • IgA nephropathy IgAN
  • Tn(+)IgAl galactose-deficient IgAl
  • anti-Tn CICs novel anti-Tn circulating immune complexes
  • Anti- Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. It has been found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgAl, releasing IgAl from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of IgAN patients. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are currently unavailable.
  • IgAN A contributing component to IgAN is the unusual nature of the glycosylation of IgAl. Unlike IgA2, IgAl contains 22 amino acids in a hinge region (HR), comprised primarily of Ser/Thr/Pro residues, in which 3- 6 of the 9 Ser/Thr residues may be modified by O- glycans 11,12 .
  • HR hinge region
  • Tn antigen GalNAcal-Ser/Thr here designated Tn(+)IgAl and also termed galactose-deficient IgAl or Gd-IgAl
  • mono- and/or di-sialyl Core 1 structures Gaip3GalNAcal-Ser/Thr here designated Tn(- )IgAl 13,14 .
  • Tn(+)IgAl any form of Tn antigen on IgAl is considered Tn(+) IgAl irrespective of healthy and IgAN patient serum.
  • Total IgAl is frequently elevated in sera of patients with IgAN 8,9,15- 19 , and some of those studies indicate that there is an accompanying elevation of the Tn(+)IgAl glycoform.
  • Tn(+)IgAl and Tn(-)IgAl the relative proportion of these two glycoforms does not appear to be statistically different in patients, but the overall elevation of IgAl in patients leads to a concomitant rise in both glycoforms 13 ’ 14 .
  • Tn(+)IgAl glycoforms 8 The emerging picture suggests that the Tn(+)IgAl glycoform associates with anti-IgAl specific IgG, IgA, or IgM antibodies to form large macromolecular immune complexes, depositing in the mesangium and ultimately responsible for disease pathology 8,25-29 .
  • Tn antigen glycomimetics can dissociate these anti-Tn CICs to release Tn(+)IgAl, and also inhibit the proliferative nature of the anti-Tn CICs on primary human renal mesangial cells.
  • a key focus of the study was to define the identities, properties, and major components of the anti-Tn CICs in human serum, particularly focused on IgAN.
  • a keyadvantage of the approach taken was the affinity purification of such complexes using a Tn(+)matrix, which allowed for the identification and quantification of the anti-Tn CICs and their ability to bind the Tn(+)IgAl.
  • a key aspect of the study was the generation of unique glycoforms of IgAl, Dakiki-derived materials with or without Cosme expression representing Tn(-)IgAl and Tn(+)IgAl, respectively. Both goals were succeeded and the availability of the Tn(+)matrix and Tn(-)IgAl and Tn(+)IgAl glycoforms should be helpful in future studies to determine the interactions of IgAl with autoantibodies.
  • anti-Tn CICs anti-Tn circulating immune complexes
  • Novel anti-Tn CICs of large macromolecular assemblies were identified, predominantly containing IgM together with Tn(+)IgAl and some level of IgG. The level of such antibodies was measured, where a correlation with IgAN diagnosis was found.
  • anti-Tn CICs can be disrupted by glycomimetics, which block recognition of the Tn antigen, releasing IgAl from such complexes.
  • the glycomimetic compound blocks the proliferative activity of anti-Tn CICs toward primary human renal mesangial cells.
  • IgM, Tn(+)IgAl, and IgG form CICs of several megadaltons size, which contain predominantly IgM. There may be other proteins associated with these large molecular masses, based on BN-APAGE size estimation and Coomassie stained SDS-PAGE bands, while the smaller size CICs of -1.2 MDa may contain IgM and few IgAl molecules. It is not unexpected that the anti-Tn CICs are heterogeneous in terms of combinations of IgM, Tn(+)IgAl, and IgG as well as other apparent sub- stoichiometric level of proteins present within the complexes.
  • IgM directly interacts with the Tn antigen of IgAl within anti-Tn CICs that have been purified, since both the Tn matrix used for the pull down and IgAl itself have the Tn antigen, and the IgM does bind to the Tn antigen.
  • the anti-Tn CICs that were discovered deposit in the kidney and are responsible for IgAN pathogenesis, but the hypothesis is that elevated levels of anti-Tn CICs containing predominantly complement C3 deposit in the kidney and promote IgAN pathogenesis.
  • whether such anti-Tn CICs that have been purified are common in IgAN patients is a very important question that is the subject of future studies.
  • IgAN is characterized by mesangial deposition of IgAl with IgG and/or IgM 2 , there is a lack of information as to whether there is colocalization of Tn(+)IgAl with either IgG and/or IgM in all types of IgAN deposits. Due to the biochemical nature of the study with a limited number of patients and their serum samples, expansion of the study to a large cohort of patients was not possible, so it is not clear at this point whether the inclusion of anti-Tn IgM within anti-Tn CICs is a common characteristic in IgAN or only limited to a small number of patients.
  • IgM 44 mesangial deposits of IgM 44 .
  • IgM deposition showed a significant association with glomerular crescent, mesangial hypercellularity, segmental sclerosis, and tubular atrophy/intestinal fibrosis 45 .
  • a similar study that focused on IgM identified IgM antibody deposits in the glomerulus, along with a similar distribution of IgAl in a specimen from an IgAN patient 46 .
  • IgG, IgM, and IgAl individual antibody levels or some combinations of them, suggesting their deposition in the kidney of the IgAN patients or in a mouse model 47-51 .
  • the results indicate that the anti-Tn CICs both from IgAN patients and healthy controls binds to human renal mesangial cells preferentially, as well as to their nuclei. Binding to the nuclei is puzzling and will require further studies as to whether the CICs also contain anti-nuclear antibodies or whether the Tn- containing antigens may somewhat reside within cells, as has been proposed by others 53 . Such a finding might have implications for IgAN pathogenesis.
  • GWAS genome-wide association studies
  • anti-Tn CICs that were purified from human serum show many biochemical and biological characteristics similar to prior characterize CICs and align with evolving model of CICs known to be important in IgAN pathogenesis 8,9 ,26,65 .
  • the data unequivocally demonstrate that anti-Tn CICs that were purified contain galactose-deficient IgAl as the key component together with IgM; these complexes may also contain some IgG.
  • Tn antigenbased glycomimetics treatment followed by blue native gel analysis shows the presence of polymeric IgAl within the complexes.
