WO2018094221A1 - Assessing and treating fibrillary glomerulonephritis - Google Patents

Abstract

This document relates to materials and methods for treating fibrillary glomerulonephritis (e.g., non-amyloid fibrillary glomerulonephritis) and/or a complication associated with fibrillary glomerulonephritis (e.g., high blood pressure, phosphorus imbalance, and/or anemia). For example, methods for using one or more inhibitors of DNAJB9 polypeptide expression or activity to treat a mammal having fibrillary glomerulonephritis are provided.

Description

ASSESSING AND TREATING FIBRILLARY GLOMERULONEPHRITIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 62/424,052, filed November 18, 2016. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to materials and methods for identifying and/or treating fibrillary glomerulonephritis (e.g. , non-amyloid fibrillary glomerulonephritis). For example, this document provides materials and methods for using one or more inhibitors of DnaJ homolog subfamily B member 9 (DNAJB9) polypeptide expression or activity to treat a mammal having fibrillary glomerulonephritis.

2. Background Information

Fibrillary glomerulonephritis (FGN) affects the ability of the filtering units (e.g. , glomeruli) of the kidney, and is characterized by fibrillary deposits in the glomeruli and inflammation of the glomeruli. The pathogenesis of FGN is unknown.

SUMMARY

This document provides materials and methods for identifying and/or treating

FGN (e.g. , non-amyloid FGN). This document also provides methods and materials for treating one or more complications associated with FGN (e.g., high blood pressure, phosphorus imbalance, and/or anemia). For example, this document provides materials and methods for detecting the presence of an elevated level of DNAJB9 polypeptides

(e.g., having an elevated level of DNAJB9 polypeptides in fibrillary deposits in the glomeruli) within a mammal and identifying the mammal as having FGN. As demonstrated herein, elevated levels of DNAJB9 polypeptides are present in the glomerular deposits of patients with FGN, but not in normal glomeruli, in glomeruli of patients with renal amyloidosis, or in patients with other forms of glomerulonephritis. In some cases, this document provides materials and methods for treating FGN. For example, a mammal (e.g. , a human) identified as a mammal as having FGN can be administered one or more inhibitors of DNAJB9 expression or activity. The administered inhibitor(s) can reduce the severity of one or more symptoms of FGN within the mammal. In some cases, the administered inhibitor(s) can reduce the severity of one or more complications associated with FGN (e.g. , high blood pressure, phosphorus imbalance, and/or anemia).

In general, one aspect of this document features a method for identifying a mammal having FGN. The method includes, or consists essentially of, detecting the presence of an elevated level of DNAJB9 polypeptides in a biological sample from the mammal, and classifying the mammal as having FGN based at least in part on the presence of an elevated level of DNAJB9 polypeptides in the biological sample. The level of DNAJB9 polypeptides in the biological sample can be determined using immunohistochemistry. The level of DNAJB9 polypeptides in the biological sample can be determined using mass spectrometry. The mass spectrometry can include proteomics- based mass spectrometry assay or targeted quantification-based mass spectrometry assay. In some cases, the biological sample can include serum or urine, and the detecting the presence of an elevated level of DNAJB9 polypeptides can include detecting the presence of anti-DNAJB9 autoantibodies in the sample.

In another aspect, this document features a method for treating FGN in a mammal. In some cases, he method includes, or consists essentially of, identifying a mammal as having an elevated level of DNAJB9 polypeptides within a biological sample from the mammal, and administering an inhibitor of DNAJB9 polypeptide expression or activity to the mammal under conditions wherein the severity of a symptom of FGN is reduced. In some cases, he method includes, or consists essentially of, identifying a mammal as having an elevated level of DNAJB9 polypeptides within a biological sample from the mammal, and administering an inhibitor of DNAJB9 polypeptide expression or activity to the mammal under conditions where progression of FGN to kidney failure in the mammal is reduced. The mammal can be a human. The biological sample can be a kidney tissue sample. The biological sample can be a fluid sample (e.g., serum or urine).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1 includes mass spectrometry data (A) and immunohistochemistry images (B) of DNAJB9 expression. (A) DNAJB9 is absent from the renal glomerular amyloid proteome and is present only in FGN proteome. (B) DNAJB9 is absent in the glomeruli of normal individual (left panel) and is present in the glomeruli of a patient with (right panel).

Figure 2 is a graph of DNAJB9 abundance in FGN and other renal diseases. CR+ stands for Congo red (CR) positivity. CR- stands for CR negativity. Amyloid represents renal amyloidosis subtypes listed in the methods. NFGNGD represents non-FGN glomerular diseases. Norm-Gloms represent healthy glomerulus samples. **** stands for a Mann-Whitney rank sum test p-value of < 0.0001.

