U.S. patent application number 15/495158 was filed with the patent office on 2017-12-21 for diagnosing and treating iga nephropathy.
The applicant listed for this patent is JUNTENDO UNIVERSITY SCHOOL OF MEDICINE, DIVISION OF NEPHROLOGY, DEPARTMENT OF INTERNAL MEDICINE, THE UAB RESEARCH FOUNDATION, UNIVERSITY OF TENNESSEE RESEARCH FOUNDATION, US ARMY WALTER REED ARMY MEDICAL CENTER, CHIEF, OFFICE OF RESEARCH, MARKETING & POLICY DEV. Invention is credited to Run Fan, Bruce A. Julian, Jiri Mestecky, Zina Moldoveanu, Jan Novak, Stephen Olson, Matthew B. Renfrow, Hitoshi Suzuki, Yusuke Suzuki, Milan Tomana, Yasuhiko Tomino, Robert J. Wyatt, Zhixin Zhang.
Application Number | 20170360927 15/495158 |
Document ID | / |
Family ID | 43223348 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170360927 |
Kind Code |
A1 |
Suzuki; Hitoshi ; et
al. |
December 21, 2017 |
DIAGNOSING AND TREATING IGA NEPHROPATHY
Abstract
Provided are methods of diagnosing IgA nephropathy in a subject.
Optionally, the methods comprise isolating an IgG from the subject
and determining whether the IgG binds to a galactose-deficient
IgA1. Optionally, the methods comprise providing a biological
sample from the subject and detecting in the sample a mutation in a
IGH gene, wherein the mutation is in a nucleotide sequence encoding
a complementarity determining region 3 (CDR3) of a IGH variable
region. Optionally, the methods comprise determining a level of IgG
specific for a galactose-deficient IgA1 in the subject. Also
provided are methods of treating or reducing the risk of developing
IgA nephropathy in a subject.
Inventors: |
Suzuki; Hitoshi; (Hoover,
AL) ; Fan; Run; (Omaha, NE) ; Julian; Bruce
A.; (Birmingham, AL) ; Novak; Jan; (Pelham,
AL) ; Moldoveanu; Zina; (Birmingham, AL) ;
Zhang; Zhixin; (Omaha, NE) ; Tomana; Milan;
(Birmingham, AL) ; Mestecky; Jiri; (Birmingham,
AL) ; Wyatt; Robert J.; (Memphis, TN) ;
Tomino; Yasuhiko; (Tokyo, JP) ; Suzuki; Yusuke;
(Tokyo, JP) ; Olson; Stephen; (Rockville, MD)
; Renfrow; Matthew B.; (Birmingham, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UAB RESEARCH FOUNDATION
US ARMY WALTER REED ARMY MEDICAL CENTER, CHIEF, OFFICE OF RESEARCH,
MARKETING & POLICY DEV
JUNTENDO UNIVERSITY SCHOOL OF MEDICINE, DIVISION OF NEPHROLOGY,
DEPARTMENT OF INTERNAL MEDICINE
UNIVERSITY OF TENNESSEE RESEARCH FOUNDATION |
Birmingham
Washingon
TOKYO
MEMPHIS |
AL
DC
TN |
US
US
JP
US |
|
|
Family ID: |
43223348 |
Appl. No.: |
15/495158 |
Filed: |
April 24, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14318082 |
Jun 27, 2014 |
9655963 |
|
|
15495158 |
|
|
|
|
13321025 |
Jul 3, 2012 |
|
|
|
PCT/US10/36239 |
May 26, 2010 |
|
|
|
14318082 |
|
|
|
|
61181083 |
May 26, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
A61P 13/12 20180101; G01N 33/6893 20130101; C07K 16/4283 20130101;
C07K 2317/55 20130101; A61K 39/39541 20130101; C07K 2317/41
20130101; G01N 33/6854 20130101; G01N 2800/347 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/42 20060101 C07K016/42; G01N 33/68 20060101
G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government funding under Grant
Nos. 1 RO1 DK078244 and 1 PO1 DK61525 from the National Institutes
of Health. The government has certain rights in this invention.
Claims
1.-50. (canceled)
51. A kit for performing an immunoassay, the kit comprising: (a) an
isolated antibody specific for galactose-deficient IgA1; and (b) a
container.
52. The kit of claim 51, further comprising an IgA1 specific
antibody and/or an IgG specific antibody.
53. The kit of claim 51, further comprising an assay substrate.
54. The kit of claim 53, wherein the assay substrate comprises a
membrane.
55. The kit of claim 51, further comprising a control sample.
56-61. (canceled)
62. The kit of claim 51, wherein the antibody is specific for a
galactose-deficient hinge-region O-linked glycan of IgA1
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/181,083, filed on May 26, 2009, which is
incorporated by reference herein in its entirety.
BACKGROUND
[0003] IgA nephropathy (IgAN), also called Berger disease, was
described in 1968 based on the immunohistochemical finding of IgA-
and IgG-containing immune complexes in the glomerular mesangium of
the kidney. Proliferation of mesangial cells and expansion of the
extracellular matrix can occur from the earliest stages of the
disease, with progression to glomerular and interstitial sclerosis
resulting in development of end-stage renal disease in 30%-40% of
patients within 20 years of the estimated time of disease
onset.
[0004] The IgA in the mesangial deposits is exclusively of the IgA1
subclass and is aberrantly glycosylated, with the hinge-region
O-linked glycans being deficient in galactose (Gal). The IgA1 in
the circulation of patients with IgAN also carries Gal-deficient
O-glycans, although Gal-deficient variants are rarely found in the
IgA1 in sera from normal individuals. The production of these
variants is associated with altered expression of specific
glycosyltransferases in the IgA1-producing cells. It is the binding
of IgA1-containing immune complexes with aberrantly glycosylated
IgA1 to mesangial cells that induces the renal manifestations
characteristic of IgAN; however, the events that initiate the
disease process are most likely of extra-renal origin, as IgAN
recurs in more than 50% of patients within 2 years of kidney
transplantation.
SUMMARY
[0005] Provided are methods of diagnosing IgA nephropathy in a
subject. The methods include, for example, isolating an IgG from
the subject and determining whether the IgG binds to a
galactose-deficient IgA1. Binding of the IgG to the
galactose-deficient IgA1 indicates the subject has or is at risk
for developing IgA nephropathy.
[0006] The methods comprise providing a biological sample from the
subject and detecting in the sample a mutation in a IGH gene,
wherein the mutation is in a nucleotide sequence encoding a
complementarity determining region 3 (CDR3) of a IGH variable
region. A mutation in the nucleotide sequence compared to a control
sequence indicates the subject has or is at risk for developing IgA
nephropathy.
[0007] The methods comprise determining a level of IgG specific for
a galactose-deficient IgA1 in the subject. An increase in the level
of IgG specific for galactose-deficient IgA1 as compared to a
control indicates the subject has or is at risk for developing IgA
nephropathy. Optionally, the methods further comprise determining a
level of galactose-deficient IgA1 in the subject. An increase in
the level of galactose-deficient IgA1 as compared to a control
indicates the subject has or is at risk for developing IgA
nephropathy.
[0008] Also provided are methods of treating or reducing the risk
of developing IgA nephropathy in a subject. The methods optionally
comprise administering to the subject an agent, wherein the agent
inhibits the binding of the IgG to galactose-deficient IgA1. The
methods optionally comprise reducing a level of IgG specific for
galactose-deficient IgA1 in a subject.
[0009] Also provided are isolated antibodies or fragments thereof.
The isolated antibodies or fragments thereof are specific for a
galactose-deficient hinge-region O-glycan of IgA1. The isolated
antibodies can comprise an alanine to serine amino acid
substitution in a complementarity determining region 3 (CDR3) of an
IGH variable region.
[0010] Further provided are methods of detecting
galactose-deficient IgA1 in a subject. The methods comprise
obtaining a biological sample from the subject and utilizing an
isolated antibody or fragment thereof specific for
galactose-deficient IgA1 in an assay to detect galactose-deficient
IgA1 in the subject.
[0011] Further provided is an isolated polypeptide comprising,
consisting of, or consisting essentially of a galactose-deficient
hinge-region O-glycan of IgA1.
[0012] Also provided are kits for performing immunoassays. The kits
comprise a galactose-deficient IgA1, and a container. The kits can
further comprise an IgG specific antibody. The kits can further
comprise an assay substrate (e.g., a plate, a membrane, a well,
etc.).
[0013] Also provided are methods of creating an animal model for
IgA nephropathy. The methods comprise forming immune complexes in
vitro, wherein the immune complexes comprise galactose-deficient
IgA1 and IgG specific for galactose-deficient IgA1 and injecting
the immune complexes into the animal. Injection of the immune
complexes into the animal results in an animal model of IgA
nephropathy. Further provided are animal models of IgA nephropathy
comprising immune complexes with galactose-deficient IgA1 and IgG
specific for galactose-deficient IgA1.
DESCRIPTION OF DRAWINGS
[0014] FIGS. 1A to 1D show serum IgG from IgA nephropathy (IgAN)
patients exhibit specificity for GalNAc, binding to
galactose-deficient and desialylated IgA1. FIG. 1A shows an image
of a Western blot demonstrating that Gal-deficient IgA1 (Mce)
antigen bound serum IgG from 2 IgAN patients but serum IgG from 2
healthy controls minimally bound to the IgA1 heavy chain. After
removal of sialic acid, IgG binding increased, as it did for
binding to Helix aspersa agglutinin (HAA). N+, treated with
neuraminidase; N-, not treated with neuraminidase. FIG. 1B shows an
image of a Western blot demonstrating the glycan-specific binding
of IgG. To test glycan-specific IgG binding to
N-acetylgalactosamine (GalNAc) on IgA1, these IgA1 proteins were
used: lane 1, Gal-deficient IgA1 (Mce); lane 2, desialylated and
degalactosylated (dd)-IgA1; lane 3, enzymatically regalactosylated
dd-IgA1; and lane 4, enzymatically resialylated dd-IgA1. dd-IgA1
bound the greatest amount of HAA, with enzymatically galactosylated
or sialylated dd-IgA1 binding very little. IgG from an IgAN patient
bound to these antigens in a fashion similar to that for HAA. FIG.
1C shows an image of a Western blot. Component chains of
Gal-deficient IgA1 (Mce) were separated by SDS-PAGE under reducing
conditions and electroblotted. The membrane was then treated with
HAA to assess whether blockade with this GalNAc-specific lectin can
inhibit IgG binding. FIG. 1D shows a bar graph demonstrating the
intensity of each band as quantified by densitometry. The binding
of serum IgG from an IgAN patient to Gal-deficient IgA1 was reduced
by 66% after treatment with HAA. Conversely, blocking with serum
IgG from an IgAN patient reduced the binding of HAA to
Gal-deficient IgA1 by 60%. Binding of anti-human IgA (heavy-chain
specific) confirmed equivalent loading. Representative results from
3 experiments are shown in A-C; lanes were run on the same gel but
were noncontiguous.
[0015] FIGS. 2A to 2C show the characterization of antibodies
specific for Gal-deficient IgA1 secreted by cloned cell lines. The
levels of antigen-specific IgG produced by IgG-secreting cell lines
were measured by capture ELISA. The results are expressed as OD
measured at 490 nm. FIGS. 2A and 2B shows graphs demonstrating that
the levels of IgG directed against dd-IgA1 (2A) and Fab-IgA1 (2B)
were higher in IgAN patients than in controls. Each group, n=16.
**P<0.0001; data are shown as individual values and mean.+-.SD.
FIG. 2C shows a graph demonstrating IgG secreted by cell lines from
IgAN patients and healthy controls (each group, n=10) was tested
for binding to a hinge-region glycopeptide (HR-GalNAc-BSA) or
HR-BSA, with or without HAA blockade. IgG produced by cell lines
from IgAN patients bound to HR-GalNAc in an HAA-inhibitable
fashion. *P<0.001; data are shown as the mean.+-.SD. P values
were generated using 2-tailed Student's t test. The experiments
were repeated 3 times with similar results.
[0016] FIGS. 3A to 3C show the characterization of immune-complex
formation. FIG. 3A shows a graph demonstrating size-exclusion
chromatography and ELISA analysis of immune complexes formed in
vitro with monomeric Gal-deficient IgA1 (50 .mu.g) and monoclonal
glycan-specific IgG (50 .mu.g) from cell lines from 3 patients with
IgAN (filled circles) or 3 healthy controls (open circles). IgG and
monomeric (m) and dimeric (d) IgA1 standards were used to calibrate
the column. Glycan-specific IgG from IgAN patients exhibited more
binding to Gal-deficient IgA1 as compared with the binding of IgG
from healthy controls. Immune complexes likely contained 1 or 2
molecules of IgA1 bound to 1 molecule of IgG. Data are shown as
mean.+-.SD. FIG. 3B shows an image of a Dot-blot analysis
demonstrating that IgG secreted by cell lines from 5 of the 6 IgAN
patients exhibited high binding to Gal-deficient IgA1; cell line
no. 3081 from an IgAN patient and cells from 5 of the 6 healthy
controls exhibited low binding. FIG. 3C shows a graph demonstrating
that the findings shown in FIG. 3B were confirmed by
densitometrical analysis. P<0.01; P values were generated using
the 2-tailed Student's t test. Data are shown as individual values
and mean.+-.SD. Experiments were repeated 3 times with similar
results.
[0017] FIGS. 4A to 4D show the importance of the A to S
substitution in YCAR (SEQ ID NO:45) or YCAK (SEQ ID NO:37) sequence
of CDR3 in the binding of IgG to Gal-deficient IgA1. FIG. 4A shows
an image of a Western blot analysis using Gal-deficient IgA1 (Ale
poly) as antigen that demonstrated binding of rIgG cloned from an
IgAN patient (subject 1123) but only marginal binding of rIgG from
a healthy control (subject 9017). FIG. 4B shows an image of a
Western blot. The reduced Gal-deficient IgA1 (Mce1) (lane 1);
enzymatically desialylated Gal-deficient IgA1 (Mce1) (lane 2); and
desialylated and degalactosylated Gal-deficient IgA1 (Mce1) (lane
3) were incubated with rIgG after SDS-PAGE/Western blotting.
Removal of sialic acid and Gal in the IgA1 hinge region increased
the binding, suggesting that the rIgG bound specifically to GalNAc.
FIG. 4C shows the amino acid (aa) sequence YCSKVCRPWNYRRPYYYGMDVW
(SEQ ID NO:2) in the CDR3 of VH of IgG from an IgAN patient
(subject 1123) was reverted to the healthy control germline
counterpart sequence YCAKVCRPWNYRRPYYYGMDVW (SEQ ID NO:35) using an
overlap PCR strategy. Conversely, the aa sequence
YCARVQRYDSTGYYPLGYLDLW (SEQ ID NO:12) in the CDR3 of IgG from a
healthy control (subject 9017) was mutated to generate
YCSRVQRYDSTGYYPLGYLDLW (SEQ ID NO:36). FIG. 4D shows after the S to
A substitution was introduced in CDR3 of VH of IgG of the cells
from an IgAN patient (subject 1123), rIgG binding to Gal-deficient
IgA1 was reduced by 72%. Conversely, the A to S substitution in
CDR3 of IgG of the cells from a healthy control (subject 9017)
increased binding to Gal-deficient IgA1. Anti-human IgA (heavy
chain specific) Western blotting was used as load control. Results
were evaluated densitometrically. Representative results from 2
experiments are shown in A-D; lanes were run on the same gel but
were noncontiguous.