  • purified anti-Tn CICs from healthy control can bind to and stimulate proliferation of human renal mesangial cells; such occasional IgAl deposition may occur as seen in healthy kidney biopsies 66,67 , the interactions may be different or not sufficient to cause IgAN pathogenesis.
  • the results show that anti-Tn CICs are elevated and complement C3 is elevated within the immune complexes compared to anti-Tn CICs found in healthy control sera; such differences may be critical for driving IgAN pathogenesis.
  • Another important aspect of this study is the development of a sensitive assay to detect as well as monitor anti-Tn IgM antibodies in circulation, which has important implications for IgAN diagnosis and as a tool to follow disease progression.
  • An assay has been developed based on the findings that IgM is dominantly present within the anti-Tn CICs, which contain IgM and IgAl together.
  • the results show that IgM within the CICs can be easily detected by secondary anti-IgM antibodies, whereas IgAl and IgG cannot be directly and easily detected in the native CICs. As such, it can be reasoned that an assay targeted towards detecting IgM bound to the Tn(+)matrix would be more meaningful and sensitive.
  • glycomimetics represent promising candidates to consider for the treatment of IgAN. It has been reported that the properties of CICs (Tn containing IgAl), for example, size, components, and antigen: antibody ratio, influence the nature of the CICs deposited in the glomeruli 69,70 . Thus, it can be deduced that the anti-Tn CICs that were purified behave similarly, and that treatment of the anti-Tn CICs with a-methylGalNAc -based glycomimetics can dissociate the Tn(+)IgAl from the complex and reduce kidney deposition. Such glycomimetics may hold great potential for treatment of patients if the compounds are orally administrable and absorbable.
  • the present disclosure provides, in some aspects, methods of detecting levels of an analyte, such as an antibody, in a sample derived from a subject who has, who is expected of having, or who is at risk of developing a form of renal disease, such as IgA nephropathy or Berger’s disease.
  • the present disclosure also provide, in some aspects, compositions for use in treating a form of renal disease, such as IgA nephropathy or Berger’s disease, as well as methods of administering the composition to a subject in need thereof.
  • the present disclosure is related, in part, to the development of diagnostic methods, treatment methods, and therapeutic compositions for a form of renal disease.
  • Renal disease, or kidney disease is characterized by gradual loss of normal kidney function. Over time, gradual loss of kidney function results in end-stage renal disease (ESRD) or end-stage kidney disease.
  • ESRD end-stage renal disease
  • Treatment for ESRD requires blood dialysis and eventually kidney transplant.
  • the kidneys function by filtering waste material and excess fluid from the blood. As the kidneys fail and lose normal function, waste builds up in the body and results in a variety symptoms. Examples of symptoms of kidney failure include decreased urinary output, swelling, nausea, fatigue, shortness of breath, and in serious cases, death.
  • the present disclosure is related, in part, to kidney disease that affects glomeruli.
  • Glomeruli are small networks of blood vessels that are vital to the waste removal function of kidneys.
  • the present disclosure is related to the buildup of toxic complexes that block glomeruli function.
  • the toxic complexes are immune complexes that are elevated in individuals who have, who are suspected of having, or who are at risk of developing kidney disease.
  • the present disclosure is related to detecting levels of toxic immune complexes in a sample derived from a subject who has, who is suspecting of having, or who is at risk of developing kidney disease.
  • the present disclosure is related to administering a composition that targets toxic immune complexes in subjects who have, who are suspecting of having, or who are at risk of developing kidney disease.
  • the present disclosure is related to a form of kidney disease.
  • the form of kidney disease is any form of kidney disease that results in loss of normal kidney function.
  • the form of kidney disease is any form of kidney disease that results in loss of glomeruli function.
  • the present disclosure is related to a form of kidney disease that is characterized by increased levels of toxic immune complexes.
  • the form of kidney disease is IgA nephropathy.
  • the form of kidney disease is Berger’s disease.
  • IgA nephropathy is also called Berger’s disease.
  • IgA nephropathy is referred to as “IgAN.”
  • the present disclosure is related, in part, to the discovery by the inventors of an antibody that is elevated in subjects who have, who are suspected of having, or who are at risk of developing IgAN.
  • the antibody is an IgM antibody.
  • the IgM antibody is also referred to as immunoglobulin M and is one of several isotypes of antibodies that are produced in vertebrates.
  • antibodies function by binding to antigens.
  • Antibodies are also often characterized by the antigens they bind.
  • the IgM antibody discovered by the inventors was discovered in circulating blood of subjects who have IgAN.
  • the IgM antibody of the present disclosure was found to bind to the GalNAc-al-Ser/Thr antigen found in the hinge region of IgAl.
  • the hinge region is a stretch of antibody heavy chain between the Fab and Fc antibody portions.
  • the GalNAc-al-Ser/Thr antigen also known as the Tn antigen, is an O-glycan found in the IgAl hinge region.
  • IgAl in the blood serum from subjects who have IgAN has been found to contain aberrant Tn antigen.
  • IgM also referred to as Anti-Tn
  • the toxic immune complex forms toxic deposits in the kidneys of subjects with IgAN. In some embodiments, the toxic immune complex forms toxic deposits in the renal glomeruli mesangium of subjects with IgAN.
  • subjects with IgAN have increased levels of total IgAl, as compared to subjects without IgAN.
  • subjects with IgAN have increased levels of IgAl comprising the Tn antigen in the hinge region, as compared to subjects without IgAN.
  • subjects with IgAN have increased levels of IgM (or Anti-Tn), as compared to subjects without IgAN.
  • levels of IgM (Anti-Tn), total IgAl, or Tn antigen in a sample derived from a subject can be indicative of a diagnosis of IgAN in the subject.
  • therapeutic compositions can be used to dissociate Anti-Tn from IgAl, thereby blocking the formation of or removing toxic immune complexes from the subject.
  • the present disclosure is related, in part, to a method of detecting levels of an analyte derived from a subject who has, who is suspected of having, or who is at risk of developing a renal disease.
  • the present disclosure is related, in part, to a method of detecting levels of an analyte derived from a subject who has, who is suspected of having, or who is at risk of developing IgAN.
  • the analyte is a sample derived from a subject.
  • the analyte or sample is blood, blood serum, blood plasma, blood fraction, mucous, urine, saliva, tears, or any other tissue or fluid found in a subject.
  • the analyte or sample is blood serum derived from a subject.