Figure 3 shows a use for serum DNAJB9 in identifying FGN patients. (A) DNAJB9 concentrations are elevated in serum of FGN patients relative to proteinuric controls. (B) A receiver operating characteristic (ROC) curve for DNAJB9 as a serum marker for FGN.

DETAILED DESCRIPTION

This document provides materials and methods for using detecting elevated levels of DNAJB9 polypeptides (e.g. , elevated levels of DNAJB9 polypeptides in fibrillary glomerular deposits) to identify mammals having FGN. As described herein, the presence of an elevated level of DNAJB9 polypeptides can indicate that a mammal (e.g. , a human) has FGN. DNAJB9 polypeptides can be properly folded DNAJB9 polypeptides or misfolded DNAJB9 polypeptides. This document also provides materials and methods for treating FGN (e.g. , non-amyloid FGN) and/or a complication associated with FGN (e.g. , high blood pressure, phosphorus imbalance, and/or anemia). For example, one or more inhibitors of DNAJB9 polypeptide expression or activity can be administered to a mammal identified as having FGN (e.g. , a mammal identified as having FGN as described herein) to reduce the severity of FGN and/or to reduce a symptom of FGN. In some cases, one or more inhibitors of DNAJB9 polypeptide expression or activity can be used to reduce and/or resolve fibrillary glomerular deposits within a mammal (e.g., a human). In some cases, one or more inhibitors of DNAJB9 polypeptide expression or activity can be used to slow or prevent the progression of FGN (e.g., to kidney failure) within a mammal (e.g., a human).

The term "elevated level" as used herein with respect to a level of DNAJB9 polypeptides refers to any level that is greater than a reference level of DNAJB9 polypeptides. The term "reference level" as used herein with respect to DNAJB9 polypeptides refers to the level of DNAJB9 polypeptides typically observed in a sample (e.g., a control sample) from one or more mammals (e.g. , humans) without FGN. Control samples can include, without limitation, samples from normal (e.g., healthy) mammals, mammals with renal amyloidosis, mammals with other forms of glomerulonephritis, diabetes, monoclonal immunoglobulin disease, and thrombotic microangiopathy. In some cases, an elevated level of DNAJB9 polypeptides can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater relative to a reference level of DNAJB9 polypeptides. In some cases, when control samples have undetectable levels of DNAJB9 polypeptides, an elevated level can be a detectable level of DNAJB9 polypeptides. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an elevated level.

Any appropriate sample can be assessed to determine if a mammal (e.g. , a human) has an elevated level of DNAJB9 polypeptides. For example, biological samples such as tissue samples (e.g., kidney), and fluids (e.g., blood, serum, plasma, urine) can be obtained from a mammal and assessed for the presence of an elevated level of DNAJB9 polypeptides. In some cases, a kidney tissue sample can be obtained and assessed to determine whether or not the mammal has an elevated level of DNAJB9 polypeptides. In some cases, serum and/or urine can be used as a biological sample to determine whether or not a mammal has an elevated level of DNAJB9 polypeptides. Any appropriate method can be used to detect the presence, absence, or level of DNAJB9 polypeptides within a sample (e.g. , within glomerular fibrillary deposits). In some cases, immunohistochemistry (IHC) techniques, immunofluorescence, mass spectrometry techniques (e.g. , proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), western blotting techniques, quantitative RT-PCR techniques, and/or ELISA can be used to determine whether or not a sample contains an elevated level of DNAJB9 polypeptides. For example, the presence, absence, or level of DNAJB9 can be detected using an anti-DNAJB9 antibody (e.g. , Sigma HPA040967). In some cases, IHC and/or MS can be used to determine whether or not a particular sample contains an elevated level of DNAJB9 polypeptides. In some cases, an elevated level of DNAJB9 polypeptides can be determined by detecting the presence of anti-DNAJB9 autoantibodies in a sample (a serum and/or urine sample) from the mammal.

Once identified as having an elevated level of DNAJB9 polypeptides, a mammal can be administered or instructed to self-administer one or more inhibitors of DNAJB9 polypeptide expression or activity (e.g., one, two, three, four, five, or more DNAJB9 inhibitors). Administration of an inhibitor of DNAJB9 polypeptide expression or activity to a mammal (e.g. , human) having an elevated level of DNAJB9 polypeptides can be effective to treat FGN.

