U.S. patent application number 13/797006 was filed with the patent office on 2014-04-24 for diagnosis and treatment of autoantibody-mediated heart disease.
The applicant listed for this patent is Lizbeth Cornivelli, Myra A. Lipes. Invention is credited to Lizbeth Cornivelli, Myra A. Lipes.
Application Number | 20140112912 13/797006 |
Document ID | / |
Family ID | 39854334 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140112912 |
Kind Code |
A9 |
Lipes; Myra A. ; et
al. |
April 24, 2014 |
DIAGNOSIS AND TREATMENT OF AUTOANTIBODY-MEDIATED HEART DISEASE
Abstract
Provided herein are, inter alia, methods of diagnosing and
treating autoimmune cardiomyopathy in subjects, based upon the
detection of IgG4 antibodies to cardiac autoantigens.
Inventors: |
Lipes; Myra A.; (Brookline,
MA) ; Cornivelli; Lizbeth; (Medford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lipes; Myra A.
Cornivelli; Lizbeth |
Brookline
Medford |
MA
MA |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130202589 A1 |
August 8, 2013 |
|
|
Family ID: |
39854334 |
Appl. No.: |
13/797006 |
Filed: |
March 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13184115 |
Jul 15, 2011 |
8420330 |
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13797006 |
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Current U.S.
Class: |
424/133.1 ;
435/7.92; 604/6.04 |
Current CPC
Class: |
A61K 39/3955 20130101;
G01N 33/564 20130101; G01N 2800/325 20130101; A61P 43/00 20180101;
A61P 9/00 20180101; A61P 3/10 20180101; G01N 2333/47 20130101; A61P
3/06 20180101; A61P 9/12 20180101; G01N 2800/50 20130101; A61K
38/54 20130101; G01N 2800/52 20130101; C07K 14/515 20130101; A61P
9/10 20180101; G01N 33/6854 20130101 |
Class at
Publication: |
424/133.1 ;
435/7.92; 604/6.04 |
International
Class: |
G01N 33/68 20060101
G01N033/68; A61K 39/395 20060101 A61K039/395 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with Government support under Grant
No. RO1 HL077554 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method for diagnosing the presence of, or risk of developing,
autoimmune heart disease in a subject, the method comprising:
providing a sample comprising serum of a subject; and detecting a
level, presence, or absence in the sample of IgG autoantibodies to
cardiac troponin I (cTnI), wherein the presence of IgG
autoantibodies to cTnI, e.g., a level above a reference, indicates
that the subject has or is at risk of developing autoimmune heart
disease.
2. The method of claim 1, wherein the subject has cardiac
arrhythmia, idiopathic dilated cardiomyopathy, ischemic
cardiomyopathy or unexplained heart failure.
3. The method of claim 1, wherein autoantibodies that bind to an
epitope within residues 92-164 of human cTnI are detected.
4. The method of claim 3, wherein autoantibodies that bind to
epitopes within residues 92-101, 127-136, or 155-164 of human cTnI
are detected
5. A method for diagnosing the presence of, or risk of developing,
autoimmune heart disease in a subject, the method comprising:
providing a sample comprising cardiac tissue of a subject; and
detecting deposition of IgG autoantibodies on cardiac myocytes in
the sample, wherein the deposition of IgG autoantibodies indicates
that the subject has or is at risk of developing autoimmune heart
disease.
6. The method of claim 5, wherein the sample is an endomyocardial
biopsy.
7. The method of claim 5, wherein the subject has cardiac
arrhythmia, idiopathic dilated cardiomyopathy, ischemic
cardiomyopathy or unexplained heart failure.
8. The method of claim 5, wherein autoantibodies that bind to an
epitope within residues 92-164 of human cTnI are detected.
9. The method of claim 8, wherein autoantibodies that bind to
epitopes within residues 92-101, 127-136, or 155-164 of human cTnI
are detected
10. A method for treating autoimmune heart disease, the method
comprising: selecting a subject who has autoimmune heart disease;
and administering to the subject a therapy that depletes B
lymphocytes in the subject.
11. The method of claim 10, wherein selecting a subject comprises:
providing a sample comprising serum of a subject; detecting the
presence or absence in the sample of IgG autoantibodies to cardiac
troponin I (cTnI), wherein the presence of IgG autoantibodies to
cTnI indicates that the subject has or is at risk of developing
autoimmune cardiomyopathy; selecting a subject who has IgG
autoantibodies to cTnI.
12. The method of claim 11, wherein the therapy comprises
administration of an effective amount of rituximab or other B-cell
depleting or B-cell inactivating agents.
13. The method of claim 11, wherein the therapy comprises treating
the subject with plasmapheresis or administering intravenous
immunoglobulin.
14. A method for monitoring the efficacy of a treatment for
autoimmune heart disease, the method comprising: providing a first
sample comprising serum of a subject; detecting a level of IgG
autoantibodies to cardiac troponin I (cTnI) in the first sample,
administering a therapy to the subject; providing a subsequent
sample comprising serum of a subject; detecting a level of IgG
autoantibodies to cTnI in the subsequent sample; comparing the
level of IgG autoantibodies to cTnI in the first sample to the
level of IgG autoantibodies to cTnI in the subsequent sample,
wherein a decrease in level of IgG4 autoantibodies to cTnI from the
first to the subsequent sample indicates that the therapy is
effective.
15. The method of claim 14, wherein the therapy comprises
administration of an effective amount of rituximab or other B-cell
depleting or B-cell inactivating agents.
16. The method of claim 14, wherein the therapy comprises treating
the subject with plasmapheresis or administering intravenous
immunoglobulin.
17. The method of claim 1, wherein the IgG antibodies are IgG4
subclass.
18. The method of claim 5, wherein the IgG antibodies are IgG4
subclass.
19. The method of claim 11, wherein the IgG antibodies are IgG4
subclass.
20. The method of claim 14, wherein the IgG antibodies are IgG4
subclass.
Description
TECHNICAL FIELD
[0002] This invention relates to methods of diagnosing and treating
autoimmune mediated heart disease, e.g., cardiomyopathy or cardiac
arrhythmias, in subjects.
BACKGROUND
[0003] Cardiomyopathy is a disease that weakens and enlarges heart
muscle, and can lead to heart failure. Heart failure is the most
common hospital discharge diagnosis and accounts in the United
States. Dilated cardiomyopathy (DCM) is a relatively common
condition (estimated prevalence 1:2500) 36.5 per 100,000 in Olmsted
County, Minn. (Cetta and Michels, Ann. Med. 27, 169-173 1995) and
is the third leading cause of heart failure. The clinical course of
DCM is usually one of inexorable decline punctuated by acute
decompensation. As a result, DCM remains the most frequent
indication for cardiac transplantation. Despite the high mortality,
morbidity and costs associated with DCM, the pathophysiology of
this condition is largely unknown, and almost half do not have an
identifiable etiology and are labeled as having idiopathic DCM
(Felker, G M et al, Medicine 78(4): 270-283, 1999). Current
management of DCM provides only supportive therapy rather than
treating an underlying cause. As a result, once heart failure is
established in a patient with DCM, the expected outcome is poor,
with a 5 year mortality of about 46% (Felker et al., The New
England journal of medicine. 2000; 342:1077-1084).