  • the present disclosure is related to use of a form of the IgAl molecule that has been engineered to bind Anti-Tn with higher efficiency than naturally-occurring IgAl.
  • the IgAl comprises the Tn antigen in more regions than the hinge region alone.
  • the IgAl completely expresses Anti-Tn.
  • the method of detecting levels of Anti-Tn comprises collecting a sample or analyte from a subject.
  • the sample is contacted with the IgAl.
  • the sample and IgAl are incubated.
  • incubation is for less than 1 minute, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes.
  • the incubating is for a sufficient time for the antigen to bind to the antibody. In some embodiments, incubation occurs at room temperature.
  • incubation occurs at ambient temperature. In some embodiments, incubation occurs at 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C,28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.
  • the IgAl or any control IgAl is adhered to a substrate.
  • the substrate is a solid substrate.
  • the substrate is a gel substrate.
  • the solid substrate is a plate.
  • the solid substrate is a microwell.
  • the solid substrate is a dish.
  • the solid substrate is a bead.
  • the solid substrate is a microbead.
  • the IgAl bound to Anti-Tn is separated from the sample.
  • the step of separating requires methods that will be known to the skilled artisan.
  • the step of separating requires protein extraction, depletion of non-bound molecules, or an immunological assay.
  • the level of Anti-Tn in the sample is measured.
  • the level of Anti-Tn in the sample is measured by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the level of Anti- Tn in the sample is measured by flow cytometry.
  • the level of Anti-Tn in the sample is measured by another method of determining protein concentration.
  • the level of Anti-Tn in a sample derived from a subject who has, who is suspected of having, or who is at risk of developing IgAN is compared to a control measurement.
  • a second sample derived from the subject is contacted with a control IgAl that does not comprises the Tn antigen.
  • the sample and the second sample are treated the same way, except the sample is contacted with the IgAl and the second sample is contacted with the control IgAl.
  • contacting the second sample with the control IgAl does not result in Anti-Tn binding to the control IgAl.
  • contacting the second sample with the control IgAl results in minimal or negligible Anti-Tn binding to the control IgAl. In some embodiments, contacting the second sample with the control IgAl results in fewer Anti-Tn molecules binding the control IgAl than the number of Anti-Tn molecules binding the IgAl. In some embodiments, the level of Anti-Tn bound to the IgAl is compared to the level of Anti-Tn bound to the control IgAl. In some embodiments, a control sample derived from a subject who does not have IgAN is contacted with the IgAl.
  • the level of Anti-Tn detected in the sample contacted with the IgAl is compared to the level of Anti- Tn detected in the control sample. In some embodiments, if the level of Anti-Tn detected in the sample is higher than the level of Anti-Tn detected in the second sample and in the control sample, the subject may be diagnosed with IgAN.
  • the present disclosure relates, in part, to a method of administering a pharmaceutical composition to a subject in need thereof.
  • the pharmaceutical composition is a glycomimetic.
  • glycomimetic refers to a drug-like compound which mimic the structure and function of native carbohydrates.
  • the glycomimetic is a compound that incorporates N- acetylgalactosamine (GalNAc).
  • the glycomimetic is a compound that incorporates a GalNAc derivative.
  • the glycomimetic is a chemical compound based on the structure of GalNAc.
  • the glycomimetic is able to inhibit formation of the toxic immune complexes comprises of antibodies to GalNAc within the IgAl molecule. In some embodiments, the glycomimetic is able to block an antibody that binds the Tn antigen within the IgAl molecule. In some embodiments, the glycomimetic is able to block Anti-Tn from binding the Tn antigen within the IgAl molecule. In some embodiments, the glycomimetic is able to dissociate a toxic immune complex that has already formed. In some embodiments, the glycomimetic is able to dissociate Anti-Tn from the IgAl molecule.
  • the glycomimetic is of a class of glycomimetics that feature the GalNAc structure. In some embodiments, the glycomimetic has biological activity of inhibiting toxic immune complexes in IgAN. In some embodiments, the glycomimetic is a-methylGalNAc. In some embodiments, the glycomimetic is DiaGalNAc. In some embodiments, the glycomimetic is synthesized by combining two aGalNAc molecules to form a dimer.
  • the glycomimetic is an aGalNAc monosaccharide or disaccharide. In some embodiments, the glycomimetic is an aGalNAc disaccharide. In some embodiments, the glycomimetic is a modified aGalNAc, wherein the aGalNAc is modified to change the -OH group(s) and/or N-acetyl group. In some embodiments, the aGalNAc disaccharide further comprises linkers linking the two aGalNAc molecules. In some embodiments, the aGalNAc disaccharide comprises a carbohydrate or noncarbohydrate linker. In some embodiments, the linker is a flexible or non-flexible linker.
  • the linker is a cleavable or stable linker.
  • the glycomimetic comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers.
  • the glycomimetic further comprises one or more constituents on the aGalNAc.
  • the constituent is a carbohydrate or a non-carbohydrate constituent.
  • the present disclosure is related to a method of administering to a subject a glycomimetic that features the GalNAc structure. In some embodiments, the present disclosure is related to a method of administering to a subject a- methylGalNAc or a similar molecule. In some embodiments, the present disclosure is related to a method of administering to a subject a-methylGalNAc. In some embodiments, the present disclosure is related to administering to a subject DiaGalNAc or a similar molecule. In some embodiments, the present disclosure is related to administering to a subject DiaGalNAc. In some embodiments, the glycomimetic is administered as a pharmaceutical composition.
  • the glycomimetic binds to an antibody that is elevated in subjects who have IgAN. In some embodiments, the glycomimetic binds Anti-Tn. In some embodiments, the glycomimetic is able to dissociate a toxic immune complex. In some embodiments, the glycomimetic inhibits proliferation of mesangial cells. In some embodiments, the glycomimetic is used for treatment of IgAN. In some embodiments, the glycomimetic reduces symptoms of IgAN by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the glycomimetic reduces the risk of developing IgAN disease by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • a “subject” to which analyte detection or administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • a human z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • patient refers to a human subject in need of treatment of a disease.
  • the subject is a human who has, is suspected of having, or who is at risk of developing IgAN. In some embodiments, the subject has a genetic risk factor for developing IgAN. In some embodiments, the subject suspects that they are at risk of developing IgAN. In some embodiments, the subject is unaware that they have or are at risk of developing IgAN. In some embodiments, the subject was previously diagnosed with IgAN and are seeking a confirmatory diagnosis. In some embodiments, the subject is experiencing symptoms of kidney disease. In some embodiments, the subject is experiencing early symptoms of kidney disease. In some embodiments, the subject is experiencing symptoms associated with kidney disease.
