U.S. patent application number 14/700042 was filed with the patent office on 2015-09-17 for prostatitis-associated antigens and methods of use thereof.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Mark Stuart Anderson, Jason J. Devoss, Lawrence H. Fong, Yafei Hou.
Application Number | 20150260730 14/700042 |
Document ID | / |
Family ID | 42936501 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150260730 |
Kind Code |
A1 |
Fong; Lawrence H. ; et
al. |
September 17, 2015 |
PROSTATITIS-ASSOCIATED ANTIGENS AND METHODS OF USE THEREOF
Abstract
The present disclosure provides prostatitis-associated antigens,
and compositions comprising the antigens. The present disclosure
provides diagnostic methods, generally involving assaying the level
of an immune response specific for a prostatitis-associated antigen
in an individual. Kits suitable for use in performing such
diagnostic assays are also provided. The present disclosure further
provides methods of treating prostatitis, methods of treating
prostate cancer, and methods of treating benign prostatic
hyperplasia.
Inventors: |
Fong; Lawrence H.; (Palo
Alto, CA) ; Hou; Yafei; (Mountain View, CA) ;
Devoss; Jason J.; (San Francisco, CA) ; Anderson;
Mark Stuart; (Larkspur, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
42936501 |
Appl. No.: |
14/700042 |
Filed: |
April 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13262752 |
Dec 2, 2011 |
9046529 |
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PCT/US2010/029174 |
Mar 30, 2010 |
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14700042 |
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61168506 |
Apr 10, 2009 |
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Current U.S.
Class: |
435/7.4 ;
435/7.1; 435/7.92; 436/501 |
Current CPC
Class: |
G01N 2333/96433
20130101; G01N 2800/342 20130101; A61K 39/00 20130101; A61P 13/08
20180101; G01N 33/6854 20130101; G01N 2333/47 20130101; A61P 37/02
20180101; A61P 35/00 20180101; C07K 14/47 20130101; G01N 33/6893
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
numbers R01 CA102303 and U19 AI056388, awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1.-23. (canceled)
24. A method of diagnosing an individual as having prostatitis, the
method comprising: assaying the level of antibody specific for a
human semenogelin polypeptide in a biological sample obtained from
the individual, wherein said assaying comprises contacting antibody
present in the biological sample with human semenogelin polypeptide
comprising a detectable label; identifying the individual as having
prostatitis when the level of antibody specific for the human
semenogelin polypeptide is greater than a normal control level; and
outputting a report indicating the level of antibody specific for
the human semenogelin polypeptide.
25. The method of claim 30, wherein said biological sample is
serum, blood, or plasma.
26. The method of claim 30, wherein said individual is a human
male.
27. The method of claim 30, wherein said individual has a higher
than normal level of prostate-specific antigen.
28. The method of claim 30, further comprising assaying the level
of prostate specific antigen (PSA) in the biological sample,
wherein the level of PSA provides for diagnosis of prostate
cancer.
29. The method of claim 30, wherein the detectable label is a
radioactive isotope.
30. The method of claim 30, wherein the detectable label is a
fluorescent label.
31. The method of claim 30, wherein the detectable label is an
enzyme or enzyme modulator that generates a product that produces a
detectable signal.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/262,752, filed Dec. 2, 2011, which is a
national stage filing under 35 U.S.C. .sctn.371 of International
Patent Application PCT/US2010/029174, filed Mar. 30, 2010, which
claims benefit, under 35 U.S.C. .sctn.119(e), of U.S. Provisional
Patent Application No. 61/168,506, filed Apr. 10, 2009. The
contents of U.S. patent application Ser. No. 13/262,752,
International Patent Application PCT/US2010/029174, and U.S. Patent
Application No. 61/168,506 are incorporated herein by reference in
their entirety.
BACKGROUND
[0003] Prostatitis is a highly prevalent disease for men. While
acute prostatitis is commonly caused by bacterial infection, most
patients with chronic prostatitis/chronic pelvic pain syndrome
(CPPS) have no evidence of urinary tract infection. Moreover, a
significant proportion of men can have non-infectious chronic
prostatitis in the form of asymptomatic inflammatory prostatitis
that is diagnosed on prostate biopsy performed to evaluate for
prostate cancer. CPPS is in fact the most common non-malignant
diagnosis in patients being evaluated for elevated serum levels of
prostate specific antigen (PSA), a biomarker used to screen for
prostate cancer. The diagnosis of this syndrome currently relies on
reported pain in the perineum, rectum, and/or prostate by affected
men. CPPS often relapses and remits without clear triggers. Therapy
for CPPS is non-specific and usually involves empiric treatment
with antibiotics of unclear efficacy. The etiology of CPPS is
unknown.