In some cases, the materials and methods provided herein can be used to reduce the symptoms of FGN. Symptoms of FGN can include, without limitation, blood in the urine (hematuria), protein in the urine (proteinuria), swelling of legs and/or feet, anemia, and elevated blood pressure. In some cases, the materials and methods provided herein can be used to reduce a complication associated with FGN (e.g., inflammation in the glomeruli). In some cases, the materials and methods provided herein can be used to slow or prevent the progression of FGN. In some cases, one or more inhibitors of DNAJB9 polypeptide expression or activity can be used to slow or prevent the progression of FGN to kidney failure.

When treating FGN as described herein, the FGN can be any FGN. The FGN can be non-proliferative or proliferative. FGN can be a primary FGN (e.g. , occurring spontaneously) or a secondary FGN (e.g. , caused by another disease, hepatitis C, solid malignancy, hematologic malignancy, monoclonal gammopathy, or autoimmune disease). FGN can be non-amyloid (e.g. , Congo red-negative) or amyloid (e.g. , Congo red- positive). Examples of FGN that can be treated as described herein include, without limitation, non-amyloid FGN, amyloid FGN, and immunotactoid glomerulopathy. In some cases, FGN treated as described herein can be non-amyloid FGN.

Any type of mammal FGN or a complication of FGN can be treated as described herein. For example, humans and other primates such as monkeys having FGN or a complication of FGN and an elevated level of DNAJB9 polypeptides (e.g. , in glomerular fibrillary deposits) can be treated with one or more inhibitors of DNAJB9 polypeptide expression or activity as described herein. In some cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated with one or more inhibitors of DNAJB9 polypeptide expression or activity as described herein.

Example of inhibitors of DNAJB9 polypeptide expression or activity include, without limitation, nucleic acid molecules designed to induce RNA interference (e.g., a siRNA molecule or a shRNA molecule), antisense molecules, miRNAs, and antibodies (e.g., antibodies (e.g. , monoclonal antibodies) that can neutralize DNAJB9 (e.g. , circulating DNAJB) and/or clear DNAJB9 from glomerular deposits).

In some cases, an inhibitor of DNAJB9 polypeptide expression or activity can be readily designed based upon the nucleic acid and/or polypeptide sequences of DNAJB9. Examples of DNAJB 9 nucleic acids include, without limitation, the human DNAJB9 sequences set forth in GenBank^® Accession Nos. NM_012328 (Version NM_012328.2; GI: 313482783), CR533475 (Version CR533475.1 ; GI: 49065375), and KJ891596 (Version KJ891596.1 ; GI: 649101389). Examples of DNAJB9 polypeptides include, without limitation, the human DNAJB9 polypeptides having the amino acid sequence set forth in National Center for Biotechnology Information (NCBI) GenBank^® Accession Nos: CAG38506 (Version CAG38506.1), EAL24382 (Version EAL24382.1), and AAH28912 (Version AAH28912.1).

A human DNAJB9 polypeptide can have a sequence that deviates from a wild type DNAJB9 polypeptide sequence, sometimes referred to as a variant sequence. A DNAJB9 polypeptide can have at least 80 (e.g., at least 85, at least 90, at least 95, at least 98, or at least 99) percent sequence identity to a wild type DNAJB9 polypeptide sequence. A DNAJB9 polypeptide can have one or more (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g., substitutions) relative to a wild type DNAJB9 polypeptide sequence. In some cases, a DNAJB9 polypeptide can have a substitution of the arginine residue at position 68 (Arg68). For example, a DNAJB9 polypeptide can have an Arg68His (R68H) substitution.

In some cases, one or more inhibitors of DNAJB9 polypeptide expression or activity can be formulated into a pharmaceutically acceptable composition for administration to a mammal having an elevated level of DNAJB9 polypeptides. For example, a therapeutically effective amount of an inhibitor of DNAJB9 polypeptide expression or activity can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

A pharmaceutically acceptable composition including one or more inhibitors of DNAJB9 polypeptide expression or activity can be administered locally or systemically. For example, a composition containing an inhibitor of DNAJB9 polypeptide expression or activity can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human). A pharmaceutical composition containing one or more inhibitors of DNAJB9 polypeptide expression or activity can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled administration. When being administered orally, a pharmaceutical composition containing one or more DNAJB9 inhibitors can be in the form of a pill, tablet, or capsule.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: DNAJB9 is present in FGN renal biopsies