  • compositions described herein can be prepared by any method known in the art.
  • An exemplary method include contacting a glycomimetic described herein with a carrier or excipient and one or more additional ingredients, if necessary, then packing the product in a dose.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • compositions refers to a pharmacologically inactive material used together with a pharmacologically active material to formulate the pharmaceutical compositions.
  • Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers. Any one of the compositions provided in the present application may include a pharmaceutically acceptable excipient or carrier.
  • the present disclosure is related, in part, to administering a pharmaceutical composition to a subject in need thereof.
  • the pharmaceutical compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal
  • topical as
  • Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply e.g., via blood and/or lymph supply
  • direct administration e.g., direct administration to an affected site.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
  • the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
  • Tn(+)matrix a high density Tn antigen matrix
  • Asialo- BSM Asialo-bovine submaxillary mucin
  • mucin containing a high amount of Tn antigen along its backbone of primarily Ser, Thr, and Pro residues and potentially mimicking the hinge region of IgAl in many respects 17,30 . It is possible that the Tn(+)matrix could be used to affinity purify anti-Tn antibodies from human sera (FIG. 7A).
  • Tn(+)matrix specifically interacts with a defined anti-Tn antibody and lectin in control studies (FIGs. 7B-7C).
  • the matrix was used to affinity purify anti-Tn antibodies that are potentially present in human sera.
  • the bound materials were resolved on SDS-PAGE, and Coomassie stained. 3-4 major Coomassie stained bands were observed only in the material that bound to the Tn(+)matrix.
  • the protein sizes ranged between ⁇ 25 to 75 kDa (FIG. 3A, upper panel, elution lane), suggesting heavy chains and light chains of antibodies.
  • the same preparation was immunoblotted with specific antibodies, which demonstrated that the bound material contains IgM, IgA, and IgG (FIG. 3A, bottom panels).
  • Tn(+)matrix- purified anti-Tn antibodies could be further characterized from the sera of 10 different healthy individuals (FIG. 1), using appropriate controls as indicated on the top of the Coomassie stained gel. These results demonstrate that the purified anti-Tn antibodies from each sample are predominantly IgM (75 kDa), along with lesser amounts and varying proportions of IgG (50 kDa) and/or IgA (55 kDa) (FIG. 3B). Immunoblots confirmed that IgM is present in all samples along with varying amounts of IgG; the Tn(+)IgAl glycoform was also present in all samples (FIG. 3B, bottom panels). To further confirm that the identified proteins are immunoglobulins (FIG.
  • proteomic analyses by mass spectrometry was performed on individual sera samples from 5 controls (Cl, C2, C3, C6, and C7). The results demonstrate that the Coomassie- stained bands represent IgM, IgG, and IgA, as expected (FIG. 8). The results of proteomic analyses demonstrate that additional proteins are present, including the consistent presence of complement C3.
  • Tn glycopeptide array was prepared as a first approach, where small glycopeptides contain the Tn antigen in the context of IgAl hinge region (Tn(+)IgAl glycopeptides), or within non-IgAl peptides as listed (FIG. 2). Affinity-purified antibodies were applied from sera of 10 healthy individuals and detected for the presence of bound IgM.
  • the secreted IgAl was purified from both WT Dakiki cells and CosmcKO B cells. SDS-PAGE immunoblot analysis of the purified IgAl shows the KO cell line produces the Tn(+)IgAl glycoform, which is recognizable by the lectin VVA and anti-Tn antibodies from human sera.
  • normal WT-IgAl Tn(-)IgAl glycoform
  • Tn(-)IgAl glycoform lacks the Tn antigen and is not bound by reagents that bind the Tn antigen (FIG. 3G). It was then questioned whether Tn(+)IgAl could inhibit binding of affinity-purified anti-Tn antibodies to the Tn glycopeptide array, as observed in FIG. 3D and 3E.
  • Tn(+)IgAl but not WT-IgAl, can inhibit the binding of the anti-Tn antibodies to the Tn glycopeptide array (FIG. 3H).
  • anti-Tn IgM antibodies can specifically interact with Tn(+)IgAl.
  • Example 2 Elevated level of anti-Tn antibodies in sera of patients with IgAN
  • Serum IgAl levels were elevated in IgAN patients as compared to healthy controls as expected (FIG. 4F, left), but the percentage of Tn positivity of IgAl in serum did not change in both groups (FIG. 4F, right), suggesting an increased amounts of Tn(+)IgAl in the circulation of the IgAN patient.
  • Example 3 Characterization of Circulating anti-Tn macromolecular immune complexes (anti-Tn CICs) purified from human sera
  • CICs circulating immune complexes
  • Tn(+)IgAl Tn(+)IgAl
  • BN-APAGE blue native-agarose polyacrylamide gel electrophoresis
  • IgM- containing anti-Tn immune complexes migrated predominantly as large molecular weight species of 1.2 MDa and above, with significantly higher molecular weight species (several megadaltons) than control IgM, which migrated at the expected size of ⁇ 0.9 MDa (FIG. 5A).
  • ReBaGs6 was used, which is an anti-Tn mouse IgM that binds to Tn(+)matrix 35 .
  • This control antibody was purified using a similar approach on the Tn(+)matrix; the bound ReBaGs6 IgM had a predicted normal size when resolved on BN- APAGE, demonstrating that no artificial complexes are formed by possible leakage of Asialo-BSM from the Tn(+)matrix (FIG. 5B).
  • BN-APAGE was used to analyze the sizes of affinity-purified anti-Tn antibodies as well as total IgM (similar concentration used as in FIG. 5A) from controls (C2, C3, and C6). These CICs also showed similar behavior of anti-Tn IgM immune complexes from patients (FIG.
  • Anti-Tn CICs contain IgM and IgAl and the Tn antigen-based glycomimetics disrupt CICs
  • the anti-Tn IgM immune complexes can be dissociated from Tn(+)IgAl using compounds that mimic the Tn antigen- glycomimetics.
  • the anti-Tn CICs were affinity-purified from the sera of IgAN patients; the anti-Tn CICs were treated with mock, a-methylGalNAc (glycomimetic compound, aGalNAc), or a-methylGlcNAc (control compound, aGlcNAc), followed by BN-APAGE- WB and probing for IgM, and IgA.