SUMMARY
[0004] The present invention is based, at least in part, on the
discovery of the presence of IgG4 subclass autoantibodies to
cardiac troponin I in human patients who have autoimmune heart
disease, e.g., arrhythmia and/or cardiomyopathy, and that such
patient(s) can benefit from immunotherapy. Accordingly, provided
herein are methods for diagnosing and treating autoimmune heart
disease in a subject.
[0005] In one aspect, provided herein are methods for diagnosing
autoimmune heart disease in a subject, the method comprising:
providing a sample from a subject who has heart disease; and
detecting a level, or the presence or absence, of IgG
autoantibodies, e.g., autoantibodies of the IgG1, IgG2, IgG3, or
IgG4 subclass to cardiac troponin I (cTnI) in the sample, wherein
the presence of the IgG autoantibodies indicates that the subject
has autoimmune heart disease. In some embodiments, the methods
include determining whether the anti-cTnI antibodies are
predominantly (at least 50%, e.g., at least 60%, 70%, 80%, or 90%)
IgG4 subclass, wherein the presence of predominantly IgG4 subclass
anti-cTnI antibodies indicates that the subject has autoimmune
heart disease. In some embodiments, the methods include determining
the level of IgG4 subclass autoantibodies to cTnI in the sample,
and comparing the level to a reference level, wherein the presence
of a level of IgG4 autoantibodies to cTnI above the reference level
indicates that the subject has autoimmune heart disease. Reference
levels can be determined using epidemiological and biostatistical
methods known in the art. For example, the reference level can
represent a threshold level, above which a subject has, or has an
increased risk of developing, autoimmune heart disease.
[0006] In some embodiments, the sample comprises serum from the
subject. In some embodiments, the sample comprises cardiac tissue,
e.g., from a biopsy sample, e.g., an endomyocardial biopsy sample,
and the methods include detecting IgG autoantibodies, e.g., IgG4
subclass specific deposition on the surface of cardiac myocytes
from the subject.
[0007] In some embodiments, the subject has cardiac arrhythmia or
idiopathic dilated cardiomyopathy. In some embodiments,
autoantibodies that bind to epitopes within residues 127-164 of
human cTnI are detected. In some embodiments, autoantibodies that
bind to an epitope within residues 127-136, 92-101, or 155-164 of
human cTnI are detected.
[0008] In one aspect, methods for treating autoimmune heart
disease, the method comprising: selecting a subject who has
autoimmune heart disease; and administering to the subject a
therapy that depletes B lymphocytes in the subject.
[0009] In some embodiments, the subject is selected by detecting
the presence of IgG4 autoantibodies to cardiac troponin I (cTnI) in
the subject.
[0010] In another aspect, the invention provides methods for
monitoring the efficacy of a treatment for autoimmune heart
disease. The methods include providing a first sample comprising
serum of a subject; detecting a level of IgG, e.g., IgG1, IgG2,
IgG3, or IgG4, autoantibodies to cardiac troponin I (cTnI) in the
first sample, administering a therapy to the subject; providing a
subsequent sample comprising serum of a subject; detecting a level
of IgG autoantibodies to cTnI in the subsequent sample; and
comparing the level of IgG, e.g., IgG4, autoantibodies to cTnI in
the first sample to the level of IgG, e.g., IgG4, autoantibodies to
cTnI in the subsequent sample. A decrease in level of IgG, e.g.,
IgG4, autoantibodies to cTnI from the first to the subsequent
sample indicates that the therapy is effective.
[0011] In some embodiments, the therapy is or includes
administration of an effective amount of a treatment that reduces
numbers of antibody-producing B cells, e.g., and anti-CD20
antibody, e.g., rituximab; in some embodiments, the therapy is or
includes administration of 375 mg/m.sup.2 of rituximab i.v. weekly
for four weeks. The therapy can also be or include treating the
subject with plasmapheresis or administering intravenous
immunoglobulin.
[0012] As used herein, "treatment" means any manner in which one or
more of the symptoms of a disease or disorder are ameliorated or
otherwise beneficially altered. As used herein, amelioration of the
symptoms of a particular disorder refers to any lessening, whether
permanent or temporary, lasting or transient that can be attributed
to or associated with treatment by the compositions and methods of
the present invention.
[0013] The terms "effective amount" and "effective to treat," as
used herein, refer to an amount or a concentration of one or more
compounds or a pharmaceutical composition described herein utilized
for a period of time (including acute or chronic administration and
periodic or continuous administration) that is effective for
treating autoimmunity-mediated heart disease, e.g., for reducing
one or more symptoms or clinical signs, and returning the subject
to normal or more normal cardiac function. In addition, in some
embodiments the methods of treatment may reduce levels of IgG,
e.g., IgG1, IgG2, IgG3, or IgG4 autoantibodies, e.g., circulating
levels of IgG autoantibodies.
[0014] Effective amounts of one or more compounds or a
pharmaceutical composition for use in the present invention include
amounts that treat autoimmune-mediated heart disease, e.g., prevent
or delay the onset, delay or halt the progression, ameliorate the
effects of, or generally improve the prognosis of a subject
diagnosed with e.g., autoimmune-mediated heart disease. For
example, in the treatment of autoimmune-mediated heart disease, a
compound which improves survival or cardiac function, or decreases
the level of IgG, e.g., IgG4, autoantibodies to cardiac troponin I
to any degree or delays or arrests any symptom of
autoimmune-mediated heart disease would be therapeutically
effective. Since advanced heart failure with irreversible heart
damage may already be present at onset of therapy, a
therapeutically effective amount of a compound is not required to
cure a disease but will provide a treatment for a disease. The term
"subject" is used throughout the specification to describe an
animal, human or non-human, to whom treatment according to the
methods of the present invention is provided. Veterinary and
non-veterinary applications are contemplated. The term includes,
but is not limited to, mammals, e.g., humans, other primates, pigs,
rodents such as mice and rats, rabbits, guinea pigs, hamsters,
cows, horses, cats, dogs, sheep and goats. Typical subjects include
humans, farm animals, and domestic pets such as cats and dogs.
[0015] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0016] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0017] FIG. 1A is a pair of immunoblots showing that a subject with
cardiomyopathy had autoantibodies to cardiac troponin I, and that
these cTnI autoantibodies can be greatly reduced or removed from
the serum by pre-absorption ("competition") with recombinant human
troponin I protein.
[0018] FIG. 1B is a trio of immunofluorescence images showing that
serum from the patient shown in 1A (left panel; green in original)
stains normal human ventricle muscle tissue in a pattern identical
to that of a commercial ventricle monoclonal antibody to cardiac
troponin I (shown in the middle panel; red in original). The right
panel shows a merged image (yellow in original). These
co-localization studies confirm that serum samples from the patient
and the anti-cTnI mAb identify the same protein, i.e., cTnI.
[0019] FIG. 1C is an image of an immunoblot showing that, in the
same patient shown in FIG. 1A, cTnI autoantibodies are
predominantly of the IgG4 subclass (top row). This is in contrast
to the expected IgG1 cTnI autoantibodies from a patient with
giant-cell myocarditis, which is considered to be primarily a
T-cell mediated autoimmune condition (shown in the middle row). No
autoantibodies were detected in healthy control serum.
[0020] FIG. 1D shows that although IgG4 is a rare subclass and
comprised only 3.4% of the patient's total circulating IgGs (data
not shown; normal range of serum IgG4=3-6% of total IgG), the
patient's cardiac immune deposits contained predominantly
IgG4-subclass antibodies.