[0018] FIGS. 5A to 5E show serum levels of IgG specific for
Gal-deficient IgA1 are elevated in patients with IgAN. FIG. 5A
shows an image of dot-blot assay. Gal-deficient IgA1 (Ale) placed
in 96-well plates with PVDF membranes was incubated with normalized
concentrations of serum IgG from IgAN patients, disease controls,
and healthy controls; a representative example from 3 experiments
is shown (20 samples from each group). The rIgG from an IgAN
patient served as a positive control. Serum IgG from IgAN patients
bound more to Gal-deficient IgA1 compared with the IgG from disease
controls or healthy controls. FIG. 5B shows a scatter plot with the
intensity of signal in each well measured by densitometry; the
intensity of rIgG bound to Gal-deficient IgA was assigned a value
of 100%. Serum IgG from IgAN patients has significantly higher
reactivity to Gal-deficient IgA1 compared with that from healthy
(P<0.0001) and disease controls (P<0.0001). Serum IgG from 54
of the 60 patients with IgAN showed values greater than the 90th
percentile of the values for healthy controls. Wilcoxon's rank-sum
test was used for 2-sample comparison. Data are shown as individual
values and the mean.+-.SD. FIG. 5C shows a graph showing ROC for
serum IgG binding to Gal-deficient IgA1. The area under the curve
is 0.9644. These data indicate a sensitivity of 88.3% and a
specificity of 95.0% (P<0.0001; 95% CI, 0.928-1.00). The value
of specificity is plotted as 1-specificity on the x axis. FIGS. 5D
and 5E show scatter plots demonstrating the intensity of IgG
binding to Gal-deficient IgA1 correlated with the UP/Cr ratio (5D)
(P<0.0001) as well as with urinary IgA-IgG immune complexes (5E)
(P=0.0082) in contemporaneously (i.e., within 30 days of renal
biopsy) collected urine samples. UlgA-IgG IC/Cr, urinary excretion
of IgA-IgG immune complexes/creatinine ratio.
[0019] FIGS. 6A-6C show the characterization of a passive murine
model of IgAN using immune complexes formed between Gal-deficient
IgA1 and anti-glycan IgG. FIG. 6A shows a graph demonstrating that
immune complexes were formed from Gal-deficient IgA1 (Ale) and
anti-glycan IgG in vitro. FIG. 6B shows a scanning electron
microscopic image of a red blood cell in the urine of mice injected
with the immune complexes. FIG. 6C shows microscopic images of the
immune complexes (stained for human IgA, human IgG, and murine C3)
deposited in the renal mesangium of the passive murine model of
IgAN.
[0020] FIG. 7 shows transmission electron microscopic images of
glomeruli of mice injected with Gal-deficient IgA1-IgG complexes.
The images showed electron-dense immunodeposits (bottom right) in
the mesangium and evidence of podocyte injury (podocyte effacement,
microvilli formation; top right) and the presence of a red blod
cell in Bowman's urinary space (top left), concurrent with
hematuria and proteinuria.
[0021] FIG. 8 shows an immunologically-mediated strategy for
preventing the formation of large, nephritogenic immune complexes
in IgAN. Antigenic glycan determinants in the hinge region of IgA1
are covered with monovalent, high-affinity single chain (sc)-Abs or
other monovalent antibody fragments (such as Fab or Fv) that
prevent naturally-occurring anti-GalNAc antibodies from
cross-linking polymeric Gd-IgA1 molecules. Only small,
non-nephritogenic complexes are formed.
[0022] FIG. 9 shows two immunologically-mediated strategies for
preventing the formation of large nephritogenic immune complexes in
IgA nephropathy. In strategy 1, antigenic determinants in the
hinge-region glycans are covered with monovalent, high-affinity Fv
or Fab fragments of antibodies that prevent naturally-occurring
anti-GalNAc IgG or IgA1 antibodies from cross-linking Gal-deficient
polymeric IgA molecules. In strategy 2, a synthetic glycopeptide
with a single GalNAc residue (to prevent cross-linking) is
recognized by naturally occurring IgG (or IgA1) anti-GalNAc
antibodies that cannot cross-link Gal-deficient polymeric IgA1. In
both cases, small, non-nephritogenic complexes are formed.
DETAILED DESCRIPTION
[0023] Provided herein are methods of diagnosing IgA nephropathy
(IgAN) in a subject. The methods comprise isolating an IgG from the
subject and determining whether the IgG binds to a
galactose-deficient IgA1. Binding of the IgG to the
galactose-deficient IgA1 indicates the subject has or is at risk of
developing IgA nephropathy. The IgG can, for example, be isolated
from a B cell. The B cell can be isolated from a population of
peripheral blood mononuclear cells (PBMCs). Optionally, the B cell
is immortalized. The B cell can, for example, be immortalized by
transformation with an Epstein-Barr virus (EBV).
[0024] Optionally, determining binding of the IgG to the
galactose-deficient IgA1 comprises performing an assay from the
group consisting of a Western blot, an enzyme-linked immunosorbent
assay (ELISA), an immunoaffinity assay, and a dot-blot assay.
[0025] Optionally, the method of diagnosing IgA nephropathy in a
subject comprises providing a biological sample from the subject
and detecting in the sample a mutation in an IGH gene, wherein the
mutation is in a nucleotide sequence encoding a complementarity
determining region 3 (CDR3) of an IGH variable region. A mutation
in the nucleotide sequence compared to a control sequence indicates
the subject has or is at risk of developing nephropathy. The
mutation in the nucleotide sequence can be a somatic mutation
(i.e., spontaneously occurring), or alternatively, the mutation can
be a genetic mutation (i.e., passed down generationally from
parents to offspring).
[0026] As used herein a biological sample is a sample derived from
a subject and includes, but is not limited to, any cell, tissue or
biological fluid. For example, the sample can be a tissue biopsy,
blood or components thereof, bone marrow, urine, saliva, tissue
infiltrate and the like. The biological fluid may be a cell culture
medium or supernatant of cultured cells from a subject. Optionally,
the biological sample contains cerebral spinal fluid.
[0027] Optionally, the biological sample comprises a genetic
sample. The genetic sample comprises a nucleic acid, preferably RNA
and/or DNA. A genetic sample may be obtained using any known
technique including those described in Ausubel et al., Current
Protocols in Molecular Biology (John Wiley & Sons, Inc., New
York, 1999); Sambrook et al., Molecular Cloning: A Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor (2001); and Hames and Higgins, Nucleic Acid Hybridization
(1984). The nucleic acid may be purified from whole cells using DNA
or RNA purification techniques. The genetic sample may also be
amplified using PCR or in vivo techniques requiring subcloning. The
genetic sample can be obtained by isolating mRNA from the cells of
the biological sample and reverse transcribing the RNA into DNA in
order to create cDNA (Khan et al. Biochem. Biophys. Acta 1423:17-28
(1999)).
[0028] The genetic sample can be analyzed for the presence or
absence of a particular mutation. Thus, determining whether the
CDR3 of the IGH variable region nucleotide sequence comprises a
mutation can, for example, be carried out by a method selected from
the list consisting of sequencing, PCR, RT-PCR, quantitative PCR,
one step PCR, restriction fragment length polymorphism,
hybridization techniques, Northern blot, microarray technology,
gene chip, in situ hybridization, DNA microarray technology, and
the like. Alternatively, determining whether the CDR3 of the IGH
variable region amino acid sequence comprises a mutation can, for
example, be carried out by Western Blot or protein sequencing. The
analytical techniques to determine whether the CDR3 of the IGH
variable region nucleotide sequence or amino acid sequences
comprise a mutation are known. See., e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (2001).
[0029] As used throughout, the term mutation includes one or more
deletions, insertions, or substitutions of one or more amino acids
or nucleotides. Thus, in the provided methods, the mutation can be
a deletion, insertion, or substitution. Optionally, the mutation is
a deletion or substitution. By way of example, an insertion or
deletion can result in an alteration of the reading frame of the
gene, which alters the function of the gene. A point mutation or
substitution can, for example, result in a mutation, e.g., a
missense mutation, or a nonsense mutation, that alters the function
of a gene. For example, the function of a gene can be altered in
that the gene is no longer transcribed at wild-type levels.
Alternatively, the amino acid sequence encoded by the gene no
longer functions at control levels.
[0030] Optionally, the mutation in the IGH gene comprises one or
more nucleotide substitutions resulting in an alanine to serine
amino acid substitution in a YCAR (SEQ ID NO:45) or a YCAK (SEQ ID
NO:37) amino acid sequence encoded by the IGH gene. Optionally, the
nucleotide sequence encoding the CDR3 of the IGH variable region
encodes an amino acid sequence comprising SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
[0031] The method of diagnosing or characterizing IgA nephropathy
in a subject optionally comprises determining a level of IgG
specific for a galactose-deficient IgA1 in the subject. An increase
in the level of IgG specific for galactose-deficient IgA1 as
compared to a control indicates the subject has or is at risk of
developing IgA nephropathy, or, if increased over a previous level,
the same subject may indicate a progression in the IgA nepropathy
and/or a need for a change in medication. Alternatively, if the
level of IgG is compared to a subject with IgA nephropathy and the
level is within the range of one or more subjects with IgA
nephropathy, this indicates the subject has IgA nephropathy.
Optionally, the method comprises determining a level of
galactose-deficient IgA1 in the subject. An increase in the level
of galactose-deficient IgA1 as compared to a control indicates the
subject has or is at risk of developing IgA nephropathy, or, if
increased over a previous level, the same subject may indicate a
progression in the IgA nephropathy and/or a need for a change in
medication. Alternatively, if the level of galactose-deficient IgA1
is compared to a subject with IgA nephropathy and the level is
within the range of one or more subjects with IgA nephropathy, this
indicates the subject has IgA nephropathy. A control can comprise a
sample or known value from a subject or group of subjects that does
not have IgA nephropathy. Alternatively, the control can comprise a
sample or known value from the same subject prior to or early in
the onset of IgA nephropathy.
[0032] Serum levels of galactose-deficient IgA1 are measured in
reference to a standard galactose-deficient IgA1 myeloma protein.
The amount of such protein that generates a certain OD value is
defined as one unit. The result of measurement for IgA nephropathy
patients, disease controls, and healthy controls are then compared,
and depending on normality of distribution, a cut off value is
defined, such as 90.sup.th percentile of values for healthy
controls. The results are further tested by receiver-operating
characteristic (ROC)-curve analysis to define significance,
sensitivity, and specificity at certain cut off values.
[0033] When performing an assay as described in Suzuki et al., J.
Clin. Invest. 118:629-39 (2008), the standard galactose-deficient
IgA1 myeloma protein is galactose-deficient IgA1 (Ale). The level
of galactose-deficient IgA1 indicating a diagnosis of IgA
nephropathy can, for example, be at least 95 units per milliliter
(U/ml). Optionally, the level of galactose-deficient IgA1
indicating a diagnosis of IgA nephropathy can be in the range of
about 95 U/ml to about 200 U/ml. Optionally, the level of
galactose-deficient IgA1 can be about 110 U/ml to about 175 U/ml.
Optionally, the level of galactose-deficient IgA1 can be about 130
U/ml to about 155 U/ml.
[0034] When performing an assay as described in Moldoveanu et al.,
Kidney Int. 71:134-8 (2007), the standard galactose-deficient IgA1
myeloma protein is galactose deficient IgA1 (Mce). The level of
galactose-deficient IgA1 indicating a diagnosis of IgA nephropathy
can, for example, be at least 1000 units per milliliter (U/ml).
Optionally, the level of galactose-deficient IgA1 indicating a
diagnosis of IgA nephropathy can be in the range of about 1000 U/ml
to about 8000 U/ml. Optionally, the level of galactose-deficient
IgA1 can be about 1500 U/ml to about 4000 U/ml. Optionally, the
level of galactose-deficient IgA1 can be about 1800 U/ml to about
2500 U/ml.
[0035] The level of IgG specific for galactose-deficient IgA1 can
be measured, for example, by dot blot assay with
galactose-deficient IgA1 (or its Fab fragment) as an antigen.
Standard IgG (such as recombinant monoclonal IgG or standard
polyclonal serum IgG) serve as a standard to calibrate measurement.
The results of the measurement for IgA nephropathy patients,
disease controls, and healthy controls are then compared, and
depending on normality of distribution, a cut off value is defined,
such as 90.sup.th percentile of values for healthy controls. The
results are further tested by receiver-operating characterisitic
(ROC)-curve analysis to define significance, sensitivity, and
specificity at certain cut off values. For example, when dot-blot
membranes were coated with galactose-deficient IgA1 (Ale) and a
recombinant monoclonal IgG was used as the standard, the 90.sup.th
percentile for normal healthy controls was defined as about 17
units per 0.5 .mu.g serum IgG (density of binding determined by
densitometry and expressed as a percentage of binding of 0.5 .mu.g
of recombinant monoclonal IgG standard set to 100 units), thus 34
units per 1 .mu.g serum IgG.
[0036] When performing an assay as described in Suzuki et al., J.
Clin. Invest. 118:629-39 (2008), the serum level of IgG specific
for a galactose-deficient IgA1 indicating a diagnosis of IgA
nephropathy can, for example, be at least 35 units per 1 .mu.g
serum IgG (U/.mu.g). Optionally, the serum level of IgG specific
for galactose-deficient IgA1 indicating a diagnosis of IgA
nephropathy can be in the range of about 35 U/.mu.g to about 100
U/.mu.g. Optionally, the serum level of IgG specific for
galactose-deficient IgA1 can be about 50 U/.mu.g to about 85
U/.mu.g. Optionally, the serum level of IgG specific for
galactose-deficient IgA1 can be about 60 U/.mu.g to about 75
U/.mu.g.
[0037] Optionally, the IgG or galactose-deficient IgA1 is isolated
from the subject. The IgG or galactose-deficient IgA1 can, for
example, be isolated from a B cell. Optionally, the B cell is
isolated from a population of peripheral blood mononuclear cells
(PBMCs). The B cell can be immortalized. Optionally, the B cell is
immortalized by transformation with an Epstein-Barr virus.
[0038] Determining the level of IgG specific for
galactose-deficient IgA1 or the level of galactose-deficient IgA1
comprises, for example, performing an assay from the group
consisting of a Western blot, an enzyme-linked immunosorbent assay
(ELISA), an immunoaffinity assay, and a dot-blot assay. However,
other assay systems can be used.
[0039] Further provided are methods of treating or reducing the
risk of developing IgA nephropathy in a subject. The method
optionally comprises administering to the subject an agent, wherein
the agent inhibits the binding of the IgG (and/or IgA1) to
galactose-deficient IgA1. Inhibiting the binding of IgG and
galactose-deficient IgA1 can comprise interfering with the
formation of and/or reducing the size of the immune complexes
formed by IgG and galactose-deficient IgA1 in subjects comprising
IgA nephropathy. Optionally, the agent is selected from the group
consisting of a small molecule, a polypeptide, an inhibitory
nucleic acid molecule, a peptidomimetic, or a combination thereof.
Optionally, the agent can be a polypeptide. The polypeptide can,
for example, comprise the hinge region of IgA1 to be used as a
competitive inhibitor to block binding of IgG with
galactose-deficient IgA1. Optionally, the polypeptide can, for
example, comprise a glycopeptide with a single GalNAc residue. The
glycopeptide is recognized by the IgG specific for
galactose-deficient IgA1, thus, preventing binding and the
formation of immune complexes comprised of the IgG specific for
galactose-deficient IgA1 and galactose-deficient IgA1. Optionally,
the polypeptide can comprise an antibody. The antibody can be
specific for the galactose-deficient hinge-region O-linked glycans
of IgA1 to be used as a competitive inhibitor. Optionally, the
antibody is a single-chain antibody (sc-Ab), a high affinity Fv
antibody fragment, or a Fab antibody fragment specific for the
hinge-region O-linked gylcans of IgA1.
[0040] The method of treating or reducing the risk of developing
IgA nephropathy in a subject optionally comprises reducing a level
of IgG specific for galactose-deficient IgA1 in the subject.
Optionally, reducing the level of IgG specific for
galactose-deficient IgA1 in the subject comprises the use of
plasmapheresis. Optionally, reducing the level of IgG specific for
galactose-deficient IgA1 in the subject comprises administering to
the subject an agent that reduces the level of IgG in the subject.
The agent can be selected from the group consisting of a small
molecule, a polypeptide, an inhibitory nucleic acid molecule, a
peptidomimetic, or a combination thereof. Optionally, an inhibitory
nucleic acid molecule can be selected from the group consisting of
a short interfering RNA (siRNA) molecule, a microRNA (miRNA)
molecule, or an antisense nucleic acid molecule. The inhibitory
nucleic acid molecule can, for example, target the gene encoding
the IGH variable region of the IgG or the mRNA for the IGH variable
region of the IgG in the subject to reduce the level of IgG
specific for galactose-deficient IgA1.