[0004] Spontaneous prostatitis has been described in several aged
rat strains and even in the aged nonobese diabetic (NOD) mice, but
the mechanism by which these strains render the host susceptible to
prostatitis is unclear. To date, none of the prostate antigens
identified in these mouse models has been shown to be relevant for
the human disease.
[0005] There is a need in the art for improved methods for
diagnosing CPPS.
LITERATURE
[0006] U.S. Patent Publication No. 2008/0268463; U.S. Patent
Publication No. 2009/0060842.
SUMMARY OF THE INVENTION
[0007] The present disclosure provides prostatitis-associated
antigens, and compositions comprising the antigens. The present
disclosure provides diagnostic methods, generally involving
assaying the level of an immune response specific for a
prostatitis-associated antigen in an individual. Kits suitable for
use in performing such diagnostic assays are also provided. The
present disclosure further provides methods of treating
prostatitis, methods of treating prostate cancer, and methods of
treating benign prostatic hyperplasia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A-1C depict the spontaneous development of autoimmune
prostatitis in the Aire-KO mouse.
[0009] FIGS. 2A-2D depict the presence of autoantibodies to SVS2 in
Aire-KO mice.
[0010] FIGS. 3A and 3B depict the expression profile of SVS2 in
various tissues.
[0011] FIGS. 4A-4D depict spontaneous T cells responses to SVS2 in
Aire-KO mice.
[0012] FIGS. 5A-5C depict induction of SVS2-specific antibody and T
cell response in Aire-sufficient B6 mice.
[0013] FIGS. 6A-6D depicts induction of prostatitis in mice with
SVS2 immunization.
[0014] FIGS. 7A-7C depict autoimmune response to human semenogelin
in prostatitis patients.
[0015] FIG. 8 depicts the presence of prostate-specific
autoantibodies in Aire-deficient male mice.
[0016] FIGS. 9A-9E depict amino acid sequences of human semenogelin
I isoform a (FIG. 9A), human semenogelin I isoform b (FIG. 9B),
human semenogelin II (FIG. 9C), mouse SVS2 (FIG. 9D), and rat SVS2
(FIG. 9E).
[0017] FIG. 10 is a graph depicting survival rates for wild-type
(WT-B6) male mice or Aire.sup.-/- (AIRE-ko) male mice implanted
with Tramp-c2 tumor cells.
[0018] FIG. 11 depicts expression of Sg11 and Sg12 in human BPH and
prostate cancer cells.
DEFINITIONS
[0019] As used herein, a "biological sample" encompasses a variety
of sample types obtained from an individual and can be used in a
diagnostic or monitoring assay. The term "biological sample"
encompasses semen, blood, and other liquid samples of biological
origin, solid tissue samples such as a biopsy specimen or tissue
cultures or cells derived therefrom and the progeny thereof. The
term "biological sample" also includes samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents; washed; or enrichment for certain cell
populations, such as splenocytes, CD4.sup.+ T lymphocytes,
CD8.sup.+ T lymphocytes, macrophages, tumor cells, peripheral blood
mononuclear cells (PBMC), cancer cells, and the like. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, seminal fluid, semen,
tissue samples, organs, bone marrow, blood, plasma, serum, and the
like.
[0020] The terms "polypeptide," "peptide," and "protein," used
interchangeably herein, refer to a polymeric form of amino acids of
any length, which can include coded and non-coded amino acids,
chemically or biochemically modified or derivatized amino acids,
and polypeptides having modified peptide backbones. The term
includes fusion proteins, including, but not limited to, fusion
proteins with a heterologous amino acid sequence, fusions with
heterologous and homologous leader sequences, with or without
N-terminal methionine residues; immunologically tagged proteins;
and the like. NH.sub.2 refers to the free amino group present at
the amino terminus of a polypeptide. COOH refers to the free
carboxyl group present at the carboxyl terminus of a polypeptide.
In keeping with standard polypeptide nomenclature, J. Biol. Chem.,
243 (1969), 3552-59 is used.
[0021] As used herein, the terms "label" and "detectable label"
refer to a molecule capable of detection, including, but not
limited to, radioactive isotopes, fluorescers, chemiluminescers,
chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin,
avidin, streptavidin, haptens, and the like), intercalating dyes,
and the like. The term "fluorescer" refers to a substance or a
portion thereof which is capable of exhibiting fluorescence in the
detectable range.
[0022] As used herein the term "isolated" is meant to describe a
polynucleotide, a polypeptide, or a cell that is in an environment
different from that in which the polynucleotide, the polypeptide,
or the cell naturally occurs. An isolated genetically modified host
cell may be present in a mixed population of genetically modified
host cells. An isolated polypeptide will in some embodiments be
synthetic. "Synthetic polypeptides" are assembled from amino acids,
and are chemically synthesized in vitro, e.g., cell-free chemical
synthesis, using procedures known to those skilled in the art. An
isolated polypeptide will in some embodiments be purified.