  • DiaGalNAc z.e., GalNAc dimer
  • a control DiaGlcNAc z.e., GlcNAc dimer
  • the DiaGalNAc exhibited higher inhibition compared to a-methylGalNAc in terms of anti- Tn antibody binding to the array, as listed (FIG. 2, FIG. 12A).
  • the IC50s were calculated using an ELISA based approach, where Tn glycopeptides were used from IgAl (ID 18 and 19, listed in FIG. 2), and performed the inhibition experiments using the glycomimetics (z.e. a- methylGalNAc and DiaGalNAc).
  • FIGS. 12B-12C A higher inhibition of anti-Tn CICs binding with DiaGalNAc treatment was observed (FIGs. 12B-12C). Together, the result indicates that the anti-Tn CICs are composed of at least IgM and IgA, and Tn antigen glycomimetics specifically dissociate Tn(+)IgAl from the complexes.
  • Example 5 Glycomimetic treatment inhibits the proliferative nature of anti-Tn CICs on primary human mesangial cells
  • anti-Tn CICs binding to mesangial cell surfaces were also analyzed, using flow cytometric analysis; next the cells were stained with anti-Tn CICs and analyzed the binding, and it was observed significant binding not only with the predominant component of anti-Tn CICs (IgM), but also with IgAl, and IgG (FIGs. 6D-6E) to mesangial cells (except IgAl in the case of healthy control) indicating that anti-Tn CICs can interact with the mesangial cell surface.
  • IgM the predominant component of anti-Tn CICs
  • IgAl IgAl
  • IgG IgGs. 6D-6E
  • anti-Tn CICs purified from IgAN patients are different in terms of amounts of complement C3.
  • the significantly higher amount of complement C3 within anti-Tn CICs may explain the differential mesangial cell proliferation by anti-Tn CICs from IgAN compared to healthy control (FIGs. 61, 6G, 6Q).
  • anti-Tn CICs that were purified from IgAN patient serum contain IgM, Tn(+)IgAl, IgG, and complement C3, suggesting a pathogenic nature of anti-Tn CICs of IgAN patients in their serum.
  • Example 6 Diagnostic for IgA Nephropathy
  • the present disclosure is based, in part, on the discovery of IgM antibodies in human blood that bind to the carbohydrate antigen described herein, termed Tn (GalNAc-al- Ser/Thr).
  • Tn carbohydrate antigen described herein
  • This antigen is found in subjects with IgAN and is elevated in their blood compared to healthy individuals.
  • These anti-Tn antibodies can bind to the immunoglobulin IgAl, a portion of which has the Tn antigen in the hinge region, the so-called “hinge-region glycans.”
  • the formation of a complex between this IgM and the IgAl containing the Tn antigen in the hinge-region glycans is presumed to cause IgAN.
  • IgA nephropathy can lead to end-stage kidney disease, sometimes called ESRD, which means the kidneys no longer work well enough to keep a person healthy.
  • ESRD end-stage kidney disease
  • the definitive diagnostic is a renal biopsy by a nephrologist and a pathologist finding of deposits of IgAl in the kidney biopsy. The disease slowly progresses over several years before the pathology becomes evident, usually as blood in the urine.
  • the diagnostic method described herein required several innovative and novel steps.
  • a form of IgAl was engineered to express completely the Tn antigen IgAl(Tn+) as a test reagent. This is different from a natural source of to IgAl expressed by the commercially available Dakiki cells and which lacks the Tn antigen IgAl(Tn-).
  • the IgM discovered in the blood of patients can bind to the IgAl(Tn+) but not natural IgAl(Tn-).
  • a microbead assay has been established to capture anti-Tn IgM antibodies from small amounts of serum and using flow cytometry to measure the amount of IgM that recognizes the Tn antigen.
  • the amount of anti-Tn IgM is significantly elevated in the sera of patients with IgAN compared to sera from healthy individuals.
  • a microbead assay has also been established in which the IgAl(Tn+) or the IgAl(Tn-) proteins are affixed to beads, the anti-Tn IgM in blood that bind to the former but not the latter is measured. Elevations in anti-Tn IgM and/or antiIgA l(Tn+) are diagnostic for IgAN.
  • This new diagnostic using either flow cytometry or ELISA, can be useful preclinically, before symptoms of IgAN become apparent. Also, this new diagnostic could be used to monitor treatment of patients, who are often treated by dialysis to remove antibodies, and often given kidney transplants. Materials related to Examples 1-6
  • a Tn (+) matrix (desialylated bovine submaxillary mucin (Asialo-BSM)) resins were prepared as previously described 35 . Briefly, ⁇ 2 ml of coupled BSM resins (50% slurry) were desialylated using 50 mU neuraminidase (Cat#10269611001, Roche) in 50 mM sodium acetate (pH 5.0) for 1 h at 37°C (10 rpm). The beads were washed 3x with 10 ml of PBS, and desialylated one more time to completely remove the remaining sialic acid. The prepared desialylated BSM (Asialo-BSM) beads (Tn(+)matrix beads) were kept at 4°C in PBS for future uses. Beads alone were prepared with no proteins in parallel.
  • Tn(+)matrix beads 50% slurry in PBS
  • the preparation of Tn(+)matrix beads was washed 6x with 5 ml of chilled 1 M NaCl on a column (Cat#10561284, PierceTM) dropwise (1 ml/min), and the bound material was eluted by 5 ml of chilled 0.1 M glycine-NaOH, pH 10.5 and eluted sample was immediately neutralized with chilled glycine-HCl.
  • the signals were analyzed on an AmershamTM Imager 600 (GE Healthcare Life Sciences) using SuperSignalTM West Pico chemiluminescent substrate (Cat#34578, Thermo Scientific).
  • the membranes were blocked with 5% (w/vol) BSA (Cat#B Pl 600-1, Fraction V, Fisher BioReagentsTM) in TBST for 1 h at RT, and incubated with biotinylated VVA (Cat#B- 1235-2, Vector Laboratories, diluted to 0.5 mg/ml) in TBST containing 0.5% BSA for 1 h at RT.
  • HRP-labeled streptavidin (Cat#SA-5014, Vector Laboratories) at 1:10,000 dilution in TBST containing 0.5% BSA was used for detection. ⁇ 5 ml of Tn(+)matrix beads (50% slurry in PBS) or control beads were incubated with purified anti-Tn antibodies diluted to 1 mg/ml for IgM staining; 4 mg/ml for IgG and IgA staining in PBS for 1 h on ice.