[0041] Further provided are isolated antibodies or fragments
thereof specific for a galactose-deficient hinge region O-linked
glycans of IgA1. Optionally, the isolated antibody or fragment
thereof comprises an alanine to serine amino acid substitution in
the YCAR (SEQ ID NO:45) or a YCAK (SEQ ID NO:37) amino acid
sequence encoded in the complementarity determining region 3 (CDR3)
of an IGH variable region amino acid sequence. Optionally, the
antibody comprises a monoclonal antibody. Also provided is a
hybridoma cell line capable of producing the monoclonal antibody
described herein. Optionally, the isolated antibodies or fragments
thereof specific for the galactose-deficient hinge region O-linked
glycans of IgA1 are produced by a cell line transfected with a
vector encoding the antibody. The transfected cell line can, for
example, comprise a primary cell line or an immortalized cell line.
As defined herein, the term antibody includes, but is not limited
to, fragments of the antibody, single-chain antibodies, conjugates
of antibody fragments, chimeric antibodies, and hybrid
antibodies.
[0042] Further provided herein are methods of detecting
galactose-deficient IgA1 in a subject. The methods comprise
obtaining a biological sample from the subject and utilizing an
isolated antibody specific for galactose-deficient IgA1 to detect
galactose-deficient IgA1 in the subject. An assay to detect
galactose-deficient IgA1 in the subject can be selected from the
group consisting of a Western blot, an enzyme-linked immunosorbent
assay (ELISA), an immunoaffinity assay, an immunofluorescence
assay, and a dot-blot assay. However, other assays can be used.
[0043] Also provided herein are isolated polypeptides comprising
galactose-deficient hinge-region O-linked glycans of IgA1 or a
fragment thereof. Optionally, the polypeptide consists of or
consists essentially of the galactose-deficient hinge-region
O-linked glycans of IgA1 or a fragment thereof. The isolated
polypeptide comprising the galactose-deficient hinge-region
O-linked glycans of IgA1 can comprise the amino acid sequence
CHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHR (SEQ ID NO:34).
Fragments include, for example, CHVKHYTNPS (SEQ ID NO:38),
VTVPCPVPST (SEQ ID NO:39), STPPTPSPST (SEQ ID NO:40), TPPTPSPSCC
(SEQ ID NO:41), and VPSTPPTPSP (SEQ ID NO:42). Optionally, the
fragment blocks binding of IgG specific for galactose-deficient
IgA1 to galactose-deficient IgA1. Consequently, the polypeptide
fragment thereof can be used as a competitive inhibitor of binding
for IgG specific for galactose-deficient IgA1 binding to
galactose-deficient IgA1.
[0044] Also provided are kits for performing immunoassays described
herein. The kits comprise a galactose-deficient IgA1 and a
container. Optionally, the kit further comprises an IgG specific
antibody. The kit can further comprise an assay substrate (e.g., a
plate, a membrane, and a well). Optionally, the kit can further
comprise a control sample. The control sample can be from a patient
with IgAN.
[0045] Optionally, the kit can comprise the isolated antibodies
described herein and a container. The kit can further comprise an
IgA1 specific antibody. Optionally, the kit can comprise as assay
substrate. Optionally, the kit can further comprise a control
sample. The control sample can comprise galactose-deficient
IgA1.
[0046] Also provided are methods of creating an animal model of IgA
nephropathy. The methods comprise forming immune complexes in
vitro, wherein the immune complexes comprise galactose-deficient
IgA1 and IgG specific for galactose-deficient IgA1 and injecting
the immune complexes into the animal. Injection of the immune
complexes into the animal results in an animal model of IgA
nephropathy. Optionally, the animal model comprises a mouse model.
Optionally, the mouse is a nude mouse. Optionally, the immune
complexes are deposited in the renal mesangium of the animal
model.
[0047] By nude mouse it is meant that the mouse contains mutations
in both copies of the "nu" gene. The nude mouse does not contain a
thymus, rendering the mouse incapable of producing T cells.
Therefore, the nude mouse cannot reject tumors or transplants of
cells from humans or other animals.
[0048] Also provided herein are animal models of IgA nephropathy
comprising immune complexes with galactose-deficient IgA1 and IgG
specific for galactose-deficient IgA1. Optionally, the animal model
is produced by the methods disclosed herein. Optionally, the animal
model is a mouse model.
[0049] As used herein, the term antibody encompasses whole
immunoglobulin (i.e., an intact antibody) of any class. Native
antibodies are usually heterotetrameric glycoproteins, composed of
two identical light (L) chains and two identical heavy (H) chains.
Typically, each light chain is linked to a heavy chain by one
covalent disulfide bond, while the number of disulfide linkages
varies between the heavy chains of different immunoglobulin
isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a
variable domain (V(H)) followed by a number of constant domains.
Each light chain has a variable domain at one end (V(L)) and a
constant domain at its other end; the constant domain of the light
chain is aligned with the first constant domain of the heavy chain,
and the light chain variable domain is aligned with the variable
domain of the heavy chain. Particular amino acid residues are
believed to form an interface between the light and heavy chain
variable domains. The light chains of antibodies from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains. Depending on the
amino acid sequence of the constant domain of their heavy chains,
immunoglobulins can be assigned to different classes. There are
five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,
and several of these may be further divided into subclasses
(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
The heavy chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. Although antibodies are described throughout,
fragments of antibodies, single-chain antibodies, conjugates of
antibody fragments, chimeric antibodies, and hybrid antibodies can
be used in the methods described herein.
[0050] The term variable is used herein to describe certain
portions of the antibody domains that differ in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not usually evenly distributed through the variable
domains of antibodies. It is typically concentrated in three
segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of the
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a .beta.-sheet configuration, connected
by three CDRs, which form loops connecting, and in some cases
forming part of, the .beta.-sheet structure. The CDRs in each chain
are held together in close proximity by the FR regions and, with
the CDRs from the other chain, contribute to the formation of the
antigen binding site of antibodies. The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody-dependent cellular toxicity.
[0051] As used herein, the term epitope is meant to include any
determinant capable of specific interaction with the provided
antibodies. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
Identification of the epitope that the antibody recognizes is
performed as follows. First, various partial structures of the
target molecule that the monoclonal antibody recognizes are
prepared. The partial structures are prepared by preparing partial
peptides of the molecule. Such peptides are prepared by, for
example, known oligopeptide synthesis technique or by incorporating
DNA encoding the desired partial polypeptide in a suitable
expression plasmid. The expression plasmid is delivered to a
suitable host, such as E. coli, to produce the peptides. For
example, a series of polypeptides having appropriately reduced
lengths, working from the C- or N-terminus of the target molecule,
can be prepared by established genetic engineering techniques. By
establishing which fragments react with the antibody, the epitope
region is identified. The epitope is more closely identified by
synthesizing a variety of smaller peptides or mutants of the
peptides using established oligopeptide synthesis techniques. The
smaller peptides are used, for example, in a competitive inhibition
assay to determine whether a specific peptide interferes with
binding of the antibody to the target molecule. If so, the peptide
is the epitope to which the antibody binds. Commercially available
kits, such as the SPOTs Kit (Genosys Biotechnologies, Inc.; The
Woodlands, Tex.) and a series of multipin peptide synthesis kits
based on the multipin synthesis method (Chiron Corporation,
Emeryville, Calif.) may be used to obtain a large variety of
oligopeptides.
[0052] The term antibody or fragments thereof can also encompass
chimeric antibodies and hybrid antibodies, with dual or multiple
antigen or epitope specificities, and fragments, such as
F(ab').sub.2, Fab', Fab and the like, including hybrid fragments.
Thus, fragments of the antibodies that retain the ability to bind
their specific antigens are provided. For example, fragments of
antibodies which maintain galactose-deficient IgA1 binding activity
are included within the meaning of the term antibody or fragment
thereof. Such antibodies and fragments can be made by techniques
known in the art and can be screened for specificity and activity
according to general methods for producing antibodies and screening
antibodies for specificity and activity (See Harlow and Lane.
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York (1988)).
[0053] Conjugates of antibody fragments and antigen binding
proteins (single chain antibodies) can be used in the methods
taught herein. Such conjugates of antigen binding proteins are
described, for example, in U.S. Pat. No. 4,704,692, the contents of
which are hereby incorporated by reference in their entirety.
[0054] Optionally, the antibody is a monoclonal antibody. The term
monoclonal antibody as used herein refers to an antibody from a
substantially homogeneous population of antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies may be prepared
using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 256:495 (1975) or Harlow and Lane, Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, New York
(1988). In a hybridoma method, a mouse or other appropriate host
animal is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The immunizing agent can
be a galactose-deficient IgA1 or an immunogenic fragment
thereof.
[0055] Generally, either peripheral blood lymphocytes (PBLs) are
used in methods of producing monoclonal antibodies if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, pp. 59-103 (1986)). Immortalized cell lines are usually
transformed mammalian cells, including myeloma cells of rodent,
bovine, equine, and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells may be cultured in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium") substances
that prevent the growth of HGPRT-deficient cells.
[0056] Immortalized cell lines useful here are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. Immortalized cell lines include murine
myeloma lines, which can be obtained, for instance, from the Salk
Institute Cell Distribution Center; San Diego, Calif. and the
American Type Culture Collection; Rockville, Md. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, Marcel Dekker, Inc., New York (1987)
pp. 51-63).
[0057] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against galactose-deficient IgA1 or selected epitopes
thereof. The binding specificity of monoclonal antibodies produced
by the hybridoma cells can be determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and
assays are known in the art, and are described further in Harlow
and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York (1988).
[0058] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution or FACS sorting procedures
and grown by standard methods. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0059] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0060] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies can be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells can serve as a preferred source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which
are then transfected into host cells such as simian COS cells,
Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma
cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567) or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody provided herein, or can be substituted for the variable
domains of one antigen-combining site of an antibody to create a
chimeric bivalent antibody comprising one antigen-combining site
having specificity for galactose-deficient IgA1 and another
antigen-combining site having specificity for a different
antigen.
[0061] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348, U.S. Pat. No. 4,342,566, and Harlow and Lane,
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, (1988). Papain digestion of antibodies typically produces
two identical antigen binding fragments, called Fab fragments, each
with a single antigen binding site, and a residual Fc fragment.
Pepsin treatment yields a fragment, called the F(ab').sub.2
fragment that has two antigen combining sites and is still capable
of cross-linking antigen.
[0062] The Fab fragments produced in the antibody digestion can
also contain the constant domains of the light chain and the first
constant domain of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus
of the heavy chain domain including one or more cysteines from the
antibody hinge region. The F(ab').sub.2 fragment is a bivalent
fragment comprising two Fab' fragments linked by a disulfide bridge
at the hinge region. Fab'-SH is the designation herein for Fab' in
which the cysteine residue(s) of the constant domains bear a free
thiol group.
[0063] One method of producing proteins comprising the provided
antibodies or polypeptides is to link two or more peptides or
polypeptides together by protein chemistry techniques. For example,
peptides or polypeptides can be chemically synthesized using
currently available laboratory equipment using either Fmoc
(9-fluorenylmethyl-oxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry (Applied Biosystems, Inc.; Foster City, Calif.). Those of
skill in the art readily appreciate that a peptide or polypeptide
corresponding to the antibody provided herein, for example, can be
synthesized by standard chemical reactions. For example, a peptide
or polypeptide can be synthesized and not cleaved from its
synthesis resin whereas the other fragment of an antibody can be
synthesized and subsequently cleaved from the resin, thereby
exposing a terminal group that is functionally blocked on the other
fragment. By peptide condensation reactions, these two fragments
can be covalently joined via a peptide bond at their carboxyl and
amino termini, respectively, to form an antibody, or fragment
thereof. (Grant G A (1992) Synthetic Peptides: A User Guide. W.H.
Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993)
Principles of Peptide Synthesis. Springer Verlag Inc., NY).
Alternatively, the peptide or polypeptide can by independently
synthesized in vivo. Once isolated, these independent peptides or
polypeptides may be linked to form an antibody or fragment thereof
via similar peptide condensation reactions.
[0064] For example, enzymatic ligation of cloned or synthetic
peptide segments can allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776 779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide a thioester with another unprotected peptide
segment containing an amino terminal Cys residue to give a
thioester linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site. Application of this
native chemical ligation method to the total synthesis of a protein
molecule is illustrated by the preparation of human interleukin 8
(IL-8) (Baggiolini et al., FEBS Lett. 307:97-101 (1992); Clark et
al., J. Biol. Chem. 269:16075 (1994); Clark et al., Biochemistry
30:3128 (1991); Rajarathnam et al., Biochemistry 33:6623-30
(1994)).
[0065] Alternatively, unprotected peptide segments can be
chemically linked where the bond formed between the peptide
segments as a result of the chemical ligation is an unnatural (non
peptide) bond (Schnolzer et al., Science 256:221 (1992)). This
technique has been used to synthesize analogs of protein domains as
well as large amounts of relatively pure proteins with full
biological activity (deLisle et al., Techniques in Protein
Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
[0066] The provided polypeptide fragments can be recombinant
proteins obtained by cloning nucleic acids encoding the polypeptide
in an expression system capable of producing the polypeptide
fragments thereof, such as a bacterial, adenovirus or baculovirus
expression system. For example, one can determine the active domain
of an antibody from a specific hybridoma that can cause a
biological effect associated with the interaction of the antibody
with galactose-deficient IgA1. For example, amino acids found to
not contribute to either the activity or the binding specificity or
affinity of the antibody can be deleted without a loss in the
respective activity.
[0067] The provided fragments, whether attached to other sequences,
can also include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the fragment is not
significantly altered or impaired compared to the nonmodified
antibody or epitope. These modifications can provide for some
additional property, such as to remove or add amino acids capable
of disulfide bonding, to increase its longevity, to alter its
secretory characteristics, and the like. In any case, the fragment
can possess a bioactive property, such as binding activity,
regulation of binding at the binding domain, and the like.
Functional or active regions may be identified by mutagenesis of a
specific region of the protein, followed by expression and testing
of the expressed polypeptide. Such methods can include site
specific mutagenesis of the nucleic acid encoding the antigen.
(Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)).
[0068] Further provided herein is a humanized or human version of
the antibody. Optionally, the antibody modulates the activity of
the galactose-deficient IgA1 molecule by inhibiting binding of IgG
to the galactose-deficient IgA1 molecule. Optionally, the humanized
or human antibody comprises at least one complementarity
determining region (CDR) of an antibody having the same epitope
specificity as an antibody produced by the hybridoma cell line
disclosed herein. For example, the antibody can comprise one or all
CDRs of an antibody having the same epitope specificity as an
antibody produced by the hybridoma cell line.
[0069] Optionally, the humanized or human antibody can comprise at
least one residue of the framework region of the monoclonal
antibody produced by a disclosed hybridoma cell line. Humanized and
human antibodies can be made using methods known to a skilled
artesian; for example, the human antibody can be produced using a
germ-line mutant animal or by a phage display library.
[0070] Antibodies can also be generated in other species and
humanized for administration to humans. Alternatively, fully human
antibodies can also be made by immunizing a mouse or other species
capable of making a fully human antibody (e.g., mice genetically
modified to produce human antibodies) and screening clones that
bind galactose-deficient IgA1. See, e.g., Lonberg and Huszar, Int.
Rev. Immunol. 13:65-93, (1995), which is incorporated herein by
reference in its entirety for methods of producing fully human
antibodies. As used herein, the term humanized and human in
relation to antibodies, relate to any antibody which is expected to
elicit a therapeutically tolerable weak immunogenic response in a
human subject. Thus, the terms include fully humanized or fully
human as well as partially humanized or partially human.
[0071] Humanized forms of non-human (e.g., murine) antibodies or
fragments thereof are chimeric immunoglobulins, immunoglobulin
chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2,
or other antigen-binding subsequences of antibodies) which contain
minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a CDR of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues that are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin (Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)).
[0072] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source that is non-human. These
non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the methods
described in Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-327 (1988); or Verhoeyen et al., Science
239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. Accordingly,
such humanized antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0073] The nucleotide sequences encoding the provided antibodies
can be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). These nucleotide sequences can also be modified, or
humanized, for example, by substituting the coding sequence for
human heavy and light chain constant domains in place of the
homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567).