[0021] FIG. 1E shows that the patient's IgG4 autoantibody titers
increased over time; this was consistent with the worsening disease
and decrease in cardiac function seen clinically.
[0022] FIG. 1F is a photomicrograph showing immunogold staining of
IgG autoantibodies deposited on tissue from an endocardial biopsy
from subject H105 (left and middle) and control stain (right).
[0023] FIG. 2A is an immunoblot (top) and cholesterol assay
(bottom) showing the biochemical localization of TnI to a
cardiomyocyte lipid-rich membrane fraction (#5) rich enriched in
caveolin 3, Na/K ATPase and other signaling proteins that play a
crucial role in cardiac contractility.
[0024] FIG. 2B is an immunoblot showing that the patient's cTnI can
immunoprecipitate cTnI from the same membrane fraction #5 isolated
from heart cells.
[0025] FIG. 2C is a line graph of myocyte length and a pair of bar
graphs showing contractility and calcium transients in the presence
("Pt") or healthy control ("Control") immunoglobulins. Treatment
with the patient's autoantibodies impaired the contractility of
isolated cardiomyocytes.
[0026] FIG. 3A is a pair of line graphs showing the changes in B
cell numbers and cTnI autoantibody titers in response to Rituximab
therapy administered in November-December 2009.
[0027] FIG. 3B is a trio of bar graphs showing that Rituximab
therapy, administered in November-December 2009 resulted in a
sustained decrease in the patient's anti-cTnI IgG4 autoantibodies
(top graph), but did not affect his GAD65 (IgG1) autoantibodies
(middle graph) or protective anti-measles antibodies (bottom
graph).
[0028] FIG. 4 is a western blot and a dot plot showing that the TnI
autoantibody-positive patients contain autoatibodies that are
predominantly of the IgG4 subclass. As expected these
autoantibodies are not detected in serum from healthy control
subjects.
[0029] FIG. 5 is a bar graph showing detection of autoantibodies to
cardiac myosin or troponin I in a cohort of cardiomyopathy
patients.
[0030] FIG. 6 is dot blot data demonstrating that the
autoantibodies found in subjects recognized specific epitopes in
human cardiac troponin I protein (SEQ ID NO:1).
DETAILED DESCRIPTION
[0031] The importance of autoimmunity in the pathogenesis of heart
disease has been uncertain because of a lack of reliable
serological assays that can define autoimmune heart disorders with
the same sensitivity and specificity that are available for other
autoimmune diseases. Data described herein suggest that patients
with heart disease who have high-affinity autoantibodies to cardiac
troponin I as detected by fluid-phase radioimmunoassay represent a
distinct heart failure phenotype that could be treated with
immunotherapy, e.g., B-cell targeted immunotherapy.
[0032] Accordingly, described herein are methods for diagnosing
autoimmune heart disease in subjects by detecting the presence or
absence of IgG, e.g., IgG4, autoantibodies to cardiac troponin I
(cTnI). Subjects who have been diagnosed as having autoimmune heart
disease can be treated with immunotherapy, e.g., the B-cell
targeted immunotherapy described herein. Methods for treating
autoimmune heart disease are also provided.
[0033] IgG4 Subclass Antibodies
[0034] IgG4 are the rarest of the human IgG subclasses, accounting
for only 3-6% of total serum IgG. They are strongly linked to
antibody-mediated autoimmune diseases, e.g., idiopathic membranous
nephropathy (Beck et al., N Engl J Med 2009; 361(1):11-21),
pemphigus (Bhol et al., Proceedings of the National Academy of
Sciences of the United States of America 1995; 92(11):5239-43) and
hyperparathyoidism due to antibodies against the calcium-sensing
receptor (Pallais et al., N Engl J Med 2004; 351(4):362-9). The
target antigens of IgG4-mediated autoimmune diseases are generally
expressed on the cell surface or extracellularly where they are
accessible to antibodies; for example, in pemphigus, the
autoantigen is desmoglein-3 (Stanley and Amagai, N Engl J Med 2006;
355(17):1800-10); in idiopathic membranous nephropathy, the
autoantigen is phospholipase A2 receptor (Beck et al., N Engl J Med
2009; 361(1):11-21). IgG4 subclass autoantibodies to cardiac
autoantigens have not previously been described in heart failure
patients.
[0035] Diagnostic Methods
[0036] Provided herein are methods for diagnosing whether a
subject's heart disease is related to or mediated by autoimmunity.
The diagnostic methods include detecting the presence or absence of
IgG, e.g., IgG4 autoantibodies to cTnI in a subject who has heart
disease. The presence of IgG, e.g., IgG4 subclass autoantibodies to
a cardiac autoantigen indicates that the subject has autoimmune
heart disease (e.g., cardiac arrhythmia, heart failure, or
cardiomyopathy mediated by or associated with the presence of
autoantibodies).
[0037] In some embodiments, IgG, e.g., IgG4, autoantibodies to
specific epitopes within cTnI are detected. For example,
autoantibodies that recognize epitopes within residues 127-164 or
92-164 of human cTnI can be detected using, e.g., a peptide
comprising residues 127-164 or 92-164. In some embodiments,
autoantibodies that recognize an epitope within residues 127-136,
92-101, or 155-164 of human cTnI are detected using, e.g., a
peptide comprising residues 127-136, 92-101, or 155-164. Since most
bone-fide autoantibody-mediated diseases are characterized by
high-affinity autoantibodies to a narrow range of epitopes (Vinuesa
et al., Nat Rev Immunol. 2009 December; 9(12):845-57), knowledge
that subjects have restricted recognition of cTnI is useful in
deciding which patients are most likely to be responsive to
rituximab.
[0038] In some embodiments, an initial screening is done using a
full-length cTnI, e.g., in a fluid-phase (e.g.,
radioimmunoprecipitation assay) format, to detect the presence of
anti-cTnI antibodies. This format is preferred to detect
high-affinity autoantibodies that are often involved in autoimmune
disease. A second screen for IgG, e.g., IgG4, antibodies could then
be done, e.g., by Western blotting, radioimmunprecipitation assay,
or ELISA.
[0039] Once it has been determined that a subject has autoimmune
heart disease, the information can be used in a variety of ways.
For example, a decision to administer a B-cell specific
immunomodulatory treatment, e.g., the treatment described herein,
can be made.
[0040] The methods described herein are useful in a wide variety of
clinical contexts. For example, the methods can be used for
diagnosing subjects in hospitals and outpatient clinics, as well as
the Emergency Department. The methods can be carried out on-site or
in an off-site laboratory.
[0041] Cardiac Troponin I
[0042] Cardiac troponin I (cTnI) polypeptides or immunogenic
fragments thereof and nucleic acids encoding cardiac TnI
polypeptides or immunogenic fragments thereof are useful in the
methods described herein. Exemplary cardiac TnI amino acid
sequences can be found at, e.g., Genbank Accession Nos.
NP.sub.--000354.4 (human; set forth as SEQ ID NO:1 in FIG. 5) and
NP.sub.--033432.1 (mouse). Exemplary cardiac TnI nucleic acid
sequences can be found at Genbank Accession Nos. NM.sub.--000363.4
(human) and NM.sub.--009406.3 (mouse). Immunogenic fragments can
include amino acids 92-164, 127-164, 127-136, 92-101, or 155-164 of
SEQ ID NO:1.