Mass spectrometry and immunohistochemistry were utilized for detecting FGN in renal tissues. For mass spectrometry, H&E stained FFPE tissue sections of each case were mounted on a special Director slide (OncoplexDX, Rockville, MD) and examined for presence of amyloid in renal glomeruli. Stained slides were loaded on a LMD apparatus (Lieca, Wetzlar, Germany) and renal glomeruli were visualized under florescent light for dissection. For non-amyloid tissues, hematoxylin and eosin (H&E) stained slides were loaded on a LMD apparatus and renal glomeruli were visualized under bright field. Multiple independent dissections (replicates), each configured to capture material from an area of 60,000 M2, were performed for each case. Material from each replicate dissection was captured in a tube containing sample buffer containing 35 of Tris/EDTA/0.002% Zwittergent. Proteins were extracted from the captured FFPE fragments using heat and sonication. Extracted proteins were digested with trypsin and resulting peptides were analyzed on either a QExactive mass spectrometer (Thermo- Fisher Scientific, Waltham, MA) or LTQ-Velos mass spectrometer (Thermo-Fisher Scientific, Waltham, MA) or LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA) using liquid chromatography-assisted tandem mass spectrometry (LC- MS/MS).

LC-MS/MS data from each patient sample was analyzed using multiple database search engines configured to match MS/MS against a composite protein sequence database containing SwissProt human proteome (downloaded 08/2012) and common contaminants (e.g. cotton, etc.). Reversed protein sequences were utilized to estimate false discovery rates. The search engines were configured to derive tryptic peptides from the sequence database and look for the following variable modifications: oxidation of methionine (+15.994 Da) and formation of n-terminal pyroglutamic acid (-17.023 Da). Scaffold software processed the results and assembled protein identifications with at least two confident (identification probability > 0.9) and unique peptide matches.

Immunohistochemistry was performed on four micron sections cut from formalin- fixed, paraffin-embedded blocks which were placed on "charged" slides; slides were then dried and melted in a 62°C oven for twenty minutes. Slides were placed on a Ventana BenchMark XT (Ventana Medical Systems Inc.) for staining. IHC slides were stained with Anti DNAJB9 rabbit polyclonal antibody (cat# #HPA040967; Sigma, 1/75 titer). The staining protocol includes on-line deparaffinization, HIER (Heat Induced Epitope Retrieval) with Ventana Cell Conditioning 1 for 32 minutes, and primary antibody incubation for 32 minutes at 37°C. Antigen-antibody reactions were visualized using Ventana OptiView™ Universal DAB Detection and OptiView Amplification Kits.

Counterstaining was performed online using Ventana Hematoxylin II for 8 minutes, followed by bluing reagent for 4 minutes. Protein DNAJB9 was identified by mass spectrometry in the glomerular deposits of patients with FGN. This protein was not detected by mass spectrometry in normal glomeruli, in glomeruli of patients with renal amyloidosis, or in patients with other forms of glomerulonephritis (Figure 1A). The presence of abundant DNAJB9 in the glomeruli of FGN was confirmed by immunohistochemistry (Figure IB).

The relative expression of the DNAJB9 in FGN renal biopsies, normal renal biopsies, and renal biopsies of various glomerulonephropathies was examined using a shotgun proteomics-based mass spectrometry assay (Figure 2).

These results demonstrate that an elevated level of DNAJB9 polypeptides can be used to identify mammals having FGN. These results also demonstrate that inhibitors of DNAJB9 polypeptide expression or activity can be used to treat FGN.

Example 2: DNAJB9 is present in serum of FGN patients

DNAJB9 levels were measured in serum of FGN patients and proteinuric controls using an immuno-MRM assay. The concentration of DNAJB9 in serum from FGN patients was significantly higher than the concentration of DNAJB9 in serum from healthy controls (Figure 3A).

A ROC analysis was performed to examine the sensitivity and specificity of DNAJB9 as a serum marker for FGN. A ROC curve demonstrates good sensitivity and specificity (Figure 3B) with data from the ROC analysis shown in the table below.

Cutoff Sensitivity% 95% CI Specificity% 95% CI Likelihood ratio

> 0.023 100 71.51% to 100% 6.667 0.1686% to 31.95% 1.071

> 0.038 100 71.51% to 100% 13.33 1.658% to 40.46% 1.154

> 0.0405 100 71.51% to 100% 20 4.331% to 48.09% 1.25

> 0.105 100 71.51% to 100% 26.67 7.787% to 55.1% 1.364

> 0.18 100 71.51% to 100% 33.33 11.82% to 61.62% 1.5

> 0.218 100 71.51% to 100% 40 16.34% to 67.71% 1.667

> 0.2625 100 71.51% to 100% 46.67 21.27% to 73.41% 1.875

> 0.2815 100 71.51% to 100% 53.33 26.59% to 78.73% 2.143

> 0.2965 100 71.51% to 100% 60 32.29% to 83.66% 2.5

> 0.327 100 71.51% to 100% 66.67 38.38% to 88.18% 3

> 0.3595 90.91 58.72% to 99.77% 66.67 38.38% to 88.18% 2.727

> 0.386 90.91 58.72% to 99.77% 73.33 44.9% to 92.21% 3.409 > 0.4565 90.91 58.72% to 99.77% 80 51.91% to 95.67% 4.545