The nucleotide sequences encoding any of the provided antibodies
can be expressed in appropriate host cells. These include
prokaryotic host cells including, but not limited to, E. coli,
Bacillus subtilus, other enterobacteriaceae such as Salmonella
typhimurium or Serratia marcesans, and various Pseudomonas species.
Eukaryotic host cells can also be utilized. These include, but are
not limited to, yeast cells (for example, Saccharomyces cerevisiae
and Pichia pastoris), and mammalian cells such as VERO cells, HeLa
cells, Chinese hamster ovary (CHO) cells, W138 cells, BHK cells,
COS-7 cells, 293T cells and MDCK cells. The antibodies produced by
these cells can be purified from the culture medium and assayed for
binding, activity, specificity or any other property of the
monoclonal antibodies by utilizing the methods set forth herein and
standard in the art.
[0074] Transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in
the absence of endogenous immunoglobulin production can be
employed. For example, it has been described that the homozygous
deletion of the antibody heavy chain joining region (J(H)) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-255
(1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et
al., Year in Immuno. 7:33 (1993)). Human antibodies can also be
produced in phage display libraries (Hoogenboom et al., J. Mol.
Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).
The techniques of Cole et al. and Boerner et al. are also available
for the preparation of human monoclonal antibodies (Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, ed., p. 77
(1985); Boerner et al., J. Immunol. 147(1):86-95 (1991)).
[0075] Provided herein is an antibody, a humanized antibody, heavy
and light chain immunoglobulins of an antibody, CDRs of the
antibody, and certain truncations of these antibodies or
immunoglobulins that perform the functions of the full length
antibody or immunoglobulin. For example, the nucleic acid sequence
coding for the antibodies can be altered. As such, nucleic acids
that encode the polypeptide sequences, variants, and fragments of
thereof are disclosed. These sequences include all degenerate
sequences related to a specific protein sequence, i.e., all nucleic
acids having a sequence that encodes one particular protein
sequence as well as all nucleic acids, including degenerate nucleic
acids, encoding the disclosed variants and derivatives of the
protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequences.
[0076] As with all peptides, polypeptides, and proteins, including
fragments thereof, it is understood that additional modifications
in the amino acid sequence of the antibodies can occur that do not
alter the nature or function of the peptides, polypeptides, or
proteins. Such modifications include conservative amino acids
substitutions and are discussed in greater detail below.
[0077] The isolated antibodies or fragments thereof provided herein
have a desired function. The isolated antibody or fragment thereof
binds a specific epitope of the galactose-deficient IgA1. Binding
of the epitope can, for example, treat or reduce the risk of
developing IgA nephropathy.
[0078] The antibodies described herein can be further modified and
varied so long as the desired function is maintained. It is
understood that one way to define any known modifications and
derivatives or those that might arise, of the disclosed nucleic
acid sequences and proteins herein is through defining the
modifications and derivatives in terms of identity to specific
known sequences. Specifically disclosed are polypeptides which have
at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 ,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
percent identity to the antibodies or fragments thereof provided
herein. Those of skill in the art readily understand how to
determine the identity of two polypeptides. For example, the
identity can be calculated after aligning the two sequences so that
the identity is at its highest level.
[0079] Another way of calculating identity can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman, Adv. Appl. Math 2:482 (1981), by the identity alignment
algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. USA 85: 2444 (1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by inspection.
[0080] The same types of identity can be obtained for nucleic acids
by, for example, the algorithms disclosed in Zuker, Science
244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA
86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306
(1989), which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity and to be disclosed herein.
[0081] Protein modifications include amino acid sequence
modifications. Modifications in amino acid sequence may arise
naturally as allelic variations (e.g., due to genetic
polymorphism), may arise due to environmental influence (e.g.,
exposure to ultraviolet radiation), or may be produced by human
intervention (e.g., by mutagenesis of cloned DNA sequences), such
as induced point, deletion, insertion, and substitution mutants.
These modifications can result in changes in the amino acid
sequence, provide silent mutations, modify a restriction site, or
provide other specific mutations. Amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional, or deletional modifications. Insertions include amino
and/or terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Deletions are characterized by the removal of one or more amino
acid residues from the protein sequence. Typically, no more than
about from 2 to 6 residues are deleted at any one site within the
protein molecule. Amino acid substitutions are typically of single
residues, but can occur at a number of different locations at once;
insertions usually will be on the order of about from 1 to 10 amino
acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e., a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations may or
may not place the sequence out of reading frame and may or may not
create complementary regions that could produce secondary mRNA
structure. Substitutional modifications are those in which at least
one residue has been removed and a different residue is inserted in
its place. Conservative substitutions generally are made in
accordance with the following Table 1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Amino Acid
Substitutions (others are known in the art) Ala Ser, Gly, Cys Arg
Lys, Gln, Met, Ile Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys
Ser, Met, Thr Gln Asn, Lys, Glu, Asp Glu Asp, Asn, Gln Gly Pro, Ala
His Asn, Gln Ile Leu, Val, Met Leu Ile, Val, Met Lys Arg, Gln, Met,
Ile Met Leu, Ile, Val Phe Met, Leu, Tyr, Trp, His Ser Thr, Met, Cys
Thr Ser, Met, Val Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met
Non-conservative mutations can be made as well (e.g., proline for
glycine).
[0082] Modifications, including the specific amino acid
substitutions, are made by known methods. By way of example,
modifications are made by site specific mutagenesis of nucleotides
in the DNA encoding the protein, thereby producing DNA encoding the
modification, and thereafter expressing the DNA in recombinant cell
culture. Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are well known,
for example M13 primer mutagenesis and PCR mutagenesis.
[0083] As used herein, an inhibitory nucleic acid molecule can also
be a short-interfering RNA (siRNA) molecule or a micro-RNA (miRNA)
molecule. A 21-25 nucleotide siRNA or miRNA molecule can, for
example, be produced from an expression vector by transcription of
a short-hairpin RNA (shRNA) sequence, a 60-80 nucleotide precursor
sequence, which is subsequently processed by the cellular RNAi
machinery to produce either an siRNA or miRNA molecule.
Alternatively, a 21-25 nucleotide siRNA or miRNA molecule can, for
example, be synthesized chemically. Chemical synthesis of siRNA or
miRNA molecules is commercially available from such corporations as
Dharmacon, Inc. (Lafayette, Colo.), Qiagen (Valencia, Calif.), and
Ambion (Austin, Tex.). A siRNA molecule preferably binds a unique
sequence within the target mRNA with exact complementarity and
results in the degradation of the target mRNA molecule. A siRNA
molecule can bind anywhere within the target mRNA molecule. A miRNA
molecule preferably binds a unique sequence within the target mRNA
with exact or less than exact complementarity and results in the
translational repression of the target mRNA molecule. A miRNA
molecule can bind anywhere within the target mRNA sequence, but
preferably binds within the 3' untranslated region of the target
mRNA molecule. Methods of delivering siRNA or miRNA molecules are
known in the art. See, e.g., Oh and Park, Adv. Drug. Deliv. Rev.
61(10):850-62 (2009); Gondi and Rao, J. Cell Physiol. 220(2):285-91
(2009); and Whitehead et al., Nat. Rev. Drug. Discov. 8(2):129-38
(2009).
[0084] As used herein, an inhibitory nucleic acid molecule can also
be an antisense nucleic acid molecule. Antisense nucleic acid
molecules can, for example, be transcribed from an expression
vector to produce an RNA which is complementary to at least a
unique portion of the target mRNA and/or the endogenous gene which
encodes target mRNA. Hybridization of an antisense nucleic acid
under specific cellular conditions results in inhibition of target
protein expression by inhibiting transcription and/or
translation.
[0085] Provided herein are methods of treating or reducing the risk
of IgA nephropathy in a subject. Such methods include administering
an effective amount of an agent comprising a small molecule, a
polypeptide, an inhibitory nucleic acid molecule, a peptidomimetic
or a combination thereof. Optionally, the small molecules,
polypeptides, inhibitory nucleic acid molecules, and/or
peptidomimetics are contained within a pharmaceutical
composition.
[0086] Provided herein are compositions containing the provided
small molecules, polypeptides, inhibitory nucleic acid molecules,
and/or peptidomimetics and a pharmaceutically acceptable carrier
described herein. The herein provided compositions are suitable for
administration in vitro or in vivo. By pharmaceutically acceptable
carrier is meant a material that is not biologically or otherwise
undesirable, i.e., the material is administered to a subject
without causing undesirable biological effects or interacting in a
deleterious manner with the other components of the pharmaceutical
composition in which it is contained. The carrier is selected to
minimize degradation of the active ingredient and to minimize
adverse side effects in the subject.
[0087] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy, 21.sup.st Edition,
David B. Troy, ed., Lippicott Williams & Wilkins (2005).
Typically, an appropriate amount of a pharmaceutically-acceptable
salt is used in the formulation to render the formulation isotonic.
Examples of the pharmaceutically-acceptable carriers include, but
are not limited to, sterile water, saline, buffered solutions like
Ringer's solution, and dextrose solution. The pH of the solution is
generally about 5 to about 8 or from about 7 to 7.5. Other carriers
include sustained release preparations such as semipermeable
matrices of solid hydrophobic polymers containing the immunogenic
polypeptides. Matrices are in the form of shaped articles, e.g.,
films, liposomes, or microparticles. Certain carriers may be more
preferable depending upon, for instance, the route of
administration and concentration of composition being administered.
Carriers are those suitable for administration of the agent, e.g.,
the small molecule, polypeptide, inhibitory nucleic acid molecule,
and/or peptidomimetic, to humans or other subjects.
[0088] The compositions are administered in a number of ways
depending on whether local or systemic treatment is desired, and on
the area to be treated. The compositions are administered via any
of several routes of administration, including topically, orally,
parenterally, intravenously, intraperitoneally, intramuscularly,
subcutaneously, transdermally, or intrarenally.
[0089] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives are optionally present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0090] Formulations for topical administration include ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids, and
powders. Conventional pharmaceutical carriers, aqueous, powder, or
oily bases, thickeners and the like are optionally necessary or
desirable.
[0091] Compositions for oral administration include powders or
granules, suspension or solutions in water or non-aqueous media,
capsules, sachets, or tables. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders are optionally
desirable.
[0092] Optionally, the nucleic acid molecule or polypeptide is
administered by a vector comprising the nucleic acid molecule or a
nucleic acid sequence encoding the polypeptide. There are a number
of compositions and methods which can be used to deliver the
nucleic acid molecules and/or polypeptides to cells, either in
vitro or in vivo via, for example, expression vectors. These
methods and compositions can largely be broken down into two
classes: viral based delivery systems and non-viral based deliver
systems. Such methods are well known in the art and readily
adaptable for use with the compositions and methods described
herein.
[0093] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids into the cell without
degradation and include a promoter yielding expression of the
nucleic acid molecule and/or polypeptide in the cells into which it
is delivered. Viral vectors are, for example, Adenovirus,
Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus,
Sindbis, and other RNA viruses, including these viruses with the
HIV backbone. Also preferred are any viral families which share the
properties of these viruses which make them suitable for use as
vectors. Retroviral vectors, in general are described by Coffin et
al., Retroviruses, Cold Spring Harbor Laboratory Press (1997),
which is incorporated by reference herein for the vectors and
methods of making them. The construction of replication-defective
adenoviruses has been described (Berkner et al., J. Virol.
61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83
(1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et
al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques
15:868-72 (1993)). The benefit and the use of these viruses as
vectors is that they are limited in the extent to which they can
spread to other cell types, since they can replicate within an
initial infected cell, but are unable to form new infections viral
particles. Recombinant adenoviruses have been shown to achieve high
efficiency after direct, in vivo delivery to airway epithelium,
hepatocytes, vascular endothelium, CNS parenchyma, and a number of
other tissue sites. Other useful systems include, for example,
replicating and host-restricted non-replicating vaccinia virus
vectors.
[0094] The provided polypeptides and/or nucleic acid molecules can
be delivered via virus like particles. Virus like particles (VLPs)
consist of viral protein(s) derived from the structural proteins of
a virus. Methods for making and using virus like particles are
described in, for example, Garcea and Gissmann, Current Opinion in
Biotechnology 15:513-7 (2004).
[0095] The provided polypeptides can be delivered by subviral dense
bodies (DBs). DBs transport proteins into target cells by membrane
fusion. Methods for making and using DBs are described in, for
example, Pepperl-Klindworth et al., Gene Therapy 10:278-84
(2003).
[0096] The provided polypeptides can be delivered by tegument
aggregates. Methods for making and using tegument aggregates are
described in International Publication No. WO 2006/110728.
[0097] Non-viral based delivery methods can include expression
vectors comprising nucleic acid molecules and nucleic acid
sequences encoding polypeptides, wherein the nucleic acids are
operably linked to an expression control sequence. Suitable vector
backbones include, for example, those routinely used in the art
such as plasmids, artificial chromosomes, BACs, YACs, or PACs.
Numerous vectors and expression systems are commercially available
from such corporations as Novagen (Madison, Wis.), Clonetech (Pal
Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life
Technologies (Carlsbad, Calif.). Vectors typically contain one or
more regulatory regions. Regulatory regions include, without
limitation, promoter sequences, enhancer sequences, response
elements, protein recognition sites, inducible elements, protein
binding sequences, 5' and 3' untranslated regions (UTRs),
transcriptional start sites, termination sequences, polyadenylation
sequences, and introns.
[0098] Preferred promoters controlling transcription from vectors
in mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis B virus, and most
preferably cytomegalovirus (CMV), or from heterologous mammalian
promoters, e.g. .beta.-actin promoter or EF1.alpha. promoter, or
from hybrid or chimeric promoters (e.g., CMV promoter fused to the
.beta.-actin promoter). Of course, promoters from the host cell or
related species are also useful herein.
[0099] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' or 3' to the transcription unit. Furthermore,
enhancers can be within an intron as well as within the coding
sequence itself. They are usually between 10 and 300 base pairs
(bp) in length, and they function in cis. Enhancers usually
function to increase transcription from nearby promoters. Enhancers
can also contain response elements that mediate the regulation of
transcription. While many enhancer sequences are known from
mammalian genes (globin, elastase, albumin, fetoprotein, and
insulin), typically one will use an enhancer from a eukaryotic cell
virus for general expression. Preferred examples are the SV40
enhancer on the late side of the replication origin, the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0100] The promoter and/or the enhancer can be inducible (e.g.
chemically or physically regulated). A chemically regulated
promoter and/or enhancer can, for example, be regulated by the
presence of alcohol, tetracycline, a steroid, or a metal. A
physically regulated promoter and/or enhancer can, for example, be
regulated by environmental factors, such as temperature and light.
Optionally, the promoter and/or enhancer region can act as a
constitutive promoter and/or enhancer to maximize the expression of
the region of the transcription unit to be transcribed. In certain
vectors, the promoter and/or enhancer region can be active in a
cell type specific manner. Optionally, in certain vectors, the
promoter and/or enhancer region can be active in all eukaryotic
cells, independent of cell type. Preferred promoters of this type
are the CMV promoter, the SV40 promoter, the .beta.-actin promoter,
the EF1.alpha. promoter, and the retroviral long terminal repeat
(LTR).
[0101] The vectors also can include, for example, origins of
replication and/or markers. A marker gene can confer a selectable
phenotype, e.g., antibiotic resistance, on a cell. The marker
product is used to determine if the vector has been delivered to
the cell and once delivered is being expressed. Examples of
selectable markers for mammalian cells are dihydrofolate reductase
(DHFR), thymidine kinase, neomycin, neomycin analog G418,
hygromycin, puromycin, and blasticidin. When such selectable
markers are successfully transferred into a mammalian host cell,
the transformed mammalian host cell can survive if placed under
selective pressure. Examples of other markers include, for example,
the E. coli lacZ gene, green fluorescent protein (GFP), and
luciferase. In addition, an expression vector can include a tag
sequence designed to facilitate manipulation or detection (e.g.,
purification or localization) of the expressed polypeptide. Tag
sequences, such as GFP, glutathione S-transferase (GST),
polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak; New
Haven, Conn.) sequences typically are expressed as a fusion with
the encoded polypeptide. Such tags can be inserted anywhere within
the polypeptide including at either the carboxyl or amino
terminus.