[0043] A nucleic acid encoding a mammalian, e.g., human, cTnI amino
acid sequence can be amplified from human cDNA by conventional PCR
techniques, using primers upstream and downstream of the coding
sequence.
[0044] One method for producing cTnI polypeptides or fragments
thereof for use in the invention is recombinant expression, which
typically involves in vitro translation and transcription from a
recombinant nucleic acid expression vector encoding a cTnI cDNA or
portion thereof. Guidance concerning recombinant DNA technology can
be found in numerous well-known references, including Sambrook et
al., 2001, "Molecular Cloning--A Laboratory Manual," 3d Ed. Cold
Spring Harbor Press; and Ausubel et al. (eds.), 2002, "Short
Protocols in Molecular Biology," John Wiley & Sons, Inc.
[0045] Purification of recombinant cTnI polypeptides or fragments
thereof can be performed by conventional methods and is within
ordinary skill in the art. The purification can include two or more
steps, and one step can be affinity chromatography employing
anti-cTnI antibodies covalently linked to a solid phase
chromatography support (beads) such as crosslinked agarose or
polyacrylamide. Other useful purification steps include gel
filtration chromatography and ion exchange chromatography. Purified
cTnI polypeptides and fragments thereof are also commercially
available (e.g., from Sigma-Genosys, Biolegend, or Abcam).
[0046] Detecting Autoantibodies
[0047] Methods known in the art or described herein can be used to
detect the presence or absence of IgG, e.g., IgG4 autoantibodies to
cardiac autoantigens. In some embodiments, serum samples from
subjects are contacted with a cardiac autoantigen, e.g., cTnI or
alpha-myosin heavy chain polypeptide, or an immunogenic fragment
thereof, for a sufficient amount of time and under conditions that
allow binding of the cardiac antigens to any autoantibodies in the
serum samples. Binding occurs between the cTnI polypeptide or
fragment thereof and the autoantibodies are then detected and, in
some embodiments, quantified. In some embodiments, the
autoantibodies are isolated and the presence of IgG, e.g., IgG4
subclass antibodies is detected, and, in some embodiments,
quantified.
[0048] In some embodiments, biopsy samples are contacted with
subclass-specific binding reagents, e.g., antibodies that bind
specifically to IgG4 (and/or optionally one or more other
subclasses, e.g., IgG1, IgG2, or IgG3), and the presence (and
optionally, quantity) of IgG4 antibodies.
[0049] For example, enzyme-linked immunosorbent assay (ELISA) can
be used to detect the presence of autoantibodies in the serum of
subjects. ELISA can detect autoantibodies that bind to antigens
immobilized on solid support (e.g., a multi-well plate) by using
enzyme-linked secondary antibodies, such as goat anti-human Ig Abs,
and enzyme substrates that change color in the presence of
enzyme-labeled antibodies. Fluid-phase radioimmunoassays (RIA) can
also be used to detect the presence of autoantibodies. For example,
the gene for the antigen can be cloned into an expression vector,
and in vitro translation can be carried out with
[.sup.35S]methionine to produce radiolabeled antigens.
Antibody-bound radiolabeled antigens can be separated from free
radiolabeled antigens with, e.g., protein A-Sepharose or protein
G-Sepharose beads, which bind to the antibodies. To detect the
presence of a specific subclass of autoantibodies, e.g., IgG1,
IgG2, IgG3 or IgG4 antibodies, anti-IgG1, IgG2, IgG3, or IgG4
antibodies can be used. For example, mouse anti-human IgG4
antibodies (available from, e.g., Sigma) bound to Sepharose beads
can be employed in RIA to detect subclass-specific autoantibodies
in samples from a human subject using radioactive-labeled antigens
and detecting specific immunocomplexes using a liquid scintillation
counter. Other methods known in the art or described herein can be
used to detect the presence of autoantibodies.
[0050] Subjects
[0051] The presence of autoimmune heart disease can be detected in
a subject with cardiomyopathy, e.g., ischemic, dilated,
hypertrophic, idiopathic, or restrictive cardiomyopathy; a subject
with unexplained cardiac arrhythmias; or a subject with heart
failure, e.g., unexplained heart failure; using methods described
herein. Those of ordinary skill in the art would be able to
determine whether a subject has cardiomyopathy, cardiac arrhythmia,
or heart failure, using methods and knowledge known in the art
(see, e.g., Wynne and Braunwald, "The cardiomyopathies and
myocarditides." In: Braunwald E., Zipes D. P., P. L, eds. Heart
disease: a textbook of cardiovascular medicine. 6th ed.
Philadelphia: W.B. Saunders Company; 2001: 1751-806).
[0052] In addition, the presence of autoimmune heart disease can be
detected using the methods described herein in subjects who are
asymptomatic, or subjects who are asymptomatic relatives of
affected patients.
[0053] Therapeutic Methods
[0054] Data described herein suggest that a subject who suffers
autoimmune-mediated heart disease will benefit from immunotherapy.
In particular, these subjects may benefit from agents that target B
lymphocytes, which make antibodies. Accordingly, the present
therapeutic methods can include selecting a subject who has
autoimmune heart disease, and administering to the subject an
effective amount of an immunotherapy. In some embodiments, the
methods include administering a compound that decreases B
lymphocytes, e.g., employing a direct depletion approach by
engagement of B cell surface molecules, e.g., CD19, CD20, or CD200,
e.g., with an anti-CD20 monoclonal antibody (e.g., rituximab), an
anti-CD19 monoclonal antibody (e.g., XmAb5574 (MOR208; Xencor,
Monrovia Calif.) or MDX-1342 (Medarex, Princeton, N.J.)), an
anti-CD22 monoclonal antibody (e.g., epratuzumab); or an indirect
attrition by inhibition of survival by neutralization of B
lymphocyte stimulator (BLyS), a potent B cell survival factor
(e.g., using anti-BLyS/BAFF agents such as belimumab or atacicept).
In some embodiments, the methods include administering, in addition
to or as an alternative to rituximab, Azathioprine; a steroid such
as cyclosporine or methylprednisolone; Immunoadsorption
(IAS)/plasmapheresis. See, e.g., Engel et al., Pharmacol Rev. 2011
March; 63(1):127-56. Epub 2011 Jan. 18; Stohl and Looney, Clin
Immunol. 2006 October; 121(1):1-12. Epub 2006 May 11; Stummvoll et
al., Atheroscler Suppl. 2009 Dec. 29; 10(5):110-3; and Vaughan et
al., Int J Biochem Cell Biol. 2011 March; 43(3):280-5. Epub 2010
Dec. 13.
[0055] In patients with rheumatoid arthritis treated with
rituximab, clinical relapse was preceded by a rise in autoantibody
levels and B cells reappeared at a mean of 8 months after treatment
(Leandro et al., Arthritis Rheum 2006; 54(2):613-20). In contrast,
in severe pemphigus associated with IgG4 autoantibodies, 86% of
subjects treated with rituximab remained in complete remission
almost after mean follow-up of 34 months suggesting that in some
autoimmune conditions, remissions can be sustained (Joly et al., N
Engl J Med 2007; 357(6):545-52). The use of rituximab for
cardiomyopathy has not previously been described.