  • the beads were washed 3x with 1 ml of chilled 1 M NaCl each time using centrifugation at 150 x g for 30 seconds at 4°C, and incubated with respective Alexa Fluor* 488-goat anti-human IgM (Cat#A-21215, Thermo Fisher Scientific), goat anti-human IgG (Cat#A-l 1013, Thermo Fisher Scientific) or FITC- labeled mouse anti-human IgAl (Cat#9130-02, Southern Biotech) at 1:400 dilution in PBS for 1 h on ice in the dark.
  • Alexa Fluor* 488-goat anti-human IgM Cat#A-21215, Thermo Fisher Scientific
  • goat anti-human IgG Cat#A-l 1013, Thermo Fisher Scientific
  • FITC- labeled mouse anti-human IgAl Cat#9130-02, Southern Biotech
  • the beads were washed twice with chilled 1 ml of PBS, and analyzed using a microscope (AMG EVOS FL digital inverted microscope, Fisher Scientific, xlOO magnification).
  • Isotype human IgM (Cat#31146, diluted to 1 mg/ml), human IgG (Cat#31154, diluted to 4 mg/ml), or human IgA (Cat#31148, diluted to 4 mg/ml) from Invitrogen in PBS were used as controls.
  • purified anti-Tn antibodies were preincubated with 20 mM a-methylGalNAc (Cat#sc-222088, Santa Cruz) in PBS for 30 min on ice.
  • the Tn glycopeptide microarray was prepared as previously described 77 . Briefly, the glycopeptides printed on the microarray is as listed (FIG. 2). Purified anti-Tn antibodies (diluted to 10 mg/ml for IgM, and 40 mg/ml for IgG and IgA detection) in TSM binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaC12, 2 mM MgC12, with 1% BSA and 0.05% Tween-20) were added to the respective array slides for 1 h at RT.
  • TSM binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaC12, 2 mM MgC12, with 1% BSA and 0.05% Tween-20
  • Tn(+)IgAl produced from CosmcKO Dakiki cells was utilized (See Generation of CosmcKO Dakiki cells using CRISPR-Cas9 system for generating the antibody') to inhibit anti-Tn antibodies binding to microarray and Tn(-)IgAl produced from Dakiki cells as a positive control.
  • Purified anti-Tn antibodies (Cl, 10 mg/ml) in TSM binding buffer was preincubated with 1 mg of purified Tn(+)IgAl, or Tn(-)IgAl for 30 min at RT.
  • Alexa Fluor* 647-labeled goat anti-human IgM antibody (Cat#A-21249, Thermo Fisher Scientific, diluted at 1:400) in TSM binding buffer was used for detection.
  • purified anti-Tn antibodies C3, 10 mg/ml in TSM binding buffer was preincubated with 20 mM a- methylGalNAc, or a-methylGlcNAc (Cat#M0257, Sigma), or DiaGalNAc, or DiaGlcNAc synthesized as described in (Synthesis of GalNAc and GlcNAc Dimer (DiaGalNAc and DiaGlcNAc)) for 30 min at RT; after 1 h incubation with the solution on the array, Alexa Fluor* 488-labeled goat anti-human IgM (Cat#A-21249, Thermo Fisher Scientific, diluted at 1:400) in TSM binding buffer was used for detection. Heat map and correlation map of binding preferences were analyzed using GLAD (Glycan Array Dashboard).
  • CosmcKO cells Human B cell line (Dakiki) was purchased from American Type Culture Collection (TIB -206TM, ATCC)) and cultured in RPMI 1640 medium (Corning * ) supplemented with 20% (vol/vol) fetal bovine serum and 200 units/ml penicillin-streptomycin (Fisher Scientific) at 37°C and 5% CO2.
  • the sgRNA sequence (Dharmacon) to target Cosme gene is described in FIGs. 10A-10E. Dakiki cells were infected by spinoculation. Tn positivity serves as a cell surface marker of CosmcKO.
  • Cells were first selected using drug and then sorted for Tn positive population using ReBaGs6 on a cell sorter (Beckman Coulter, MoFlo Astrios EQs Sorter). The resultant homogenous cell population was termed CosmcKO cells.
  • CosmcKO cells First, whole-cell extracts from WT were analyzed and CosmcKO Dakiki cells which were resolved on SDS-PAGE and immunoblotted for Cosme, T-synthase, and "-actin using anti-Cosmc antibody (Cat#sc-271829, H-10, Santa Cruz), anti- T-synthase antibody (Cat#sc- 100745, F-31, Santa Cruz), or anti-"-actin antibody (Cat#sc- 47778, C4, Santa Cruz) at 1:1000 dilution in TBST with 1% nonfat milk.
  • Anti-Cosmc antibody Cat#sc-271829, H-10, Santa Cruz
  • anti- T-synthase antibody Cat#sc- 100745, F-31, Santa Cruz
  • anti-actin antibody Cat#sc- 47778, C4, Santa Cruz
  • Biotinylated PNA (Cat#B- 1075-5, Vector Laboratories, diluted to 1 pg/ml), or HPA (Cat#L6512, Sigma, diluted to 1 pg/ml) in TBST containing 1% BSA were used as primary reagents. Secondary detection was performed with HRP-labeled streptavidin at 1:10,000 dilution in TBST containing 0.5 % BSA.
  • T-synthase enzyme activity was fluorescently assayed using 4- Methylumbelliferyl 2-acetamido-2- deoxy-a-D-galactopyranoside (Cat#EM04782, Biosynth Carbosynth®, 4MU-a-GalNAc) as an acceptor substrate and UDP-Galactose (MU06699, Biosynth Carbosynth®) as donor sugar as described previously 79 .
  • 4- Methylumbelliferyl 2-acetamido-2- deoxy-a-D-galactopyranoside Cat#EM04782, Biosynth Carbosynth®, 4MU-a-GalNAc
  • UDP-Galactose MU06699, Biosynth Carbosynth®
  • a- mannosidase activity was assayed using 4-Methylumbelliferyl a-D-mannopyranoside (Cat#M3657, Sigma-Aldrich, 4MU-a-Man) as an acceptor substrate.
  • the T-synthase activity was normalized as a ratio of T-synthase activity/ a-Mannosidase activity in Dakiki WT cells.