[0023] By "purified" is meant a compound of interest (e.g., a
polypeptide) has been separated from components that accompany it
in nature. "Purified" can also be used to refer to a compound of
interest (e.g., a polypeptide) separated from components that can
accompany it during manufacture (e.g., in chemical synthesis). In
some embodiments, a compound (e.g., a polypeptide) is substantially
pure when it is at least 50% to 60%, by weight, free from organic
molecules with which it is naturally associated or with which it is
associated during manufacture. In some embodiments, the preparation
is at least 75%, at least 90%, at least 95%, at least 98%, or at
least 99%, by weight, of the compound of interest. Thus, e.g., a
subject polypeptide that is "purified" is present in a composition
where the polypeptide is present in an amount of at least 75%, at
least 90%, at least 95%, at least 98%, or at least 99%, by weight,
of the composition. A substantially pure polypeptide can be
obtained, for example, by extraction from a natural source, by
recombinant production in a genetically modified host cell, by
chemically synthesis, or by a combination of purification and
chemical modification. A substantially pure compound can also be
obtained by, for example, enriching a sample having a compound that
binds an antibody of interest. Purity can be measured by any
appropriate method, e.g., chromatography, mass spectroscopy, high
performance liquid chromatography analysis, etc.
[0024] An "antigen" is defined herein to include any substance that
may be specifically bound by an antibody molecule or a T cell
receptor. An "immunogen" is an antigen that is capable of
initiating lymphocyte activation resulting in an antigen-specific
immune response.
[0025] By "epitope" is meant a site on an antigen to which specific
B cells and/or T cells respond. The term is also used
interchangeably with "antigenic determinant" or "antigenic
determinant site." B cell epitope sites on proteins,
polysaccharides, or other biopolymers may be composed of moieties
from different parts of the macromolecule that have been brought
together by folding. Epitopes of this kind are referred to as
conformational or discontinuous epitopes, since the site is
composed of segments of the polymer that are discontinuous in the
linear sequence but are continuous in the folded conformation(s).
Epitopes that are composed of single segments of biopolymers or
other molecules are termed continuous or linear epitopes. T cell
epitopes are generally linear peptides. Antibodies that recognize
the same epitope can be identified in a simple immunoassay showing
the ability of one antibody to block the binding of another
antibody to a target antigen.
[0026] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts.
[0027] Monoclonal antibodies included hybrid and recombinant
antibodies (e.g. "humanized" antibodies) regardless of species of
origin or immunoglobulin class or subclass designation, as well as
antibody fragments (e.g., Fab, F(ab').sub.2, and Fv), so long as
they are capable of binding specifically to a target antigen as
described herein. Cabilly, et al., U.S. Pat. No. 4,816,567; Mage
& Lamoyi, in Monoclonal Antibody Production Techniques and
Applications, pp. 79-97 (Marcel Dekker, Inc., New York, 1987).
[0028] Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from such a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies of the invention may be made
using the hybridoma method first described by Kohler &
Milstein, Nature 256:495 (1975), or may be made by recombinant DNA
methods. Cabilly, et al., U.S. Pat. No. 4,816,567.
[0029] The terms "subject," "individual," "host," and "patient" are
used interchangeably herein to refer to a mammal, including, but
not limited to, murines (rats, mice), felines, non-human primates
(e.g., simians), humans, canines, ungulates, etc.
[0030] The terms "treatment," "treating," "treat," and the like are
used herein to generally refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete
stabilization or cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, e.g., a human, and includes:
(a) preventing the disease or symptom from occurring in a subject
which may be predisposed to the disease or symptom but has not yet
been diagnosed as having it; (b) inhibiting the disease symptom,
i.e., arresting its development; or (c) relieving the disease
symptom, i.e., causing regression of the disease or symptom.
[0031] As used herein in the context of patient response to
treatment with an immunomodulatory treatment regimen, the terms
"beneficial response," "beneficial patient response," and
"clinically beneficial response," "clinical benefit," and the like,
are used interchangeably and include partial response (PR),
complete response (CR), and stabilization of disease (SD).
[0032] Beneficial response to treatment with an immunomodulatory
treatment regimen can be assessed according to whether an
individual patient experiences a desirable change in disease
status. Examples of desirable change in disease status in
prostatitis include a decrease in inflammatory response; decrease
in urination frequency; lessening of pain in urination; loss of
pelvic, groin or back pain; and a decrease in the level of
prostate-specific antigen. Continued increase in the
above-mentioned symptoms indicates a lack of beneficial response to
treatment.