[0056] The treatment methods described herein can be combined with
other immunotherapy, e.g., plasmapheresis to remove the
autoantibodies typically with supplemental administration of
intravenous immunoglobulin (IVIg). Two methods are typically used
in plasmapheresis to separate plasma from blood cells: membrane
filtration and extracorporeal centrifugation. Both techniques are
designed to remove large molecular weight substances, such as
antibodies, from the plasma. IVIg is a mixture of proteins
containing y-globulins, predominantly IgG, to provide passive,
temporary humoral immunity against disease. Other treatments for
heart disease can also be administered.
[0057] Subject Selection
[0058] The present treatments methods can be used to treat subjects
who have autoimmune heart disease, e.g., subjects whose heart
disease has been diagnosed as mediated by or related to
autoimmunity, e.g., by a method described herein. Thus the methods
can include detecting the presence of autoimmune heart disease in a
subject using a method described herein, and administering to the
subject a therapeutically effective amount of an agent described
herein, e.g., agents that target B lymphocytes, e.g.,
rituximab.
[0059] Monitoring of Treatment
[0060] Effectiveness of the treatment methods described herein can
be determined by monitoring changes in the cardiac symptoms,
characteristic features, or parameters of the subject being
treated. For the methods described herein, it may be useful to
monitor the level of IgG, e.g., IgG4, autoantibodies to cTnI in the
subject, e.g., before, throughout, and after the treatment.
Evaluation by endomyocardial biopsy could include examination for
one or both of total IgG and IgG subclass deposition.
[0061] Pharmaceutical Compositions and Methods of
Administration
[0062] The compounds and compositions described herein can be
administered to a subject, e.g., a subject identified as being in
need of treatment, using a systemic route of administration.
Systemic routes of administration can include, but are not limited
to, parenteral routes of administration, e.g., intravenous
injection, intramuscular injection, and intraperitoneal injection;
enteral routes of administration, e.g., administration by the oral
route, lozenges, compressed tablets, pills, tablets, capsules,
drops (e.g., ear drops), syrups, suspensions and emulsions;
transdermal routes of administration; and inhalation (e.g., nasal
sprays).
[0063] In some embodiments, the modes of administration described
above may be combined in any order and can be simultaneous or
interspersed.
[0064] Pharmaceutical compositions typically include a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes saline, solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration.
[0065] Pharmaceutical compositions are typically formulated to be
compatible with its intended route of administration. Examples of
routes of administration include parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration.
[0066] Methods of formulating suitable pharmaceutical compositions
are known in the art, see, e.g., the books in the series Drugs and
the Pharmaceutical Sciences: a Series of Textbooks and Monographs
(Dekker, NY). For example, solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0067] Pharmaceutical compositions suitable for injectable use can
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0068] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying, which yield a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0069] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0070] Systemic administration of a therapeutic compound as
described herein can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0071] In one embodiment, the therapeutic compounds are prepared
with carriers that will protect the therapeutic compounds against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Such formulations
can be prepared using standard techniques, or obtained
commercially, e.g., from Alza Corporation and Nova Pharmaceuticals,
Inc. Liposomal suspensions (including liposomes targeted to
selected cells with monoclonal antibodies to cellular antigens) can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0072] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0073] The compositions described herein can be administered one
from one or more times per day to one or more times per week;
including once every other day. The skilled artisan will appreciate
that certain factors may influence the dosage and timing required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of the therapeutic compounds described herein can
include a single treatment or a series of treatments.
[0074] Dosage, toxicity and therapeutic efficacy of the therapeutic
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). The dose
ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio LD50/ED50. Compounds
which exhibit high therapeutic indices are preferred.
[0075] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0076] For compounds known in the art (e.g., rituximab),
practitioners might generally use in humans and non-human subjects
the recommended (e.g., FDA approved) dosage for that compound.
EXAMPLES
[0077] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Identification of IgG4 Autoantibodies to cTnI in Human Patients
[0078] Evidence that autoimmune mechanisms alone can cause T1D
heart disease is exemplified by a patient with T1D since age 12 yrs
("H-105") who, at age 17 yrs, presented with severe arrhythmias and
unexplained dilated cardiomyopathy with normal coronary
angiography. The family history was notable for the presence of
autoimmune diseases in multiple family members including a parent
with T1D and thyroiditis.
[0079] A complete clinical evaluation of the patient was performed
to define the etiology of his cardiomyopathy, including EKG, chest
X-ray, blood work, echocardiography, and cardiac MRI.
[0080] Blotting was performed to detect autoantibodies. 2.5 ug/lane
of total SDS lysates from human heart, skeletal muscle and liver or
0.25 ug/lane of purified human cTnI (Life Diagnostics) were
separated in a 10% SDS-PAGE gel and transferred to nitrocellulose
paper. The blots were incubated overnight with control or patient
sera diluted 1:1000, followed by incubation with
peroxidase-conjugated F(ab')2 fragment of goat anti-human IgG
(Jackson ImmunoResearch Laboratories Inc). Blots were developed
using Western Lighting Chemiluminescence Reagent Plus (Perkin
Elmer). The patient was found to have high titers of autoantibodies
directed against cTnI (FIG. 1A).
[0081] Indirect immuno-fluorescent analysis was also performed with
the patient's serum. Frozen sections from normal human donor heart
embedded in OCT were fixed in 2% paraformaldehyde and permeabilized
with TX-100/PBS. After blocking, sections were incubated overnight
with 1:100 dilutions of serum from the patient or 1:250 dilution of
mouse monoclonal anti Troponin I (SIGMA), followed by detection
with FITC-conjugated mouse F(ab').sub.2 anti-human IgG or FITC
conjugated F(ab').sub.2 goat-anti mouse IgG (Jackson ImmunoResearch
Laboratories Inc.). For identification of nuclei slides were
stained with Hoechst 33258 and mounted using Fluorogel (Electron
Microscopy Science). Images were taken with an Axiocam camera
linked to a Zeiss Axioscop 2 microscope. The results showed that
the patient's autoantibodies stained the surface of normal hearts
in a pattern indistinguishable from that seen with a commercial
monoclonal antibody against human troponin I (FIG. 1B).
[0082] The circulating (serum) anti-troponin antibodies were
subjected to subclass analysis as follows. 0.25 ug/lane of purified
human cTnI (Life Diagnostics) were separated in a 10% SDS-PAGE gel
and transferred to nitrocellulose paper. The blots were cut into
strips and incubated with patients or controls sera diluted 1:1000.
Individual strips were incubated with mouse antibodies against the
four human IgG subclasses (Zymed laboratories). Blots were
developed using Western Lighting Chemilumineiscence Reagent Plus
(Perkin Elmer) The results showed that the antibodies were
predominantly of the IgG4 subclass (FIG. 1C).
[0083] Standard hematoxylin and eosin staining of an endomyocardial
biopsy revealed the anticipated myocyte hypertrophy and
interstitial fibrosis compatible with an advanced cardiomyopathy.
No other etiology was identified by the biopsy to account for the
patient's condition, including myocarditis or iron deposition.