  • CosmcKO Dakiki cells were established as described above. Purification of IgAl from Dakiki cell line was previously described 35 . Purified IgAl with or without extended O-glycans were analyzed by SDS- PAGE-Westem blot as described in “ Affinity purification of putative anti-Tn antibodies and Western blot” with some modifications. Western blot was performed using the purified anti-Tn antibodies (10 mg/ml) in TBST containing 0.5% BSA as primary antibody. VVA lectin blotting was performed as described above.
  • Asialo-BSM 100 mg
  • Helix pomatia agglutinin HPA, Cat#L3382, Sigma, 1 mg
  • Polybead * carboxylate 6.0 micron microspheres Cat#17141, Polysciences, Inc., 2 x 10 7 beads/ml
  • Asialo-BSM microbeads were incubated with 100 ml of serum (diluted at 1:100 in PBS) for 1 h on ice.
  • the beads were washed twice with 1 ml of chilled IM NaCl (beads pelleted 850 x g for 30 sec), and incubated with 100 ml of Alexa Fluor* 488 labeled goat anti- human IgM (m chain) antibody at 1:400 dilution in PBS for 1 h on ice in the dark.
  • ⁇ 5 x 10 4 HPA microbeads were incubated with 100 ml of serum (diluted at 1:100 in PBS) for 1 h on ice.
  • Circulating immunocomplexes were resolved using BN-APAGE system as previously described 34 .
  • Samples and unstained native markers (Cat#LC0725, Invitrogen), were mixed with the sample buffer (0.5% Coomassie blue G-250 and 50 mM 8- aminocaproic acid in 10 mM Bis-Tris, pH 7.5, at final concentration) just before use, and electrophoresis was performed using lx anode buffer (50 mM Bis-Tris HC1, pH 7.0) and lx cathode buffer (50 mM Tricine, 15 mM Bis-Tris, 0.0015% G-250, pH 7.0) for at ⁇ 18 h using relatively low voltage and low current (e.g., 15-20 V, ⁇ 2mA, for four gels).
  • lx anode buffer 50 mM Bis-Tris HC1, pH 7.0
  • lx cathode buffer 50 mM Tricine, 15 mM Bis-Tris, 0.0015% G-
  • the blocked membranes were incubated with HRP- labeled goat antihuman IgM (m chain) antibody, or goat anti-mouse IgM (m chain) antibody (Cat#l 15-035- 020, Jackson ImmunoResearch Laboratories, Inc.) at 1 : 10,000 dilution in TBST containing 1% non-fat milk for 1 h at RT, and the signals were detected onto the autoradiography films (Cat#1141J52, HyBlot CL # , Thomas Scientific) using SuperSignalTM West Pico Chemiluminescent Substrate. Immunodepletion experiment
  • purified anti-Tn CICs ( ⁇ 0.1 mg) was pretreated with a-methylGalNAc (100 mM), a-methylGlcNAc (100 mM), or mock in PBS for 2 h at 4°C on a rotator (10 rpm) and the samples were analyzed by BN-APAGE-WB and probed for IgM and IgA.
  • IgAl glycopeptides (ID18, or ID19 as listed in FIG. 2, 0.01 mg/well) were immobilized using immobilization buffer (NaHCO3/Na2CO3, pH 9.6) in a 96-well plate (Thermo Fisher Scientific, PolySorp) overnight at 4°C. The plate was washed 3x with TSM containing 0.05 % tween-20 (TSMT) and added 5 % (w/v) BSA in TSMT for 1 h at RT.
  • immobilization buffer NaHCO3/Na2CO3, pH 9.6
  • the plate was incubated with 1 mg/ml of purified anti-Tn CICs from IgAN serum (P3, and pooled with P1-P10; Pmix) or healthy donors (C3, and pooled C1-C10; Cmix) in TSMT containing 0.5 % BSA for 1 h at RT.
  • the plate was washed 3x with TSMT and incubated for 1 h with HRP-conjugated goat anti-human IgM at 1:1,000 dilution in TSMT containing 0.5% BSA at RT.
  • the plate was washed 3x with TSMT and developed using TMB substrate solution (Cat# abl71523, Abeam) for 30 min, and the reaction was stopped with IN HC1.
  • HRMC Human renal mesangial cells
  • Cells were washed 3x with chilled PBS, and incubated with Alexa Fluor* 488-goat anti- human IgM, and Alexa Fluor* 633-goat anti-mouse IgG (Cat# A21052, Thermo Fisher Scientific) at 1:400 dilution in PBS for 1 h at 4°C in the dark. Cells were washed 3x with chilled PBS, and stained with DAPI (Cat#9542, Sigma, diluted to 0.1 mM) in PBS for 10 min at RT, and analyzed by confocal microscope (Zeiss Axioimager Zl, x630 magnification).
  • Anti-Tn CICs from three healthy controls C3, C6, and pooled Cl- CIO; Cmix
  • three IgAN P5, P10, and pooled P1-P10; Pmix
  • isotype control human IgM, IgG, and IgA (1 mg/ml each), or mouse IgG (Cat#0107-01, Southern Biotech, diluted to 0.5 mg/ml) in PBS were used. All images were taken in three different areas in 24-well plate.
  • HRMC cells (-5 x 10 5 ) were stained with 5 mg/ml of anti-Tn CICs purified from IgAN (P5, P10, or Pmix) or healthy control (HC) (C3, C6, or Cmix) for 1 h at 4°C. After washing 3x with 2 ml of cold PBS, cells were incubated with Alexa Fluor* 488-goat anti-human IgM (m chain), goat anti-human IgG (H+L), or FITC-labeled mouse anti-human IgAl at 1:400 dilution in PBS for 1 h on ice.
  • Alexa Fluor* 488-goat anti-human IgM m chain
  • goat anti-human IgG H+L
  • FITC-labeled mouse anti-human IgAl at 1:400 dilution in PBS for 1 h on ice.
  • Cells were cultured at -90% confluency in 96-well plate, and starved in Mesangial cell media (Cat#4201) with 0.5% FBS (Cat#0010) and 0.05x mesangial cell growth supplement (MsCGS, Cat#4252) from ScienCell for 24 h at 37°C prior to stimulation.
  • Cells were stimulated with 5% serum-Mock, 5% serum-ID with or without exogenous anti- Tn CICs (50 ng/100 ml/well, total) for 24 h at 37°C incubator.