[0102] As used herein, the terms peptide, polypeptide, or protein
are used broadly to mean two or more amino acids linked by a
peptide bond. Protein, peptide, and polypeptide are also used
herein interchangeably to refer to amino acid sequences. It should
be recognized that the term polypeptide is not used herein to
suggest a particular size or number of amino acids comprising the
molecule and that a peptide of the invention can contain up to
several amino acid residues or more.
[0103] As used throughout, subject can be a vertebrate, more
specifically a mammal (e.g. a human, horse, cat, dog, cow, pig,
sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles,
amphibians, fish, and any other animal. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, whether
male or female, are intended to be covered. As used herein, patient
or subject may be used interchangeably and can refer to a subject
with a disease or disorder (e.g. IgA nephropathy). The term patient
or subject includes human and veterinary subjects.
[0104] A subject at risk of developing a disease or disorder can be
genetically predisposed to the disease or disorder, e.g., have a
family history or have a mutation in a gene that causes the disease
or disorder, or show early signs or symptoms of the disease or
disorder. A subject currently with a disease or disorder has one or
more than one symptom of the disease or disorder and may have been
diagnosed with the disease or disorder.
[0105] The methods and agents as described herein are useful for
both prophylactic and therapeutic treatment. For prophylactic use,
a therapeutically effective amount of the agents described herein
are administered to a subject prior to onset (e.g., before obvious
signs of IgA nephropathy) or during early onset (e.g., upon initial
signs and symptoms of IgA nephropathy). Prophylactic administration
can occur for several days to years prior to the manifestation of
symptoms of IgA nephropathy. Prophylactic administration can be
used, for example, in the preventative treatment of subjects
diagnosed with a genetic predisposition to IgA nephropathy.
Therapeutic treatment involves administering to a subject a
therapeutically effective amount of the agents described herein
after diagnosis or development of IgA nephropathy.
[0106] According to the methods taught herein, the subject is
administered an effective amount of the agent. The terms effective
amount and effective dosage are used interchangeably. The term
effective amount is defined as any amount necessary to produce a
desired physiologic response. Effective amounts and schedules for
administering the agent may be determined empirically, and making
such determinations is within the skill in the art. The dosage
ranges for administration are those large enough to produce the
desired effect in which one or more symptoms of the disease or
disorder are affected (e.g., reduced or delayed). The dosage should
not be so large as to cause substantial adverse side effects, such
as unwanted cross-reactions, anaphylactic reactions, and the like.
Generally, the dosage will vary with the age, condition, sex, type
of disease, the extent of the disease or disorder, route of
administration, or whether other drugs are included in the regimen,
and can be determined by one of skill in the art. The dosage can be
adjusted by the individual physician in the event of any
contraindications. Dosages can vary, and can be administered in one
or more dose administrations daily, for one or several days.
Guidance can be found in the literature for appropriate dosages for
given classes of pharmaceutical products.
[0107] As used herein the terms treatment, treat, or treating
refers to a method of reducing the effects of a disease or
condition or symptom of the disease or condition. Thus in the
disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100% reduction in the severity of an
established disease or condition or symptom of the disease or
condition. For example, a method for treating a disease is
considered to be a treatment if there is a 10% reduction in one or
more symptoms of the disease in a subject as compared to a control.
Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, or any percent reduction in between 10% and 100% as
compared to untreated levels. It is understood that treatment does
not necessarily refer to a cure or complete ablation of the
disease, condition, or symptoms of the disease or condition.
[0108] As used herein, the term reducing the risk of developing a
disease or disorder refers to an action, for example,
administration of a therapeutic agent, that occurs before or at
about the same time a subject begins to show one or more symptoms
of the disease or disorder, which inhibits or delays onset or
exacerbation of one or more symptoms of the disease or disorder. As
used herein, references to decreasing, reducing, or inhibiting
include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
greater as compared to an untreated level. Such terms can include
but do not necessarily include complete elimination.
[0109] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed methods and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly
disclosed, each is specifically contemplated and described herein.
For example, if a method is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the method are discussed, each and every combination and
permutation of the method, and the modifications that are possible
are specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. This concept applies to
all aspects of this disclosure including, but not limited to, steps
in methods using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed, it is understood
that each of these additional steps can be performed with any
specific method steps or combination of method steps of the
disclosed methods, and that each such combination or subset of
combinations is specifically contemplated and should be considered
disclosed.
[0110] Publications cited herein and the material for which they
are cited are hereby specifically incorporated by reference in
their entireties.
EXAMPLES
General Methods
[0111] Human subjects. Peripheral blood was collected from a total
of 60 patients with biopsy-proven IgAN (IgA nephropathy) (mean age,
34.8.+-.12.5 years; serum creatinine, 1.3.+-.0.6 mg/dl; UP/Cr
ratio, 1.31.+-.1.60), from 40 healthy controls (mean age,
38.0.+-.16.2 years; serum creatinine, 0.9.+-.0.2 mg/dl; UP/Cr
ratio, 0.06.+-.0.06), and from 20 disease controls (patients with
biopsy-proven lupus nephritis, membranous nephritis, and minimal
change nephritic syndrome; mean age, 35.0.+-.11.4 years; serum
creatinine, 1.1.+-.0.4 mg/dl; UP/Cr ratio, 1.56.+-.1.93) (Table 2).
The IgAN patients included 16 white males and 9 white females, 1
African-American male and 2 African-American females, and 12
Japanese males and 20 Japanese females. The healthy control group
consisted of 12 white males and 12 white females, 2
African-American males and 4 African-American females, and 4
Japanese males and 6 Japanese females. All healthy controls had
normal UP/Cr ratio or dipstick test for protein, and none exhibited
microscopic hematuria. Disease controls consisted of a group of 5
white males and 1 white female and 1 African-American female, and 7
Japanese males and 6 Japanese females. The levels of IgA,
Gal-deficient IgA1, and IgG in the serum samples from the 60 IgAN,
20 disease controls, and 40 healthy control subjects were
determined by capture ELISA. For 20 of 60 patients with IgAN, urine
and blood samples were collected within 30 days of renal biopsy
(contemporaneous samples).
TABLE-US-00002 [0111] TABLE 2 Clinical characteristics of study
population. Data expressed as mean .+-. SD. SCr.sup.a is serum
creatinine concentration. UP/Cr.sup.b is urinary protein/creatinine
ratio. Serum IgG Serum IgA SCr.sup.a Cohort Age Male Fem. Race
(mg/ml) (mg/ml) (mg/dl) UP/Cr.sup.b IgAN US 28 40.0 .+-. 15.2 17 11
W25, B3 12.3 .+-. 2.7 4.4 .+-. 2.4 1.4 .+-. 0.9 1.2 .+-. 1.5 Jap.
32 30.3 .+-. 7.0 12 20 J32 12.1 .+-. 2.9 3.5 .+-. 1.1 1.2 .+-. 0.4
1.4 .+-. 1.7 Tot. 60 34.8 .+-. 12.5 29 31 W25, B3, J32 12.2 .+-.
2.8 3.9 .+-. 1.9 1.3 .+-. 0.6 1.3 .+-. 1.6 Disease US 7 33.7 .+-.
16.4 5 2 W6, B1 17.2 .+-. 6.3 3.4 .+-. 1.2 1.0 .+-. 0.3 1.5 .+-.
2.6 Cont. Jap. 13 35.7 .+-. 8.4 7 6 J13 15.5 .+-. 3.5 3.8 .+-. 1.8
1.2 .+-. 0.4 1.6 .+-. 1.7 Tot. 20 35.0 .+-. 11.4 12 8 W6, B1, J13
16.1 .+-. 4.6 3.7 .+-. 1.6 1.1 .+-. 0.4 1.6 .+-. 1.9 Healthy US 30
38.6 .+-. 17.9 14 16 W24, B6 10.4 .+-. 2.4 3.1 .+-. 1.7 1.0 .+-.
0.2 0.1 .+-. 0.1 Cont. Jap. 10 36.1 .+-. 10.0 4 6 J10 10.9 .+-. 2.9
2.8 .+-. 1.5 0.8 .+-. 0.2 0.1 .+-. 0.0 Tot. 40 38.0 .+-. 16.2 18 22
W24, B6, J10 10.5 .+-. 2.5 3.0 .+-. 1.7 0.9 .+-. 0.2 0.1 .+-.
0.1
[0112] Isolation of PBMCs, transformation with EBV, and cloning of
IgG-secreting cell lines. PBMCs from patients with IgAN and healthy
controls were isolated from heparinized peripheral blood by
Ficoll-Hypaque density gradient centrifugation. The B cell fraction
was enriched from the PBMCs by removal of adherent cells through
incubation in a plastic tissue-culture flask for 1 hour at
37.degree. C. and removal of T cells by CD3 (PanT) Dynabeads,
according to the manufacturer's instructions (Dynal; Invitrogen;
Carlsbad, Calif.). PBMCs from 16 randomly selected IgAN patients
(10 white males and 6 white females; 13 subjects had proteinuria or
microscopic hematuria at the time of study) and 16 randomly
selected white healthy controls (6 white males and 10 white
females) were then immortalized with EBV (Suzuki et al., J. Clin.
Invest. 118:629-39 (2008); Kubagawa et al., Proc. Natl. Acad. Sci.
USA 85:875-9 (1988)). To establish cell lines from the initial
EBV-immortalized PBMCs from patients with IgAN and healthy
controls, IgG-secreting cells were subcloned by limiting dilution
(using 96-well plates seeded with 5 to 10 cells per well) in RPMI
1640 supplemented with I-glutamine, 20% FCS, penicillin, and
streptomycin (Suzuki et al., J. Clin. Invest. 118:629-39 (2008)).
After several rounds of cloning and screening, IgG-producing cell
lines were generated from all 16 IgAN patients and all 16 healthy
controls. [0113] Measurement of Ig and immune-complex levels. The
isotypes of the Igs secreted by the immortalized cells were
determined by capture ELISA (Tomana et al., J. Clin. Invest.
104:73-81 (1999); Moore et al., Mol. Immunol. 44:2598-604 (2007)).
ELISA plates were coated with 1 g/ml of the F(ab').sub.2 fragment
of goat IgG specific for human IgA, IgG, or IgM (Jackson
ImmunoResearch Laboratories Inc.; West Grove, Pa.). The captured
Igs were then detected with a biotin-labeled F(ab').sub.2 fragment
of goat IgG anti-human IgA, IgG, or IgM antibody (BioSource;
Invitrogen). Avidin-horseradish peroxidase conjugate (ExtrAvidin;
Sigma-Aldrich) and the peroxidase chromogenic substrate
o-phenylenediamine-H.sub.2O.sub.2 (Sigma-Aldrich; St. Louis, Mo.)
were then added. The color reaction was stopped with 1 M sulfuric
acid, and the absorbance at 490 nm was measured using an EL312
BioKinetics Microplate Reader (BioTek; Winooski, Vt.). Standard
curves for Igs were generated from a pool of normal human sera
calibrated for all Ig isotypes (Binding Site; San Diego, Calif.).
The results were calculated using a DeltaSoft III computer program
(BioMetallics; Princeton, N.J.). Urinary IgA-IgG immune complexes
were measured using cross-capture ELISA (Matousovic et al.,
Nephrol. Dial. Transplant. 21:2478-84 (2006)). [0114] Myeloma
proteins. The IgA1 myeloma proteins that were isolated from plasma
of patients with multiple myeloma are listed in Table 3 together
with their molecular characteristics (Moore et al., Mol. Immunol.
44:2598-604 (2007)). In brief, plasma samples were precipitated
with ammonium sulfate (50% saturation). The precipitate was then
dissolved in and dialyzed against 10 mM sodium phosphate buffer (pH
7.0) prior to fractionation by ion-exchange chromatography on
DEAE-cellulose, followed by affinity chromatography using
Jacalin-agarose to capture IgA1 (Sigma-Aldrich) (Tomana et al., J.
Clin. Invest. 104:73-81 (1999)). The final purification step was
size-exclusion chromatography on columns of Sephadex G-200 or
Ultrogel AcA 22 (Amersham Biosciences; Piscataway, N.J.). As the
IgA myeloma proteins can be contaminated with IgG, the purified
protein was subjected to affinity chromatography using
staphylococcal protein G immobilized on agarose (Sigma-Aldrich).
The purity of the IgA1 preparations was assessed by SDS-PAGE and
Western blotting using an IgA1-specific monoclonal antibody (Tomana
et al., J. Clin. Invest. 104:73-81 (1999)). The molecular form of
the IgA1 proteins was assessed by size-exclusion chromatography,
SDS-PAGE under non-reducing conditions, and Western blots developed
with anti-IgA antibody.
TABLE-US-00003 [0114] TABLE 3 IgA1 myeloma proteins. Myeloma
Protein IgA isotype Molecular Form Mce IgA1 Polymer Mce1 IgA1
Polymer Ale mono IgA1 Monomer Ale poly IgA1 Polymer Fab-IgA1 IgA1
Fab frag. of IgA1cont. part of the hinge region
[0115] ELISA characterization of antigen-specific IgG antibodies.
The binding of serum IgG from IgAN patients and healthy controls,
as well as IgG secreted by EBV-immortalized cells from the same
subjects, was analyzed by ELISA using a panel of antigens: dd-IgA1,
Fab-IgA1 generated using an IgA-specific protease from Haemophilus
influenzae HK50, HR-BSA, and HRGalNAc-BSA. HR-GalNAc was
synthesized by Bachem (asterisks mark the sites with GalNAc):
V-P-S-T-P-P-*T-P-*S-P-*S-T-P-P-T-P-S-P-S-C--NH.sub.2 (SEQ ID
NO:43). The hinge-region peptide was the same peptide but with no
GalNAc. Both preparations were cross-linked to BSA.
[0116] For ELISA, flat-bottom 96-well plates (Nunc MaxiSorp;
eBioscience; San Diego, Calif.) were coated with 1 .mu.g/ml
solution of the above-mentioned antigens. Serum or culture
supernatant samples diluted in PBS were added to each well. The
amount of total IgG used for the analyses was normalized in all
samples. The captured IgG were detected with a biotin-labeled
F(ab').sub.2 fragment of goat IgG anti-human IgG antibody
(BioSource; Invitrogen). Avidin-horseradish peroxidase conjugate
(ExtrAvidin; Sigma-Aldrich) was then added, and the reaction was
developed as described before (Suzuki et al., J. Clin. Invest.
118:629-39 (2008)). [0117] SDS-PAGE and Western blotting. Serum and
culture supernatants were separated by SDS-PAGE under reducing
conditions using 4%-20% gradient slab gels (Bio-Rad). The amounts
of protein loaded were adjusted to achieve equivalent amounts of
IgA protein in each lane. The gels were blotted onto PVDF membranes
and incubated with antibody specific for IgA heavy chains (Vector
Laboratories; Burlingame, Calif.) or a biotin-labeled lectin from
Helix aspersa (HAA). HAA reacts with terminal GalNAc but not with
sialylated GalNAc or GalNAc-Gal disaccharide. Gal-deficient IgA1
myeloma proteins (Mce or Ale poly), after separation by SDS-PAGE
under reducing conditions and electroblotting onto PVDF membranes,
served as antigens for analysis of glycan-specific IgG. The bound
IgG was detected with IgG-specific antibody, and the visualization
of positive bands was accomplished by subsequent incubation of the
membrane with avidin-peroxidase conjugate, followed by enhanced
chemiluminescence detection (Pierce; Thermo Scientific; Rockford,
Ill.) (Suzuki et al., Contrib. Nephrol. 157:129-33 (2007); Moore et
al., Mol. Immunol. 44:2598-604 (2007); Moldoveanu et al., Kidney
Int. 71:1148-54 (2007)). [0118] Helix aspersa agglutinin (HAA)
inhibition. To inhibit IgG binding to Gal-deficient IgA1 (Mce)
myeloma protein or HR-GalNAc-BSA, 20 .mu.g/ml unlabeled HAA was
applied to PVDF membrane after electroblotting of IgA1 or to the
wells of ELISA plates after coating with IgA1 protein. [0119]
Immune-complex formation in vitro. IgG was isolated from
cell-culture supernatants of the IgG-secreting cell lines derived
from patients with IgAN and healthy controls by protein G affinity
chromatography (GE Healthcare; Piscataway, N.J.). These cell lines
were subcloned by limiting dilution, and clones secreting
glycan-specific IgG (binding to Gal-deficient IgA1) were selected.