[0084] The patient's endomyocardial biopsy sample was frozen in OCT
and sections were stained with mAbs against human total IgG and
IgG1, IgG2, IgG3 and IgG4. Human cardiac tissues from biopsy were
fixed overnight in Paraformaldehyde-glutaraldehyde fixative. The
tissue was cryoprotected and embedded in OCT compound (Tissue-Tek,
Sakura Finitek). Although IgG4 comprised only 3.4% of his total
circulating IgG using subclass-specific antibodies (Human IgG
Subclass Profile ELISA kit form Zymed Laboratories INC, following
the manufacturer's instructions; normal range of IgG4=3-6%), the
patient's heart biopsy sample demonstrated enriched IgG4-specific
Ab deposition (FIG. 1D).
[0085] IgG subclass specific cTnI antibodies were measured by
incubating patients or controls serum with .sup.35S-labeled human
cardiac troponin I in TBS-1% TX100 Buffer at 4.degree. C. for 24 h.
IgG subclass specific Ab bound Sepharose beads were prepared using
Biotin-labeled mouse mAbs against human IgG1, IgG2, IgG4 and IgE
(BD Pharmingen) and Sepharose 4B streptavidin beads (GE
Healthcare). Aliquots of serum were spotted in a 96-well filtration
plate (Unifilter, Whatman) and IgG subclass specific Ab beads were
added and incubated for 1 h at 4.degree. with shaking. After
incubation the plate was washed with cold TBS-TX-100 buffer, dry
and 50 ul of scintillation liquid added. The radioactivity in the
samples was determined using a Wallac MicroBeta Scintillation
Counter (Perkin Elmer). Interestingly, worsening heart function
with a progressive decrease in the patient's ejection fraction from
.about.50% to .about.30% correlated with increasing cTnI-Ab IgG4
titers over a 15-month period (FIG. 1E).
Example 2
Human Patient-Derived cTnI Autoantibodies Recognize TnI on the
Plasma Membrane of Heart Cells
[0086] Specialized biochemical membrane fractionation techniques
were performed as described in Head et al., JBC 281(36) 26391-26399
(2006). Briefly, mouse hearts were homogenized in ice-cold Buffer A
(250 mM sucrose, 20 mM Tri-HCl pH 7.4, containing freshly added
protease inhibitors) using a 40 ml glass Dounce homogenizer. The
homogenate was centrifuge at 700 g.times.20 min and the resulting
supernatant (SN1) was centrifuged at 100,000.times.g for 1 h. After
centrifugation the pellet was resuspended in 500 mM NaHCO.sub.3 pH
11 using a glass homogenizer and mixed with 1 volume of 90% sucrose
in MBS buffer (25 mM MES pH 6.5, 150 mM NaCl), and layered on a
discontinuous sucrose gradient (45, 35 and 5% sucrose in MBS
buffer). After centrifugation at 280,000.times.g for 18 h at
4.degree. C., 1 ml samples were collected from the top of the
gradient and analyzed by Western Blotting using the following
antibodies: mouse anti-caveolin 3 (BD Transduction Laboratories),
rabbit polyclonal anti ATPase beta1 (Na+/K+) (GeneTex, Inc.), mouse
anti human cTroponin I (clone C-4) (Santa Cruz Biotechnology Inc.),
rabbit anti human c Troponin C (clone H110) (Santa Cruz
Biotechnology, Inc.) and patient purified IgG4. The cholesterol
concentration for each sample was determined using the Amplex Red
Cholesterol Assay Kit (Invitrogen). A band corresponding to
cTroponin I was detected in the buoyant caveolin-3 enriched
membrane fractions (Fraction No. 5), were also co-localized with
the plasma membrane marker ATPase beta1 (Na.sup.+/K.sup.+) that was
enriched in cholesterol. Cardiac troponin I was also distributed in
heavy/non buoyant fractions (Fractions 8-12) and co-localized with
cTroponin C. Patient sera recognized the same antigen as the
commercial antibody against cTroponin I.
[0087] FIG. 2B Fractions enriched in cTnI from the NaHCO.sub.3
fractionation protocol were used for immunoprecipitation analysis.
Fraction 5 was diluted in MES buffer plus protease inhibitors (0.8
mg protein) and incubated overnight with the patient, control sera
or buffer and then with protein A/G plus beads (Pierce). The
immunocomplexes bound to the beads were separated in a 4-15%
gradient SDS-PAGE gel, transferred to nitrocellulose and probed by
western blot (WB) with mouse anti human cTroponin I (clone C-4)
(Santa Cruz Biotechnology Inc.). Patient sera but not control sera
specifically immunoprecipitated the cTroponin I present in the
caveolin enriched fraction obtained by carbonate fractionation.
[0088] The results demonstrated that troponin I is found on the
plasma membrane of heart cells, in addition to its expected
location inside heart cells (FIGS. 2A and 2B). These results are
also consistent with immunofluorescence staining and immunogold EM
studies clearly deposition of IgG4 autoantibodies on the surface of
his myocardial cells.
Example 3
Human Anti-TnI IgG4 Autoantibodies Impair Cardiac Function
[0089] cTnI Abs have been shown to cause arrhythmias and dilated
cardiomyopathy in mouse models (Okazaki et al., Nat Med 9, 1477-83
(2003)), raising the possibility of a similar pathogenic process in
this patient. "Pathogenicity assays" (functional assays) were
performed. Myocytes from adult rats were obtained by
Langendorff-perfusion with Ca.sup.2+ free Tyrode's solution.
Myocytes were culture and treated for 18 h with control or patient
sera. Ca.sup.2+ transients and mechanical properties were assessed
in the myocytes, which were continuously perfused in a heated
chamber with perfusion buffer (137 mM NaCl, 5.4 mM KCl, 0.5 mM
MgCl.sub.2, 10 mM HEPES, 5.5 mM glucose, 1.2 mM CaCl.sub.2, 0.5 mM
probenecid) and electrically stimulated at 0.5 Hz. Cell shorting
was monitored by a digital edge detection system (IonOptix) with a
sampling rate of 240 Hz. Ca.sup.2+ transients were measured in
fura-2/AM (Molecular Probes) loaded cardiomyocytes (1 uM for 15 min
at room temperature) using a dual excitation Hyperswitch
(IonOptix). Data were analyzed using the software Ionwizard
(IonOptix).
[0090] These studies were performed in infusion chambers,
visualizing heart beats of adult mouse (or rat) heart cells with
the IonOptix video detection system. The results showed that
autoantibodies from patient H-105 impaired the contractility and
calcium homeostasis of isolated heart cells (FIG. 2C).
Example 4
B-Cell Depletion Therapy for Treatment of Autoimmune Heart
Disease
[0091] Patient H105 completed five cycles of plasmapheresis
followed by administration of 2 g/kg intravenous immunoglobulin
(IVIG). The patient was subsequently treated with rituximab (375 mg
per square meter of body surface area) weekly for a total of four
weeks. Two years after this treatment the patient was rated as New
York Heart Association Class II with regard to congestive heart
failure symptoms and displayed an oxygen consumption of 19.4
ml/kg/min despite a sub-maximal effort as demonstrated by his
respiratory ratio of 0.99. The patient's estimated pulmonary artery
pressure has remained below 20 mmHg subsequent to completion of his
therapy. The rituximab therapy has had a beneficial effect on the
patient's anti-troponin I antibody titers. The treatment has also
has markedly improved this patient's symptoms and arrested the
previously rapid progression of his heart failure with
stabilization in his ejection fraction and improvement of his
oxygen consumption to 28 ml/kg/min (at his last examination in
June, 2011). There was efficient depletion of all subpopulations of
peripheral B cells, including cells with plasma cell precursor
phenotype (CD19.sup.+, CD20.sup.-, CD38.sup.+++) one month after
completing his treatment with rituximab, and this has persisted 26
months post-therapy as has the reduction in his anti-troponin
antibodies (FIG. 3A). This has been associated with a stabilization
in his ejection fraction to 25-30% (after a steady decline from 44%
to 31% over the 2-yr period preceding immunotherapy) and
improvement in his clinical symptoms.