  • Cells were fixed with 4% PFA with 0.05% Triton-X-100 in PBS for 20 min at 4°C.
  • HRMCs were starved as described above and cells were stimulated with 1, 2.5, or 5% serum with IgAN (Pmix) or healthy control (Cmix) for 24 h in a 37°C cell culture incubator.
  • IgAN IgAN
  • Cmix healthy control
  • cells were stimulated with 2.5% serum with IgAN (P1-P20) or healthy control (C1-C20) for 24 h at 37°C incubation.
  • One ml of serum with IgAN (P1-P20) or healthy control (C1-C20) was analyzed on SDS-PAGE gel, and stained with Coomassie.
  • HEK293T cells (Cat#CRL-3216TM, ATCC) were starved in Dulbecco’s Modified Eagle’s Medium (DMEM) (Cat#10-013-CV, Corning®) with 0.5% FBS for 24 h at 37°C prior to stimulation.
  • DMEM Modified Eagle’s Medium
  • Cells were stimulated with 5% serum with IgAN (mock), CICs-immunodepleted serum (ID), or exogenously adding CICs from IgAN (ID+anti-Tn CICs) for 24 h at 37°C CO2 incubator.
  • Serum and anti-Tn CICs (P10, or Pmix) were used in this assay.
  • healthy control serum and HRMCs cells were also used as described above.
  • HRP-labeled goat anti-rabbit IgG (Cat#5220-0336, KPL) at 1:10,000 dilution was used for detection.
  • the signals were detected using SuperSignalTM West Pico Chemiluminescent Substrate on an AmershamTM Imager 600.
  • Samples were loaded onto a C18 precolumn (Cl 8 PepMap 100, 300 pm x 5 mm, 5 pm, 100 A, Thermo Fisher Scientific) with 15 pl/min solvent A (0.1% FA in H2O) for 3 min and separated on a C18 analytical column (picofrit 75 pm ID x 150 mm, 3 pm, New Objective) using a linear gradient of 2% to 45% solvent B (80% acetonitrile, 0.1% FA) over 39 min at 400 nE/min.
  • the mass spectrometer was operated under following conditions: The ion source parameters were 2,100 V spray voltage and 200 °C ion transfer tube temperature.
  • MS scans were performed in the orbitrap at a resolution of 60,000 within a scan range of m/z 400 - m/z 1,600, a RF lens of 30%, AGC target of le5 for a maximum injection time of 50 ms.
  • the top 15 precursors were selected for MS 2 in a data dependent manner, within a mass range of m/z 400 - m/z 1,600 and a minimum intensity threshold of le5 and an isolation width of 2 m/z.
  • HCD was performed in stepped collision energy mode of 30% (+/- 5%) and detected in the orbitrap with a resolution of 30,000 with the first mass at m/z 120, an AGC target of 2e5 and a maximum injection of 250 ms.
  • TLC Thin layer chromatography
  • Reverse phase high performance liquid chromatography was performed using Waters (Method A) Gradient Purification System 2767 equipped with Waters 2489 UV/Vis detection module and Waters 2545 Binary Gradient Module using a C18 100A (250 x 30 mm, Phenomenex) column (PREPARATORY).
  • Waters Method A
  • Gradient Purification System 2767 equipped with Waters 2489 UV/Vis detection module and Waters 2545 Binary Gradient Module using a C18 100A (250 x 30 mm, Phenomenex) column (PREPARATORY).
  • Ultraflex II matrix-assisted laser desorption ionization time of flight mass spectrometry MALDI-TOF MS was used to analyze samples co-crystallized using super DHB matrix.
  • the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features.
  • a method of detecting levels of an antibody in a sample derived from a subject comprising: contacting the sample with an antigen, wherein the antibody, if present, binds to the antigen; and measuring the amount of antibody bound to the antigen.
  • a method of detecting levels of IgM antibody in a sample derived from a subject comprising: contacting the sample with an IgA molecule comprising a GalNAc-al-Ser/Thr antigen (antigen), wherein the IgM antibody, if present, binds to the GalNAc-al-Ser/Thr antigen; and measuring the amount of IgM antibody bound to the IgA molecule.
  • step of contacting further comprises incubating the sample with the antigen.
  • sample is blood, blood serum, blood plasma, blood fraction, saliva, mucous, urine, or a combination thereof.
  • sample is blood serum.
  • step of measuring comprises the use of flow cytometry to detect the amount of antibody bound to the antigen.
  • a method comprising: administering to a subject a glycomimetic, wherein the glycomimetic is based on the structure of N-acetylgalactosamine (GalNAc); wherein: the subject has, is suspected of having, or is at risk of developing IgA nephropathy or Berger’s disease.
  • GalNAc N-acetylgalactosamine
  • a method comprising: administering to a subject a glycomimetic, wherein the glycomimetic is a- methylGalNAc or DiaGalNAc; wherein: the subject has, is suspecting of having, or is at risk of developing IgA nephropathy or Berger’s disease.
  • the glycomimetic binds an antibody that is elevated in subjects with IgA nephropathy or Berger’s disease.
  • glycomimetic is a modified aGalNAc, wherein the aGalNAc is modified to change the - OH group(s) and/or N-acetyl group.
  • glycomimetic is an aGalNAc monosaccharide or disaccharide.
  • glycomimetic is an aGalNAc disaccharide.
  • glycomimetic comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers.
  • the glycomimetic further comprises one or more constituents on the aGalNAc.
  • glycomimetic inhibits formation of an immune complex comprising an antibody and an antigen, wherein the antibody is Anti-Tn and the antigen is an IgA comprising a GalNAc-al-Ser/Thr antigen.
  • Kiryluk, K., et al. GWAS for serum galactose-deficient IgAl implicates critical genes of the O-glycosylation pathway.

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Abstract

La présente invention concerne des méthodes et des compositions pour le diagnostic et le traitement d'une néphropathie IgA. La détection et la quantification de niveaux de sérum IgM sont réalisées au moyen d'une molécule IgA comprenant un antigène GalNAc-alpha 1-Ser/Thr. Les méthodes ELISA et basées sur la cytométrie de flux sont revendiquées. Dans les exemples, des microbilles asialo-BSM sont utilisées.
PCT/US2023/032240 2022-09-09 2023-09-08 Méthodes et compositions pour le diagnostic et le traitement d'une néphropathie iga WO2024054596A1 (fr)

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