Immune complexes were formed in vitro by mixing 50 .mu.g
Gal-deficient IgA1 (Ale mono) and 50 .mu.g purified glycan-specific
IgG and incubating the mixture overnight at 4.degree. C. The formed
complexes were fractionated by HPLC on a calibrated TSK 3000 column
(Tosoh Bioscience; South San Fransisco, Calif.), and 0.25 ml
fractions were analyzed for IgA1-IgG immune complexes using
cross-capture ELISA (Novak et al, Kidney Int. 62:465-75 (2002)).
[0120] Cloning of IGH, IG.kappa., and IG.lamda. genes. Single-cell
reverse-transcription PCR was used to amplify the V(D)J regions for
IGH, IG.kappa., and IG.lamda., genes (Wardemann et al., Science
301:1374-7 (2003)). Reverse transcription and first-round PCR were
performed with OneStep RT-PCR Kit (QIAGEN; Valencia, Calif.) under
these conditions: 50.degree. C., 30 minutes; 94.degree. C., 15
minutes; 94.degree. C., 20 seconds; 55.degree. C., 30 seconds;
72.degree. C., 1 minute for 50 cycles; 72.degree. C., 10 minutes;
and stop at 4.degree. C. Second-round PCR was performed with rTaq
DNA Polymerase (Invitrogen) under these conditions: 94.degree. C.,
3 minutes; 94.degree. C., 20 seconds; 57.degree. C. (IgH/Ig.kappa.)
or 60.degree. C. (Ig.lamda.), 30 seconds; 72.degree. C., 45 seconds
for 50 cycles; 72.degree. C., 5 minutes; and stop at 4.degree. C.
One microliter of cDNA from first-round PCR was used as the
template for the second-round PCR. The average single-cell RT-PCR
efficiency was 38.4%. Positive PCR products were purified
(QIAquick; QIAGEN) and sequenced. The resultant Ig gene sequences
were analyzed with the IgBLAST program to determine the potential
VH, DH, and JH germline gene usage and mutation analysis. The
IgBLAST program is available on the internet through the National
Center for Biotechnology Information. Restriction enzyme digestion
sites were introduced in the second round of single-cell RT-PCR.
Digested IgH, Ig.kappa., and Ig.lamda. PCR products were purified
using QIAquick PCR purification kit (QIAGEN) and directly cloned
into specific expression vectors containing human Ig 1, Ig, or Ig
constant regions. Plasmids were sequenced to confirm clones with
inserts identical to that of the original PCR products. The pl
values and CDR3 junction analysis were determined by IMGT/V-QUEST.
The corresponding DNA sequences were deposited to GenBank
(accession numbers FJ746335-FJ746360). [0121] VH CDR3 site-specific
mutagenesis. Site-directed mutagenesis was performed by 2-step PCR
to generate amplicons with mutated (IgAN patient 1123) or unmutated
(healthy control 9017) VH genes (Tiller et al., J. Immunol. Methods
329:112-24 (2008)). Primers used in PCR reverted the substitution
(S to A) in the IgAN clone or mutated (A to S) the sequence in the
clone from the healthy control (Table 4). The first PCR (PCR1)
forward primer was VH specific and contained an AgeI restriction
site. The PCR2 reverse primer was JH specific and contained the
SalI restriction site. PCR products 1 and 2 were hybridized via the
homologous region in the subsequent overlap PCR using the same
5'-AgeI VH-specific forward primer and the 3'-SalI JH-specific
reverse primer and generated the complete VDJ sequence with desired
mutations. Corresponding clones were sequenced and cloned into the
IgG expression vector for production of rIgG.
TABLE-US-00004 [0121] TABLE 4 Primer sequences for mutatgenesis.
Pat. #1123 Sense Sequence PCR1 Age1-VH3
ACTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 27) PCR2
F-mutated ATATATTACTGTGCGAAAGTGTGTCGCCCCTGG (SEQ ID NO: 28) Overlap
PCR Age1-VH3 ACTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGGAGTC (SEQ ID NO:
27) Pat. #1123 Antisense Sequence PCR1 R-mutated
AGGGGCGACACACTTTCGCACAGTAATATATGGCCG (SEQ ID NO: 29) PCR2 Sal1-JH3
CTGCGAAGTCGACGCTGAAGAGACGGTGACCATTG (SEQ ID NO: 30) Overlap PCR
Sal1-JH3 CTGCGAAGTCGACGCTGAAGAGACGGTGACCATTG (SEQ ID NO: 30) Pat.
#9017 Sense Sequence PCR1 Age1-VH3
ACTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 27) PCR2
F-mutated TGTGTATTACTGTTCCAGAGTCCAGCGCTATGATAGCACTG (SEQ ID NO: 31)
Overlap PCR Age1-VH3 ACTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGGAGTC (SEQ
ID NO: 27) Pat. #9017 Antisense Sequence PCR1 R-mutated
ATCATAGCGCTGGACTCTGGAACAGTAATACACAGCCGTG (SEQ ID NO: 32) PCR2
Sal1-JH145 CTGCGAAGTCGACGCTGAGGAGACGGTGACCAGGG (SEQ ID NO: 33)
Overlap PCR Sal1-JH145 CTGCGAAGTCGACGCTGAGGAGACGGTGACCAGGG (SEQ ID
NO: 33)
[0122] rIgG antibody production. Human embryonic kidney cells
(293H) were cultured in DMEM supplemented with 10% FBS (Ultra Low
Bovine Ig content; Gibco, Invitrogen) and cotransfected with 10
.mu.g plasmid DNA constructs encoding IgH and IgL chains by
polyethyleneimine (Sigma-Aldrich) precipitation. After 16-hour
transfection, the cell-culture medium was replaced with fresh
medium. Supernatants with secreted IgG were collected after 7 days.
[0123] Fab purification of rIgG. The Fab fragment of rIgG from an
IgAN patient was purified using the Pierce Fab Preparation Kit
(Thermo Scientific). [0124] Dot-blot analysis. Gal-deficient IgA1
(Ale poly; 0.5 .mu.g per well) was placed into the wells of a
96-well plate with PVDF membrane (MultiScreenHTS IP Filer Plate;
Millipore) and blocked with SuperBlock (Pierce; Thermo Scientific).
Serum or cell-culture supernatants (normalized to 0.5 .mu.g IgG in
each sample) were added and incubated overnight at 4.degree. C. As
a positive control, 0.5 .mu.g of rIgG from an IgAN patient was
used. The binding was detected with biotin-labeled IgG-specific
antibody, followed by subsequent incubation of the membrane with
avidin-peroxidase conjugate, and the reaction was visualized using
enhanced chemiluminescence (Pierce; Thermo Scientific), as
described above for Western blotting. Results were evaluated
densitometrically. The intensity of rIgG binding to Gal-deficient
IgA was assigned a value of 100%. [0125] Statistics. Correlations
between different parameters were analyzed by 2-tailed Student's t
test or by regression analysis. ANOVA was used to determine
differences in the characteristics among multiple groups.
Nonparametric methods, such as Spearman's rank correlation and
Wilcoxon's rank-sum test were used for the correlation and 2-sample
comparisons, respectively. Data were expressed as mean.+-.SD or
median values. P<0.05 was considered significant. These
statistical analyses were performed with StatView 5.0 software
(Abacus Concepts; Cheltenham, Gloucestershire, United Kingdom). The
ROC forGal-deficient IgA1-specific IgG levels in patients and
controls was constructed using Graph-Pad Prism, version 4.00 for
Windows (GraphPad Software; La Jolla, Calif.).
Results
Example 1
[0125] [0126] Serum IgG from IgAN patients exhibits specificity for
N-acetylgalactosamine, which results in binding with Gal-deficient
and desialylated IgA1. Binding of serum IgG from IgAN patients to
Gal-deficient IgA1 was first determined using an ELISA in which the
coated antigen was either enzymatically desialylated and
degalactosylated IgA1 (dd-IgA1) or the Fab fragment of
Gal-deficient IgA1 containing the N-terminal part of the hinge
region with O-glycans attached (Fab-IgA1) (Table 3). The levels of
serum IgG directed against dd-IgA1 and Fab-IgA1 were higher in IgAN
patients than in healthy controls (P<0.001) (Table 5). These
results, obtained using samples from 16 patients and 16 healthy
controls from the southeastern USA, were corroborated using serum
samples from 20 IgAN patients and 20 healthy controls from Japan
(P<0.0001) (Table 5).
[0127] The binding of the serum IgG to Gal-deficient IgA1 was
validated by Western blot analysis of the component chains of an
enzymatically modified IgA1 myeloma protein (Mce). In each case,
the enzymatic modification was confirmed by the binding of the
N-acetylgalactosamine-specific (GalNAc-specific) lectin, Helix
aspersa agglutinin (HAA) (Moore et al., Mol. Immunol. 44:2598-604
(2007); Moldoveanu et al., Kidney Int. 71:1148-54 (2007)). The IgG
from the sera of patients with IgAN bound to the heavy chain of the
Gal-deficient IgA1, whereas only minimal binding of the IgG from
the sera of healthy controls was observed. Removal of the sialic
acid from the Gal-deficient IgA1 by neuraminidase treatment
resulted in an increase in the binding of the serum IgG from
patients with IgAN (FIG. 1A). Desialylated and degalactosylated
(dd)-IgA1 bound greater amounts of HAA than did native
Gal-deficient IgA1, whereas enzymatically regalactosylated or
resialylated dd-IgA1 bound lower amounts of HAA than native
Gal-deficient IgA1 (FIG. 1B). The similarity between the extent of
binding of the serum IgG and HAA to each of these IgA1 preparations
suggested that the binding of serum IgG to the Gal-deficient IgA1
was dependent on the GalNAc moieties (FIG. 1B). This was confirmed
by incubation with unlabeled HAA prior to incubation with IgG
purified from the serum of an IgAN patient. The preincubation with
HAA reduced the binding of the IgG to the Gal-deficient IgA1 by 66%
(FIGS. 1C and 1D); conversely, blocking with serum IgG from an IgAN
patient reduced the binding of HAA to Gal-deficient IgA1 by 60%
(FIGS. 1C and 10). Thus, the GalNAc in the hinge region of
Gal-deficient IgA1 represents a major component of the epitope that
is recognized by the IgG specific for Galdeficient IgA1 present in
the serum of patients with IgAN.
TABLE-US-00005 TABLE 5 Serum levels of antigen-specific IgG. Cohort
from Southwestern USA Antigen IgAN (n = 16) Controls (n = 16)
dd-IgA1 2.256 .+-. 0.112* 1.995 .+-. 0.146 Fab-IgA1 2.136 .+-.
0.163* 1.724 .+-. 0.184 Cohort from Japan Antigen IgAN (n = 20)
Controls (n = 20) Fab-IgA1 2.021 .+-. 0.202** 1.532 .+-. 0.229 Data
are expressed as optical density at 490 nm and shown as means .+-.
SD. dd-IaG1, enzymatically desialylated and degalactosylated IgA1.
Fab-IgA1, Fab fragment of Gd-IgA1 containing part of the hinge
region with O-glycans. IgAN, patients with IgA nephropathy;
Controls, healthy controls. *P < 0.001, **P < 0.0001.
Example 2
[0128] Characterization of antibodies specific for Gal-deficient
IgA1 secreted by IgG-producing cell lines. To further characterize
the IgG that reacts with the Gal-deficient IgA1, IgG-producing
cells were generated by EBV immortalization of B cells isolated
from the peripheral blood of the 16 patients with IgAN and 16
healthy controls who had provided blood for measurement of serum
IgG specific for Gal-deficient IgA1 (Table 4). After subcloning of
the cells, the IgG secreted by the cell lines was characterized by
ELISA; the cells derived from IgAN patients produced antibodies
that exhibited greater binding to dd-IgA1 and Fab-IgA1 than did the
cells derived from controls (P<0.0001) (FIGS. 2A and 2B). Cell
lines were randomly selected from 10 IgAN patients and 10 healthy
controls and analyzed for the binding of the secreted IgG to a
synthetic IgA1 hinge-region peptide linked to BSA (HR-BSA) and a
synthetic IgA1 hinge-region glycopeptide linked to BSA with 3
GalNAc residues (HR-GalNAc-BSA) at sites corresponding to the major
epitopes of the Gal-deficient IgA1 myeloma protein (Thr228, Ser230,
and Ser232) (Novak et al., Contrib. Nephrol. 157:134-8 (2007)). The
IgG from the cells derived from IgAN patients did not bind the
HR-BSA but bound HR-GalNAc-BSA; moreover, the binding to
HR-GalNAc-BSA was inhibited by HAA (78%) (FIG. 2C). Thus, the
IgG-secreting cells derived from the peripheral blood of patients
with IgAN produced glycan-specific antibodies that recognize
Gal-deficient IgA1 in a GalNAc-dependent manner. These
IgG-producing cells were further subcloned to isolate single-cell
clones producing antibodies specific for Gal-deficient IgA1. 3 cell
lines were randomly selected from clones from patients with IgAN
(n=16) and 3 cell lines from clones from healthy controls (n=16)
and the cultures were scaled up to obtain sufficient amounts of
purified IgG for further characterization.
Example 3
[0128] [0129] Glycan-specific IgG forms immune complexes with
Gal-deficient IgA1. The ability of the glycan-specific antibodies
to form immune complexes with Gal-deficient IgA1 was determined in
vitro by incubation of the purified IgG proteins with a
Gal-deficient IgA1 myeloma protein (Ale mono) at a 1:1 molar ratio.
The reaction mixture was then fractionated by HPLC with the
IgA1-IgG immune complexes being identified by cross-capture ELISA
(Novak et al., Contrib. Nephrol. 157:134-8 (2007)). Incubation of
the Gal-deficient IgA1 with IgG produced by the cells derived from
IgAN patients resulted in the production of greater amounts of
immune complexes than were formed on incubation with IgG produced
by cells derived from healthy controls (FIG. 3A). Analysis of the
size and composition of the immune complexes suggested that they
were composed of 1 molecule of IgG bound to either 1 or 2 molecules
of IgA1 (FIG. 3A).
Example 4
[0129] [0130] Analyses of the IGH, IG.kappa., and IG.lamda. genes
derived from patients with IgAN. The variable regions of IGH and
IG.kappa. or IG.lamda. transcripts from single cells were amplified
in 2 rounds of nested RT-PCR reactions using specific primers
(Wardemann et al., Science 301:1374-7 (2003)). The resultant
amplicons were then purified and directly sequenced. The nucleotide
and amino acid sequences for the Ig heavy chains from subjects 1123
and 9017 are given by SEQ ID NOs:75 and 79 and SEQ ID NOs:77 and
81, respectively. The nucleotide and amino acid sequences for the
Ig light chains from subjects 1123 and 9017 are given by SEQ ID
NOs:76 and 80 and SEQ ID NOs:78 and 81, respectively. The predicted
amino acid sequences of the CDR3 of the variable region of the IGH
gene (VH genes) from the 7 IgAN patients analyzed differed
significantly from the predicted sequences for the genes of the 6
healthy controls that were analyzed (Tables 6 and 7). One of the
notable differences was that the 3' end of VH genes from cells of 6
IgAN patients included a sequence encoding YCSR (SEQ ID NO:44) or
YCSK (SEQ ID NO:48), which represented an A to S substitution as
compared with the sequence encoding YCAR (SEQ ID NO:45) that was
identified in 5 of 6 controls (Table 6). In the 1 IgAN patient
(subject 3081) who did not have the A to S substitution at this
position, there was an R to T substitution at the next position
(YCAT (SEQ ID NO:46) vs. YCAR (SEQ ID NO:45)). On dot-blot
analysis, extensive binding of the IgG secreted by the cells from
the IgAN patients to Gal-deficient IgA1 was found with 1 exception
(IgG from the clone from subject 3081; FIG. 3B). The IgG secreted
by the cells from the healthy controls either did not bind to
Gal-deficient IgA1 or exhibited significantly less binding, again
with 1 exception (IgG from the clone from subject 3070 with the
sequence YCAS (SEQ ID NO:47)) (FIG. 3B). Densitometric analysis of
these blots indicated that the IgG from IgAN patients exhibited
greater binding to Gal-deficient IgA1 than did the IgG from healthy
controls (FIG. 3C; P<0.01). Thus, the CDR3 of the VH appears to
play an important role in the binding of the glycan-specific IgG to
the Gal-deficient IgA1, and the A to S substitution found in 6 of 7
patients with IgAN appears to be associated with enhanced
binding.