[0092] Importantly, the rituximab therapy selectively depleted TnI
autoantibodies in subject H105, but did not diminish the levels of
either his type 1 diabetes-associated GAD 65 autoantibodies (GADA),
which are not postulated to play a pathogenic role in diabetes, or
the levels of his protective antibodies to measles (FIG. 3B). The
preservation of anti-microbial immunity may explain the lack of any
adverse side-effects (i.e., no increased susceptibility to
infections) from immunotherapy. These studies suggest that
rituximab therapy selectively depletes the B cells that produce
pathogenic troponin autoantibodies and underscores the safety and
therapeutic potential of using rituximab for patients with
idiopathic cardiomyopathy who test positive for troponin I
autoantibodies. The ability of rituximab to selectively reduce the
patient's IgG4 TnI autoantibodies suggests that they are derived
from short-lived autoreactive plasma cells: Selective autoantibody
depletion is noted after rituximab treatment in other IgG4-Ab human
autoimmune diseases (e.g., pemphigus, Joly et al., N Engl J Med
2007; 357:545-52) and in autoantibody-mediated autoimmune mouse
models (Huang et al., Proc Natl Acad Sci USA. 2010 Mar. 9;
107(10):4658-63. Epub 2010 Feb. 22).
Example 5
Screening Human Patients for Autoantibodies to cTnI
[0093] New screening assays using recombinantly produced human cTnI
protein in a fluid-phase format, suitable for the detection of high
affinity autoantibodies characteristic of organ-specific autoimmune
diseases, were developed and used to screen a cohort of
cardiomyopathy patients.
[0094] 1. Construction of Recombinant Human cTnI:--
[0095] Total RNA was extracted from human ventricle tissue using
Trizol (Invitrogen, USA) as per the manufacture's instructions. One
microgram of Total RNA was used to reverse-transcribe (RT-PCR) into
cDNA using Transcriptor first strand cDNA synthesis kit (Roche,
USA) according to the manufacture's protocol.
[0096] The cDNA specific to human cardiac troponin I (cTnI) was
made by PCR (FastStart High Fidelity PCR system, Roche, USA) using
the following specific primers: Forward: 5' TTG CAC TCG TCT AGA TGT
CCT CGG GGA GTC TCA AGC 3' and Reverse: 5' TAC CAC GCG TCT AGA AGC
TCA GAG AGA AGC TTT ATT 3'. Restriction sites for XbaI (underlined
in the primer sequences) were incorporated into the forward and
reverse primers in order to allow sub-cloning of the PCR products
into pSP64 Poly(A) expression vector (Promega, USA) and the
subsequent expression of the human cTnI cDNA from the SP6 promoter
present in the vector.
[0097] One hundred nanograms of cDNA were subjected to 35 cycles of
PCR amplification in a Peltier Thermal Cycler (Biorad, USA) using
the following conditions: denaturation 95.degree. C. for 30 sec,
annealing 67.4.degree. C. for 30 sec, elongation 72.degree. C. for
1 min. The PCR products (.about.837 bp) were ran on 1% agarose gel
and then purified using Qiaquick gel purification kit (Qiagen,
USA). Subsequently, the purified PCR products were digested with
XbaI (New England Biolabs, USA) and then ligated into pSP64 Poly(A)
vector at XbaI site. The ligation reaction was transformed into Top
10 competent cells (Invitrogen, USA) and selected the clones by
plating them onto LB agar plates containing ampicillin (Sigma) at
100 .mu.g/ml. Successful clones were screened for by restriction
digestion with HindIII (New England Biolabs, USA) and the
appropriate recombinant plasmids were sequenced completely to
verify that no sequence errors had been introduced and also to
confirm the orientation of the clone in order to express in vitro
from SP6 promoter. The recombinant plasmid, pSP64 Poly(A)+human
cTnI, was purified with a QIAGEN Plasmid Maxi Kit (Qiagen, USA).
This recombinant plasmid clone was used for the expression of human
cTnI protein in a cell free system.
[0098] 2. Expression of Human cTnI Using Cell Free System:--
[0099] The plasmid construct (pSP64Poly(A)+human cTnI) was used in
a TnT SP6 Quick Coupled Transcription/Translation System (Promega,
USA) to produce and label cTnI with [.sup.35S]-methionine in vitro.
The cTnI cDNA fragment was inserted into pSP64 Poly(A) in the
correct orientation to allow expression from the SP6 promoter, and
each template contained appropriate start and stop codons to ensure
accurate translation. A standard reaction mixture of 50 .mu.l
contained: quick master mix, 40 .mu.l; plasmid template, 2 .mu.g;
[.sup.355]-methionine (1000 Ci/mmol; 10 mci/ml; GE Healthcare,
USA), 2 .mu.l and made the total volume up to 50 .mu.l with the
nuclease free water. The reaction was incubated for 90 minutes at
30.degree. C. and then stored at -20.degree. C. until required.
[0100] An aliquot of each of the in vitro translation reaction
mixtures was added to SDS sample buffer and boiled for 5 minutes
before it ran on 10% Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE). Prior to drying under vacuum, gels were
soaked in fixing solution and then in 10% glycerol. Autoradiography
was carried out at -80.degree. C. Radioactivity incorporated into
the protein was determined by trichloroacetic acid (TCA)
precipitation as per the Promega's technical manual.
[0101] 3. Radioimmunoprecipitation Assay for the Detection of Human
cTnI Autoantibodies
[0102] Patient and control sera were tested for binding to
[.sup.35S]-human cTnI in radio-immunoassays as follows. For each
assay, an aliquot of in vitro translation reaction mixture
(equivalent to 48,000 counts per minute (cpm) of TCA-precipitable
material) was suspended in 120 .mu.l of immunoprecipitation buffer
containing: 20 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% (v/v) Triton
X-100; 10 .mu.g/ml aprotinin (Sigma, USA). Serum was then added to
a final dilution of 1:25 and the reaction was carried out in a
microcentrifuge tube. The samples were incubated with shaking at
room temperature for 2 hrs prior to overnight incubation with
shaking at 4.degree. C. All samples were tested in duplicates.
[0103] On the next day, 50 .mu.l (20,000 cpm) of the reaction
samples were transferred to each well of the 96 well plate
(Whatman, USA). Subsequently, 50 .mu.l of protein A/G (50% A/8% G)
Sepharose 4 Fast Flow beads (GE Healthcare, USA), prepared
according to the manufacturer's protocol, were added to each well
and incubation continued for 1 hour at 4.degree. C. The protein A/G
Sepharose-antibody complexes were then washed total of twelve times
(4 times washing with 5 minutes incubation period in between
wishes) in immunoprecipitation buffer at 4.degree. C. with
vacuum-operated 96-well plate washer (Millipore, USA). At the end
of final wash, the plate was dried under lamp for 10 min and then
added 100 .mu.l of MicroScint-20 scintillation cocktail
(PerkinElmer, USA) into each well. Immunoprecipitated radioactivity
was counted in a Wallac 1450 microbeta Trilux liquid scintillation
counter (PerkinElmer, USA). All samples were analyzed in duplicate
and the mean cpm immunoprecipitated were determined.