> 0.553 90.91 58.72% to 99.77% 86.67 59.54% to 98.34% 6.818

58.72% to

> 0.6285 90,91 93.33 68.05% to 99.83% 13.64

> 0.6945 81.82 48.22% to 97.72% 93.33 68.05% to 99.83% 12.27

> 0.779 81.82 48.22% to 97.72% 100 78.2% to 100%

> 0.8425 72.73 39.03% to 93.98% 100 78.2% to 100%

> 0.904 63.64 30.79% to 89.07% 100 78.2% to 100%

> 1.029 54.55 23.38% to 83.25% 100 78.2% to 100%

> 1.161 45.45 16.75% to 76.62% 100 78.2% to 100%

> 1.304 36.36 10.93% to 69.21% 100 78.2% to 100%

> 1.439 27.27 6.022% to 60.97% 100 78.2% to 100%

> 1.499 18.18 2.283% to 51.78% 100 78.2% to 100%

0.2299% to

> 1.872 9.091 41.28% 100 78.2% to 100%

These results demonstrate that an elevated serum level of DNAJB9 polypeptides can be used to identify mammals having FGN.

Example 3: Diagnosing FGN through a non-invasive approach and Treating FGN

A biological sample (e.g. , serum or urine) is obtained from a human suspected of having a renal condition. The obtained sample is examined for the presence of an elevated level of DNAJB9 polypeptide expression or the presence of anti-DNAJB9 autoantibodies. In some cases, an IHC assay is performed to detect the presence of an elevated level of DNAJB9 polypeptide expression the presence of anti-DNAJB9 autoantibodies. In some cases, a MS assay is performed to detect the presence of an elevated level of DNAJB9 polypeptide expression. If an elevated level of DNAJB9 polypeptide expression is detected in the sample, as compared to a control level, then the human is administered one or more inhibitors of DNAJB9 polypeptide expression or activity. The administered inhibitor(s) can reduce the severity of one or more symptoms of FGN.

Example 4: Treating FGN

A human identified as having FGN is administered one or more DNAJB9 inhibitors of DNAJB9 polypeptide expression or activity (e.g. , a DNAJB9 monoclonal antibody that can neutralize circulating DNAJB9 and/or clear DNAJB9 from glomeruli). The administered inhibitor(s) can reduce the severity of one or more symptoms of FGN.

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a mammal having fibrillary glomerulonephritis (FGN), wherein said method comprises:

detecting the presence of an elevated level of DNAJB9 polypeptides in a biological sample from said mammal, and

classifying said mammal as having said FGN based at least in part on said presence.

2. The method of claim 1, wherein said level is determined using immunohistochemistry.

3. The method of claim 1, wherein said level is determined using mass spectrometry.

4. The method of claim 3, wherein said mass spectrometry comprises proteomics-based mass spectrometry assay or targeted quantification-based mass spectrometry assay.

5. The method of claim 1, wherein said biological sample comprises serum or urine, and wherein said detecting the presence of an elevated level of DNAJB9 polypeptides comprises detecting the presence of anti-DNAJB9 autoantibodies in said sample.

6. A method for treating fibrillary glomerulonephritis (FGN) in a mammal, said method comprising:

identifying said mammal as having an elevated level of DNAJB9 polypeptides within a biological sample from said mammal, and

administering an inhibitor of DNAJB9 polypeptide expression or activity to said mammal under conditions wherein the severity of a symptom of FGN is reduced.

7. A method of treating fibrillary glomerulonephritis (FGN) in a mammal, said method comprising:

identifying said mammal as having an elevated level of DNAJB9 polypeptides within a biological sample from said mammal, and administering an inhibitor of DNAJB9 polypeptide expression or activity to said mammal under conditions wherein progression of said FGN to kidney failure in said mammal is reduced.

8. The method of any one of claims 1-7, wherein said mammal is a human.

9. The method of any one of claims 1-4 or 6-7, wherein said biological sample comprises a kidney tissue sample.

10. The method of any one of claims 1-4 or 6-7, wherein said biological sample comprises a fluid sample.

11. The method of claim 10, wherein said fluid sample is serum or urine.