TABLE-US-00006 [0130] TABLE 6 Comparison of the IgG heavy-chain
CDR3 amino acid sequences from the IgAN patients with those from
the healthy controls. Cells from IgAN patients Cell ID CDR3 (amino
acid seq.) 1023 YCSRDLAAFCSGGNCHSVAIDFW (SEQ ID NO: 1) 1123
YCSKVCRPWNYRRPYYYGMDVW (SEQ ID NO: 2) 1125 YCSRDRYYCSGGAFDYW (SEQ
ID NO: 3) 1139 YCSRKTSYPPTVGEVRGTSYYYGMDVW (SEQ ID NO: 4) 2047
YCSKTKFKGYSGFHYW (SEQ ID NO: 5) 3061 YCSRDRYGLFDYW (SEQ ID NO: 6)
3081 YCATGDYFGSGTYPIGAFDTW (SEQ ID NO: 7) Cells from healthy
controls Cell ID CDR3 (amino acid seq.) 3066 YCARDLDLW (SEQ ID NO:
8) 3070 YCASEGHLDYGGNSDAFDIW (SEQ ID NO: 9) 3064 YCARDVNITATEYYFDYW
(SEQ ID NO: 10) 8034 YCARGNDDYFDYW (SEQ ID NO: 11) 9017
YCARVQRYDSTGYYPLGYLDLW (SEQ ID NO: 12) 9035 YCAREWYSYLWDSSYYFDYW
(SEQ ID NO: 13) The amino acid sequences of CDR3 of IgG from 7 IgAN
patients and 6 controls. There were notable differences, including
a sequence YCSR (SEQ ID NO: 44) or a sequence YCSK (SEQ ID NO: 48)
with a change of A to S (bold and underlined S; excluding subject
3081, who had sequence YCAT (SEQ ID NO: 46)) in the CDR3 of heavy
chain of IgG from IgAN patients compared with the YCAR (SEQ ID NO:
45) sequence in the controls (except subject 3070; bold S).
TABLE-US-00007 TABLE 7 Repertoire and reactivity of antibodies from
B cells of patients with IgAN and healthy controls. React Ig Heavy
Chain Light Chain with Cell CDR3 (aa), CDR3 (aa), Gd- Id V D J CDR3
(nt) L Pl V J CDR3 (nt) L Pl IGA1 Cells from patients with IgAN
1023 1-46 2-15 4 (SEQ ID NO: 1), 20 5.61 2-29 .kappa.1
CMQGIHLPPTVDVF 12 6.58 .+-. (SEQ ID NO: 49) (SEQ ID NO: 14), (SEQ
ID NO: 50) 1123 3-23 1-7 6 (SEQ ID NO: 2), 19 9.49 2-11 .lamda.2
CCSYAGSYTSLF 10 13.0 + (SEQ ID NO: 51) (SEQ ID NO: 15), (SEQ ID NO:
52) 1125 3-30 3-22 4 (SEQ ID NO: 3), 14 6.44 2-8 .lamda.2
CSSYVGSNNSLF 10 13.0 + (SEQ ID NO: 53) (SEQ ID NO: 16), (SEQ ID NO:
54) 1139 3-21 3-3 6 (SEQ ID NO: 4), 24 8.83 1-40 .lamda.2
CQSYDSSLSGYVVF 12 13.0 + (SEQ ID NO: 55) (SEQ ID NO: 17), (SEQ ID
NO: 56) 2047 3-23 5-12 4 (SEQ ID NO: 5), 13 9.84 1-5 .kappa.1
CQQYNSYPWTF 9 13.0 + (SEQ ID NO: 57) (SEQ ID NO: 18), (SEQ ID NO:
58) 3061 3-11 5-12 4 (SEQ ID NO: 6), 10 6.59 1-12 .kappa.2
CQQANSFPPTGTF 11 13.0 + (SEQ ID NO: 59) (SEQ ID NO: 19), (SEQ ID
NO: 60) 3081 1-24 5-12 3 (SEQ ID NO: 7), 18 13.0 1-39 .kappa.1
CQQSYSTPRTF 9 9.25 - (SEQ ID NO: 61) (SEQ ID NO: 20), (SEQ ID NO:
62) Cells from healthy controls 3066 4-4 3-9 4 (SEQ ID NO: 8), 15
4.10 1-50 .lamda.2 CKAWDNSLNAHTVL 16 7.49 - (SEQ ID NO: 63 QAVF
(SEQ ID NO: 21), (SEQ ID NO: 64) 3070 3-7 -- 2 (SEQ ID NO: 9), 6
4.40 3-15 .kappa.5 CQQYNNWPQTF 9 13.0 - (SEQ ID NO: 65) (SEQ ID NO:
22), (SEQ ID NO: 66) 3064 4-59 4-23 3 (SEQ ID NO: 10), 17 3.92 1-39
.kappa.5 CQQSYSTPPTF 9 13.0 .+-. (SEQ ID NO: 67) (SEQ ID NO: 23),
(SEQ ID NO: 68) 8034 4-39 1-1 4 (SEQ ID NO: 11), 10 4.10 3-20
.kappa.2 CQQYGSSLYTF 9 13.0 - (SEQ ID NO: 69) (SEQ ID NO: 24), (SEQ
ID NO: 70) 9017 3-7 3-9 2 (SEQ ID NO: 12), 19 6.58 3-9 .lamda.2
CQVWDSSSDVVF 10 13.0 - (SEQ ID NO: 71) (SEQ ID NO: 25), (SEQ ID NO:
72) 9035 3-7 2-8 4 (SEQ ID NO: 13), 17 4.10 3-21 .lamda.2
CQVWDSSSDHPF 10 4.39 - (SEQ ID NO: 73) (SEQ ID NO: 26), (SEQ ID NO:
74) V, variable; D, diversity; J, joint; L, Lenght; +, high
reactivity; .+-., medium reactivity; -, no reactivity.
Example 5
[0131] The importance of the A to S substitution in the YCAR/K (SEQ
ID NO: 37) sequence of the CDR3 in the binding of IgG to
Gal-deficient IgA1. For further analyses, recombinant human IgG
(rIgG) was prepared using a single-cell PCR technique to clone the
variable regions of the heavy and light chain genes of IgG from an
IgG-secreting cell line derived from a patient with IgAN and from
an IgG-secreting cell line derived from a healthy control. The
corresponding PCR products for heavy and light chains were
subcloned into Iv and Ig.kappa. or .lamda. expression vectors,
respectively, to express rIgG1, also matching the original subclass
of the identified antibodies. Western blot analysis demonstrated
that the rIgG from the IgAN patient bound to Gal-deficient IgA1
myeloma protein (Ale poly) (FIG. 4A), and this was confirmed by
ELISA using Fab-IgA1. Furthermore, the Fab fragment of the rIgG was
purified from an IgAN patient to confirm the role of the
antigen-binding region in the interaction with Gal-deficient IgA1.
ELISA data confirmed that the Fab fragment of rIgG bound to
Fab-IgA1 in a fashion similar to that of the intact rIgG. Western
blotting against the hinge region of native IgA1, desialylated
IgA1, and dd-IgA1 myeloma proteins (Mce1) confirmed that the
binding of the rIgG to IgA1 was increased after removal of sialic
acid and Gal on the hinge region of IgA1 (FIG. 4B).
[0132] To determine whether the amino acid substitution (A to S) in
the CDR3 of the VH domain of IgG from IgAN patients affects the
binding to Gal-deficient IgA1, the VH gene of the single-cell line
from an IgAN patient (subject 1123) with the YCSKVCRPWNYRRPYYYGMDVW
(SEQ ID NO:2) sequence was reverted to the counterpart found in
most healthy controls (S to A) (FIG. 4C) using an overlap PCR
strategy (Table 4) (Tiller et al., J. Immunol. Methods 329:112-24
(2008)). Conversely, the CDR3 of the VH gene of the single-cell
line from a healthy control (subject 9017) encoding the
YCARVQRYDSTGYYPLGYLDLW (SEQ ID NO:12) sequence was mutated (A to S)
(FIG. 4C) to generate the sequence found in most of the IgAN
patients. Both mutations were confirmed by sequencing after cloning
into an IgG-expressing vector, as described above. The rIgG was
then purified and tested for binding to Gal-deficient IgA1 using
Western blotting and ELISA. The S to A change in the CDR3 of the
IgG of the IgAN patient reduced the binding of rIgG to
Gal-deficient IgA1 by 72%. Conversely, the A to S substitution in
CDR3 of the IgG of a healthy control increased binding to
Gal-deficient IgA1 to 80% of that of the rIgG of the IgAN patient
(FIG. 4D). These data were confirmed by ELISA using Fab-IgA1 as the
antigen.
Example 6
[0133] Serum levels of IgG specific for Gal-deficient IgA1 are
elevated in patients with IgAN. As patients with IgAN were found to
have higher levels of circulating IgG antibodies with specificity
for Gal-deficient IgA1, the quantitative differences were evaluated
using a novel dot-blot assay that was developed for this purpose.
The IgAN patients (n=60) were found to have elevated levels of
serum IgG specific for Gal-deficient IgA1 as compared with disease
controls (n=20) and healthy controls (n=40) (FIGS. 5A and 5B). The
relative intensity values for the serum IgG antibodies from IgAN
patients in both cohorts, disease controls, and healthy controls
were 33.2.+-.14.6, 9.9.+-.3.9, and 9.0.+-.6.8, respectively (FIG.
5B; P<0.0001). To test the reproducibility of this assay, the
same serum samples were reanalyzed twice, and the difference
between the experiments was 3.5%.+-.2.3%. Notably, 54 of the 60
patients with IgAN had mean binding values higher than the 90th
percentile of those for healthy controls. A receiver operating
characteristic (ROC)-curve analysis indicated the area under the
curve was 0.9644 (FIG. 5C; P<0.0001); when the level of serum
IgG specific for Gal-deficient IgA1 specificity reached 95.0%, the
corresponding sensitivity was 88.3%. Furthermore, for 20 IgAN
patients with urine and blood samples collected within 30 days of
renal biopsy (contemporaneous samples), possible correlations were
assessed among clinical and laboratory findings. The results of
these analyses showed that the intensity of binding of IgG to
Gal-deficient IgA1 as determined by the dot-blot analysis
correlated with proteinuria (expressed as urinary protein/urinary
creatinine [UP/Cr] ratio; FIG. 5D; P<0.0001) as well as with
urinary IgA-IgG immune complexes (expressed relative to Cr
concentration; FIG. 5E; P=0.0082).
Example 7
[0133] [0134] Measurement of serum levels of galactose-deficient
IgA1 and anti-glycan IgG antibodies pre- and post-diagnosis of IgA
nephropathy (IgAN). The Department of Defense Serum Repository was
utilized to evaluate serum levels of galactose-deficient IgA1
(Gd-IgA1) and antibodies specific for the hinge-region glyucans of
Gd-IgA1 in serially collected serum samples from service personnel
prior to IgAN diagnosis. These results were then compared to age-,
sex-, race-, and age-of-serum-sample-matched healthy controls. The
repository stores over 40 million samples, which have been
collected from active-duty soldiers approximately every one to two
years since 1985. The initial serum samples are banked at the time
of entry into the military when soldiers must pass a medical
evaluation, that includes a history, a physical examination, vital
signs, urinalysis, and laboratory testing. With normal findings, it
is presumed that the soldiers do not have even subclinical evidence
of renal disease at the time of enlistment.
[0135] In a pilot study, eight subjects with IgAN without crescents
on biopsy or clinical evidence of rapidly progressive
glomerulonephritis (RPGN) were compared to twenty-four matched
healthy controls. The earliest available sample, the second-to-last
sample, and the last sample prior to diagnosis were processed. For
subgroup analysis, the subjects were divided into 2 groups: 4
subjects with mild IgAN and 4 subjects with moderate IgAN, based on
serum creatinine concentration, quantification of proteinuria, and
level of activity on histology.
[0136] The IgAN patients had a higher mean serum Gd-IgA1 level
prior to diagnosis than did healthy controls (136.7 vs. 79.2 U/mL;
p=0.002). The Gd-IgA1 levels were measured with standard
galactose-deficient IgA1 (Ale) as described in Suzuki et al., J.
Clin. Invest. 118:629-39 (2008). For the IgAN patients, the mean
serum Gd-IgA1 levels were significantly higher than for the healthy
controls mean levels in subsets of samples at less than 1000 days
and greater than 1000 days prior to diagnosis (138.2 vs. 81.4 U/mL;
p=0.019 and 134.5 vs. 76.7U/mL; p=0.046 respectively). Based on an
ROC curve with an area of 0.731 with a threshold of 91 U/mL, the
assay achieved 67% sensitivity and 78% specificity. The mean change
in serum Gd-IgA1 levels over the change in time for the IgAN
patients was higher than for the healthy controls (0.017 vs.
0.00086 U/mL/day; p=0.006).
[0137] In a subgroup analysis, patients with moderate IgAN had a
higher mean serum Gd-IgA1 level prior to diagnosis than did
patients with mild IgAN (170.6 v. 98.2 U/mL; p=0.03).
[0138] In addition, the eight IgAN patients had a higher mean serum
level of antibody specific for the hinge-region glycans of Gd-IgA1
compared to that for the healthy controls (55 vs. 26; p=0.03). A
threshold value of 40 was 100% sensitive and specific.
[0139] The results demonstrate that the serum Gd-IgA1 level is
elevated years prior to diagnosis of IgAN and that it rises during
the interval prior to diagnosis. In addition, a higher Gd-IgA1
level may be associated with more severe IgAN.
[0140] This information can aid in the diagnosis of IgAN in
patients with urinary abnormalities or other signs of kidney
disease that are at too high a risk for renal biopsy. In addition,
this assay can distinguish between IgAN patients that will have a
benign clinical course without chronic progression to chronic
kidney damage (approximately 50% of subjects) from those that will
ultimately develop end-stage kidney disease (approximately 40% of
subjects). This information could influence the length of the
follow-up intervals and timing of medical intervention.
Example 8
[0141] Characterization of a passive model of IgAN using immune
complexes formed between Gal-deficient IgA1 and anti-glycan IgG. To
develop a passive model of IgAN, immune complexes were formed in
vitro using Gal-deficient IgA1 and anti-glycan IgG purified from a
patient with IgAN (FIG. 6A). The immune complexes were injected
intravenously into nude mice. The complexes deposited in the renal
mesangium (FIG. 6C), together with murine C3, and induced hematuria
and proteinuria. Scanning electron microscopy (EM) confirmed the
presence of red blood cells (RBC) in the urine of mice injected
with these immune complexes (FIG. 6B). Albuminuria increased by
.about.50% 24 hours after injection of the complexes, concurrently
with hematuria. Examination of the renal tissue by transmission EM
confirmed electron-dense deposits in the mesangium and showed
evidence of podocyte injury (podocyte effacement, microvilli
formation) and presence of RBC in Bowman's urinary space (FIG. 7).
In control experiments, using IgA or IgG alone or IgG from a
healthy control with Gal-deficient IgA1 that did not form
pathogenic immune complexes, no evidence of IgG or murine C3 renal
deposition or development of hematuria or proteinuria was observed.
Gal-deficient IgA1, but not fully galactosylated IgA1, deposited
only transiently and did not cause any tissue injury.
Example 9
[0141] [0142] Treatment of IgA nephropathy (IgAN) with anti-glycan
antibodies and/or glycopeptides capable of blocking
galactose-deficient IgA (Gd-IgA) binding to IgG. Blocking the
formation of nephritogenic high-molecular weight circulatory immune
complexes (CIC) from circulating Gd-IgA1 and the conversion of
active immune complexes into inactive immune complexes reduces the
deposition in the renal mesangium, thus lessening or preventing
glomerular injury. The therapeutic goal is achieved with monovalent
anti-glycan reagents, such as single-chain antibodies (sc-Abs) that
bind Gd-IgA1 as shown in FIG. 8.