[0104] The binding reactivity of each patient and control sera to
human cTnI was expressed as an antibody index calculated as: cpm
immunoprecipitated by tested serum divided by mean cpm
immunoprecipitated by all healthy control sera. The upper level of
normal for each assay was calculated using the mean antibody
index+3 standard deviations (SD) of all control sera. Patient sera
with an antibody index greater than the upper level of normal were
regarded as positive for binding to the radio-labeled cTnI used in
the assay.
[0105] In the heart failure cohort screened, about 20% of patients
with idiopathic dilated cardiomyopathy (DCM) were positive for
high-titer autoantibodies directed against cTnI (FIG. 5).
Remarkably, these patients were also distinctive in producing
predominantly IgG4 subclass anti-cTnI antibodies. Initial
characterization of the fine specificities of the autoantibodies
revealed that they target the same domain of the cTnI protein.
Patients with DCM and high circulating anti-cTnI antibodies may
represent a distinct heart failure phenotype that could respond to
B-cell targeted immunotherapy.
[0106] Mean age was 48 yrs, 61% were male, and mean EF was 30% in
HF patients. Anti-troponin Ab were detected more often in
myocarditis and idiopathic dilated cardiomyopathy compared to other
HF etiologies (20% vs. 5%, p=0.004); none were found in controls.
In HF patients, anti-myosin Ab were more common than anti-troponin
Ab (29% vs. 10%, p<0.0001) and were particularly enriched in
myocarditis subjects vs. other causes of HF (p=0.007). There was
little overlap between anti-myosin and anti-troponin Ab production,
with 87% of Ab-positive subjects having only a single Ab type
detected. See FIG. 5, suggesting that the detection of cTnI
autoantibodies defines a unique heart failure phenotype.
[0107] Cardiac autoantibodies are relatively common in HF; however,
their antigenic specificity varies with etiology. Troponin Ab are
more specific to both myocarditis and idiopathic dilated
cardiomyopathy. Differential Ab detection may be a marker or
mediator of clinical risk in a subset of inflammatory
cardiomyopathies.
Example 6
Epitope Analysis for Autoantibodies to cTnI
[0108] As a first step towards identifying the epitopes recognized
by cTnI autoantibodies in our patients, SPOTscan analysis Epitope
mapping protocol was performed as described in the company manual
(JPT Peptide Technologies, Germany) was used. This method has been
widely used in epitope mapping of autoantigens in other human
autoimmune diseases, such as SLE and Goodpasture's syndrome.
[0109] Affinity-purified IgGs from H105 and M2 (a 21-year old
patient who presented in severe heart failure with an EF of 15% and
showed the second highest levels of IgG4 cTnI antibody titers of
the cohort studied, see Example 5) were immunoblotted on Whatman 50
cellulose membranes that contained 30 covalently-bound overlapping
10-mer peptides covering the entire length of the human cTnI
protein sequence (Sigma-Genosys) (FIG. 6), as follows:
##STR00001##
[0110] These studies were remarkable in revealing that H105's
autoantibodies are essentially monospecific, recognizing only a
single peptide, cTnI.sub.127-136, whereas M2 serum recognized
cTnI.sub.92-101 and cTnI.sub.155-164. Sera from normal controls did
not react with any of the cellulose-bound cTnI peptides (FIG. 6).
These peptide sequences are distinctive in being highly conserved
across species (90-100% conservation between rodents and humans),
consistent with their important physiological roles: peptides
TnI.sub.92-101 and TnI.sub.127-136 are both part of the H2 helix
(residues 90-135 of cTnI) that binds to troponin T whereas
TnI.sub.155-164 is part of the H3 helix that binds to troponin C
(Takeda et al., Nature 2003; 424(6944):35-41). Interestingly,
sequence motifs residing in the H2 helix have also been recently
implicated in a TnI-immunization induced model of murine
myocarditis (Kava et al., Circulation 2008; 118(20):2063-72).
[0111] Immunoassays for autoantibodies based on in vitro
translation of cDNAs encoding human cardiac myosin and troponin and
immunoprecipitation were also tested with patient sera in 96-well
filtration plates. Study subjects (174 with heart failure (HF) and
74 controls) underwent comprehensive evaluation and testing for
autoantibodies and HLA-DQ/DR haplotype. HF etiology was assigned by
investigators blinded to autoimmune test results. Primary analysis
compared specific autoantibodies (Ab) according to HF etiology.
[0112] Using troponin peptide mapping techniques, the precise
epitopes recognized by autoantibodies from 7 other individuals with
high titers of troponin I autoantibodies were identified.
Interestingly, one patient (who also had type 1 diabetes) targeted
the same peptide as did patient H105 (cTnI.sub.127-136), while 4 of
8 patients reacted most strongly to a single peptide, peptide 23
(cTnI.sub.155-164), and 7 of 8 patients recognized peptides
spanning peptides 19-23 (cTnI.sub.127-164). Thus, the
autoantibodies from our cohort targeted a similar region of the TnI
protein. These findings are important because in other autoimmune
diseases, the pathogenicity of autoantibodies correlates closely
with the epitopes targeted by autoantibodies (Bhol et al.,
Proceedings of the National Academy of Sciences of the United
States of America 1995; 92(11):5239-43).
OTHER EMBODIMENTS
[0113] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
31210PRTHomo sapiens 1Met Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu
Pro Arg Pro Ala Pro1 5 10 15 Ala Pro Ile Arg Arg Arg Ser Ser Asn
Tyr Arg Ala Tyr Ala Thr Glu 20 25 30 Pro His Ala Lys Lys Lys Ser
Lys Ile Ser Ala Ser Arg Lys Leu Gln 35 40 45 Leu Lys Thr Leu Leu
Leu Gln Ile Ala Lys Gln Glu Leu Glu Arg Glu 50 55 60 Ala Glu Glu
Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys65 70 75 80 Gln
Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu Gln Asp Leu 85 90
95 Cys Arg Gln Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr
100 105 110 Asp Ile Glu Ala Lys Val Thr Lys Asn Ile Thr Glu Ile Ala
Asp Leu 115 120 125 Thr Gln Lys Ile Phe Asp Leu Arg Gly Lys Phe Lys
Arg Pro Thr Leu 130 135 140 Arg Arg Val Arg Ile Ser Ala Asp Ala Met
Met Gln Ala Leu Leu Gly145 150 155 160 Ala Arg Ala Lys Glu Ser Leu
Asp Leu Arg Ala His Leu Lys Gln Val 165 170 175 Lys Lys Glu Asp Thr
Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg 180 185 190 Lys Asn Ile
Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe 195 200 205 Glu
Ser 210 236DNAArtificial Sequencesynthetic oligonucleotide
2ttgcactcgt ctagatgtcc tcggggagtc tcaagc 36336DNAArtificial
Sequencesynthetic oligonucleotide 3taccacgcgt ctagaagctc agagagaagc
tttatt 36
* * * * *