[0143] To create sc-Abs that bind Gd-IgA1, a phage-display library
of sc-Abs obtained from immortalized B lymphocytes from IgAN
patients with active disease, patients in long-term remission, and
healthy controls is made. To make the phage-display library, cells
expressing antibodies specific for Gd-IgA1 are isolated. The
antibodies specific for Gd-IgA1 are isolated from these cells, and
the corresponding population of V.sub.H and V.sub.L regions are
cloned. These clones are then expressed as sc-Abs in a
phage-display library, and the clones with high-affinity for
Gd-IgA1 are selected.
[0144] The high-affinity sc-Abs are then expressed in vitro to
determine their ability to block binding of serum anti-glycan
antibodeies to Gd-IgA1. The selected sc-Abs are produced in a yeast
expression system. Using cultured mesangial cells (MC), the sc-Abs
are tested for their ability to block the formation of pathogenic
IgA1-containing immune complexes in the presence of anti-glycan Abs
from the sera of IgAN patients.
[0145] The high-affinity sc-Abs are then tested in the passive
murine model of IgAN for their capacity to prevent glomerular
deposition of immune complexes and renal injury. The passive murine
model of IgAN, as described above, is based on injection of immune
complexes formed from human Gd-IgA1 and anti-glycan Abs. These
immune complexes deposit in the mesangium together with murine C3
to induce pathological and clinical changes typical of human IgAN,
including hematuria and proteinuria. The high-affinity sc-Abs are
injected intravenously into the passive murine model and glomerular
deposition of the pathogenic immune complexes is monitored.
[0146] Generation of immunologically highly specific reagents that
recognize, with high selectivity, peripheral-blood B cells and
lymphoblasts expressing Gd-IgA1 on their cell surfaces is useful
for non-invasive diagnostic purposes to monitor the kinetics of
appearance and enumeration of cells ultimately producing Gd-IgA1.
Whether the sc-Abs with high affinity and specificity for Gd-IgA1
can selectively suppress differentiation of cells producing Gd-IgA1
molecules is tested using peripheral blood mononuclear cells from
IgAN patients and healthy controls stimulated in vitro with
pokeweed mitogen in the presence and absence of sc-Abs with high
specificity and affinity for Gd-IgA1. The suppression of Gd-IgA1
production is evaluated at the humoral level (secreted IgA1
antibody) and cellular level (enumeration of cells secreting
Gd-IgA1) using HAA-dependent assays previously described
(Moldoveanu et al., Kidney Int. 71:134-8 (2007); Suzuki et al., J.
Clin. Invest. 118:629-39 (2008)).
[0147] Another approach for preventing the formation of pathogenic
immune complexes is the use of synthetic glycopeptides. For
example, for the immune complexes of interest in patients with
IgAN, a single GalNAc residue prevents cross-linking of the
galactose-deficient IgA1 by an intact IgG molecule with 2
antigen-binding sites. The glycopeptide is recognized by these
naturally occurring IgG (or IgA) anti-GalNAc antibodies, thereby
inhibiting binding to the galactose-deficient polymeric IgA1. Thus,
large pathogenic immune complexes that are capable of inducing
renal injury (nephritogenic complexes) are not formed (FIG. 9, part
2). Competition for binding of the glycan-specific IgG antibodies
to the galactose-deficient IgA1 is also accomplished by using
monovalent, non-cross-linking Fab or Fv fragments of anti-glycan
antibodies to bind to the exposed GalNAc residue in the hinge
region of the galactose-deficient IgA1 (FIG. 9, part 1).
Sequence CWU 1
1
82123PRTHomo sapiens 1Tyr Cys Ser Arg Asp Leu Ala Ala Phe Cys Ser
Gly Gly Asn Cys His 1 5 10 15 Ser Val Ala Ile Asp Phe Trp 20
222PRTHomo sapiens 2Tyr Cys Ser Lys Val Cys Arg Pro Trp Asn Tyr Arg
Arg Pro Tyr Tyr 1 5 10 15 Tyr Gly Met Asp Val Trp 20 317PRTHomo
sapiens 3Tyr Cys Ser Arg Asp Arg Tyr Tyr Cys Ser Gly Gly Ala Phe
Asp Tyr 1 5 10 15 Trp 427PRTHomo sapiens 4Tyr Cys Ser Arg Lys Thr
Ser Tyr Pro Pro Thr Val Gly Glu Val Arg 1 5 10 15 Gly Thr Ser Tyr
Tyr Tyr Gly Met Asp Val Trp 20 25 516PRTHomo sapiens 5Tyr Cys Ser
Lys Thr Lys Phe Lys Gly Tyr Ser Gly Phe His Tyr Trp 1 5 10 15
613PRTHomo sapiens 6Tyr Cys Ser Arg Asp Arg Tyr Gly Leu Phe Asp Tyr
Trp 1 5 10 721PRTHomo sapiens 7Tyr Cys Ala Thr Gly Asp Tyr Phe Gly
Ser Gly Thr Tyr Pro Ile Gly 1 5 10 15 Ala Phe Asp Thr Trp 20
89PRTHomo sapiens 8Tyr Cys Ala Arg Asp Leu Asp Leu Trp 1 5
920PRTHomo sapiens 9Tyr Cys Ala Ser Glu Gly His Leu Asp Tyr Gly Gly
Asn Ser Asp Ala 1 5 10 15 Phe Asp Ile Trp 20 1018PRTHomo sapiens
10Tyr Cys Ala Arg Asp Val Asn Ile Thr Ala Thr Glu Tyr Tyr Phe Asp 1
5 10 15 Tyr Trp 1113PRTHomo sapiens 11Tyr Cys Ala Arg Gly Asn Asp
Asp Tyr Phe Asp Tyr Trp 1 5 10 1222PRTHomo sapiens 12Tyr Cys Ala
Arg Val Gln Arg Tyr Asp Ser Thr Gly Tyr Tyr Pro Leu 1 5 10 15 Gly
Tyr Leu Asp Leu Trp 20 1320PRTHomo sapiens 13Tyr Cys Ala Arg Glu
Trp Tyr Ser Tyr Leu Trp Asp Ser Ser Tyr Tyr 1 5 10 15 Phe Asp Tyr
Trp 20 1414PRTHomo sapiens 14Cys Met Gln Gly Ile His Leu Pro Pro
Thr Val Asp Val Phe 1 5 10 1512PRTHomo sapiens 15Cys Cys Ser Tyr
Ala Gly Ser Tyr Thr Ser Leu Phe 1 5 10 1612PRTHomo sapiens 16Cys
Ser Ser Tyr Val Gly Ser Asn Asn Ser Leu Phe 1 5 10 1714PRTHomo
sapiens 17Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Tyr Val Val Phe 1
5 10 1811PRTHomo sapiens 18Cys Gln Gln Tyr Asn Ser Tyr Pro Trp Thr
Phe 1 5 10 1913PRTHomo sapiens 19Cys Gln Gln Ala Asn Ser Phe Pro
Pro Thr Gly Thr Phe 1 5 10 2011PRTHomo sapiens 20Cys Gln Gln Ser
Tyr Ser Thr Pro Arg Thr Phe 1 5 10 2118PRTHomo sapiens 21Cys Lys
Ala Trp Asp Asn Ser Leu Asn Ala His Thr Val Leu Gln Ala 1 5 10 15
Val Phe 2211PRTHomo sapiens 22Cys Gln Gln Tyr Asn Asn Trp Pro Gln
Thr Phe 1 5 10 2311PRTHomo sapiens 23Cys Gln Gln Ser Tyr Ser Thr
Pro Pro Thr Phe 1 5 10 2411PRTHomo sapiens 24Cys Gln Gln Tyr Gly
Ser Ser Leu Tyr Thr Phe 1 5 10 2512PRTHomo sapiens 25Cys Gln Val
Trp Asp Ser Ser Ser Asp Val Val Phe 1 5 10 2612PRTHomo sapiens
26Cys Gln Val Trp Asp Ser Ser Ser Asp His Pro Phe 1 5 10
2741DNAArtificial SequenceSynthetic Construct 27actgcaaccg
gtgtacattc cgaggtgcag ctggtggagt c 412833DNAArtificial
SequenceSynthetic Construct 28atatattact gtgcgaaagt gtgtcgcccc tgg
332936DNAArtificial SequenceSynthetic Construct 29aggggcgaca
cactttcgca cagtaatata tggccg 363035DNAArtificial SequenceSynthetic
Construct 30ctgcgaagtc gacgctgaag agacggtgac cattg
353141DNAArtificial SequenceSynthetic Construct 31tgtgtattac
tgttccagag tccagcgcta tgatagcact g 413240DNAArtificial
SequenceSynthetic Construct 32atcatagcgc tggactctgg aacagtaata
cacagccgtg 403335DNAArtificial SequenceSynthetic Construct
33ctgcgaagtc gacgctgagg agacggtgac caggg 353447PRTHomo sapiens
34Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp Val Thr Val Pro 1
5 10 15 Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro
Pro 20 25 30 Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser Leu
His Arg 35 40 45 3522PRTHomo sapiens 35Tyr Cys Ala Lys Val Cys Arg
Pro Trp Asn Tyr Arg Arg Pro Tyr Tyr 1 5 10 15 Tyr Gly Met Asp Val
Trp 20 3622PRTHomo sapiens 36Tyr Cys Ser Arg Val Gln Arg Tyr Asp
Ser Thr Gly Tyr Tyr Pro Leu 1 5 10 15 Gly Tyr Leu Asp Leu Trp 20
374PRTHomo sapiens 37Tyr Cys Ala Lys 1 3810PRTHomo sapiens 38Cys
His Val Lys His Tyr Thr Asn Pro Ser 1 5 10 3910PRTHomo sapiens
39Val Thr Val Pro Cys Pro Val Pro Ser Thr 1 5 10 4010PRTHomo
sapiens 40Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr 1 5 10
4110PRTHomo sapiens 41Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys 1 5
10 4210PRTHomo sapiens 42Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 1
5 10 4320PRTHomo sapiens 43Val Pro Ser Thr Pro Pro Thr Pro Ser Pro
Ser Thr Pro Pro Thr Pro 1 5 10 15 Ser Pro Ser Cys 20 444PRTHomo
sapiens 44Tyr Cys Ser Arg 1 454PRTHomo sapiens 45Tyr Cys Ala Arg 1
464PRTHomo sapiens 46Tyr Cys Ala Thr 1 474PRTHomo sapiens 47Tyr Cys
Ala Ser 1 484PRTHomo sapiens 48Tyr Cys Ser Lys 14964DNAHomo sapiens
49tactgttcca gagatctggc cgctttttgt agtggtggta actgccactc tgtggcgatt
60gact 645042DNAHomo sapiens 50tgcatgcaag gtatacacct tcctcccaca
gtggacgttt tc 425166DNAHomo sapiens 51tactgttcca aagtgtgtcg
cccctggaac tatagaaggc cctactatta cggaatggac 60gtctgg 665236DNAHomo
sapiens 52tgctgctcat atgccggcag ctacacttcc ctcttc 365351DNAHomo
sapiens 53tactgttcca gagatcgtta ctattgtagt ggtggtgcct ttgactactg g
515436DNAHomo sapiens 54tgcagctcat atgtcggcag caacaattcc ctcttc
365581DNAHomo sapiens 55tactgttcca gaaagacctc ctaccccccc actgttgggg
aggtaagagg gacctcctac 60tattacggta tggacgtctg g 815642DNAHomo
sapiens 56tgccagtcct atgacagcag cctgagtggt tatgtggtat tc
425748DNAHomo sapiens 57tactgttcaa aaaccaagtt taagggatat agcggatttc
attactgg 485833DNAHomo sapiens 58tgccaacagt ataatagtta tccctggacg
ttc 335939DNAHomo sapiens 59tactgttcca gagatcgcta cggcctattt
gactactgg 396039DNAHomo sapiens 60tgtcaacagg ctaacagttt ccctcccaca
ggcactttt 396163DNAHomo sapiens 61tactgtgcaa ctggggatta ctttggttcg
gggacttacc ccataggggc ttttgatacc 60tgg 636233DNAHomo sapiens
62tgtcaacaga gttacagtac ccctcggacg ttc 336327DNAHomo sapiens
63tactgtgcga gagatctcga tctctgg 276433DNAHomo sapienst 64tgtcagcagt
ataataactg gcctcaaacg ttc 336560DNAHomo sapiens 65tactgtgcga
gcgagggaca tcttgactac ggtggtaact ccgatgcttt tgatatctgg
606633DNAHomo sapiens 66tgtcaacaga gttacagtac acctcccaca ttc
336754DNAHomo sapiens 67tactgtgcga gagacgttaa tattacggcc actgagtact
actttgacta ctgg 546854DNAHomo sapiens 68tgcaaagcat gggataacag
cctgaatgct cacacagtgc tccaggcggt attc 546939DNAHomo sapiens
69tactgtgcga gagggaacga cgactacttt gactactgg 397033DNAHomo sapiens
70tgtcagcagt atggtagctc actttacact ttt 337166DNAHomo sapiens
71tactgtgcga gagtccagcg ctatgatagc actggttact accctctggg atacctcgat
60ctctgg 667236DNAHomo sapiens 72tgtcaggtgt gggacagcag tagtgatgtg
gtattc 367360DNAHomo sapiens 73tactgtgcga gagagtggta cagctatcta
tgggactcgt cgtactactt tgactactgg 607436DNAHomo sapiens 74tgtcaggtgt
gggacagtag tagtgatcat ccattc 3675379DNAHomo sapiens 75gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgaag
cctctggatt cacctttagc agctatgcca tggcctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaaggggcg gttcaccatc tccagagaca
attccaagaa caccctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccatat attactgttc caaagtgtgt 300cgcccctgga actatagaag
gccctactat tacggaatgg acgtctgggg ccaagggaca 360atggtcaccg tctcctcag
37976331DNAHomo sapiens 76cagtctgccc tgactcagcc tcgctcagtg
tccgggtctc ctggacagtc agtcagcatc 60tcctgcactg gaaccagcag tgatgttgga
ggttataact atgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgatgtca gtaagcggcc ctcaggggtc 180cctgatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg atgaggctga ttattactgc tgctcatatg ccggcagcta
cacttccctc 300ttcggcggag ggaccaagct gaccgtccta g 33177379DNAHomo
sapiens 77gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagt agctattgga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtggccaac ataaagcaag
atggaagtga gaaatactat 180gtggactctg tgaagggccg attcaccatc
tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagtccag 300cgctatgata
gcactggtta ctaccctctg ggatacctcg atctctgggg ccgtggcacc
360ctggtcaccg tctcctcag 37978317DNAHomo sapiens 78tatgagctga
ctcagccact ctcagtgtca gtggccctgg gacagacggc caggattacc 60tgtgggggaa
acaacattgg aagtaaaaat gtgcactggt accagcagaa gccaggccag
120gcccctgtgc tggtcatcta tagggatagc aaccggccct ctgggatccc
tgagcgattc 180tctggctcca actctgggaa cacggccacc ctgaccatca
gcagagccca agccggggat 240gaggccgact attactgtca ggtgtgggac
agcagtagtg atgtggtatt cggcggaggg 300accaagctga ccgtcct
31779126PRTHomo sapiens 79Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ala Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85
90 95 Ser Lys Val Cys Arg Pro Trp Asn Tyr Arg Arg Pro Tyr Tyr Tyr
Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 120 125 80110PRTHomo sapiens 80Gln Ser Ala Leu Thr Gln Pro
Arg Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Ser Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala
Gly Ser 85 90 95 Tyr Thr Ser Leu Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 81126PRTHomo sapiens 81Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Val Gln Arg Tyr Asp Ser Thr Gly Tyr
Tyr Pro Leu Gly Tyr 100 105 110 Leu Asp Leu Trp Gly Arg Gly Thr Leu
Val Thr Val Ser Ser 115 120 125 82105PRTHomo sapiens 82Tyr Glu Leu
Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln Thr 1 5 10 15 Ala
Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His 20 25
30 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Arg
35 40 45 Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser Asn 50 55 60 Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala
Gln Ala Gly Asp 65 70 75 80 Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr
Val 100 105
* * * * *