[0033] As used herein, in the context of prostatitis, the term
"responder" refers to a patient who has prostatitis, and who
exhibits a beneficial clinical response following treatment with an
immunomodulatory treatment regimen.
[0034] As used herein, in the context of prostatitis, the term
"non-responder" refers to a patient who has prostatitis, and who
has not shown a beneficial response following treatment with an
immunomodulatory treatment regimen.
[0035] As used herein, the term "correlates," or "correlates with,"
and like terms, refers to a statistical association between
instances of two events, where events include numbers, data sets,
and the like. For example, when the events involve numbers, a
positive correlation (also referred to herein as a "direct
correlation") means that as one increases, the other increases as
well. A negative correlation (also referred to herein as an
"inverse correlation") means that as one increases, the other
decreases.
[0036] The term "substantially similar" as used in the context of
nucleic acid or amino acid sequence identity refers to two or more
sequences which have at least about 50%, at least about 60%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
98%, at least about 99%, or 100% sequence identity.
[0037] As used herein "% sequence identity" is determined using the
EMBOSS Pairwise Alignment Algorithms tool available from The
European Bioinformatics Institute (EMBL-EBI), which is part of the
European Molecular Biology Laboratory. This tool is accessible at
the website located by placing "www." in front of
"ebi.ac.uk/Tools/emboss/align/". This tool utilizes the
Needleman-Wunsch global alignment algorithm (Needleman, S. B. and
Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453; Kruskal, J. B.
(1983) An overview of sequence comparison In D. Sankoff and J. B.
Kruskal, (ed.), Time warps, string edits and macromolecules: the
theory and practice of sequence comparison, pp. 1-44 Addison
Wesley. Default settings are utilized which include Gap Open: 10.0
and Gap Extend 0.5. The default matrix "Blosum62" is utilized for
amino acid sequences and the default matrix "DNAfull" is utilized
for nucleic acid sequences.
[0038] As used herein, "autoimmune response" refers to the failure
of an organism to recognize its own constituent parts as self,
which results in an immune response against its own cells and
tissues. In certain cases, autoantibodies are generated as a part
of the autoimmune response against autoantigens. In some cases,
autoreactive immune cells, e.g., autoreactive T cells, are
generated as a part of the autoimmune response against
autoantigens.
[0039] As used herein, "immunological tolerance" refers to the
process by which the immune system does not attack an antigen. In a
healthy individual, there is adequate immunological tolerance for
all the normal self-antigens such that no immune response is
mounted unless there is an invasion of a foreign substance or a
diseased growth in the individual.
[0040] As used herein, "proteins related to SVS2" refers to
proteins that share similarities in gene structure,
three-dimensional structural and functional domains with seminal
vesicle secretory protein 2 (SVS2) in mice. In certain embodiments,
proteins related to SVS2 include mouse SVS2, rat SVS2, human
semenogelin I, human semenogelin II, and their isoforms.
"SVS2-related proteins" may also belong to a group collectively
referred as "rapidly evolving substrates for transglutaminase," as
described in Lundwall, A. et al. "A novel gene family encoding
proteins with highly differing structure because of a rapidly
evolving exon." FEBS Lett. 374 (1995): 53-56.
[0041] The terms "cancer," "neoplasm," and "tumor" are used
interchangeably herein to refer to cells which exhibit relatively
autonomous growth, so that they exhibit an aberrant growth
phenotype characterized by a significant loss of control of cell
proliferation. Cells of interest for treatment in the present
application include precancerous, malignant, pre-metastatic,
metastatic, and non-metastatic cells, as well as carcinoma in
situ.
[0042] "Cancerous phenotype" generally refers to any of a variety
of biological phenomena that are characteristic of a cancerous
cell, which phenomena can vary with the type of cancer. The
cancerous phenotype is generally identified by abnormalities in,
for example, cell growth or proliferation (e.g., uncontrolled
growth or proliferation), regulation of the cell cycle, cell
mobility, cell-cell interaction, or metastasis, etc.
[0043] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0044] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0045] Unless defined otherwise, 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. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0046] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an antibody" includes a plurality of such
antibodies and reference to "the prostatitis-associated antigen"
includes reference to one or more prostatitis-associated antigens
and equivalents thereof known to those skilled in the art, and so
forth. It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0047] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION
[0048] The present disclosure provides prostatitis-associated
antigens (PAA), and compositions comprising the antigens. The
present disclosure provides diagnostic methods, generally involving
assaying the level of an immune response specific for a PAA in an
individual. Kits suitable for use in performing such diagnostic
assays are also provided. The present disclosure further provides
methods of treating prostatitis.
[0049] The present disclosure relates to the observation that male
individuals affected by chronic prostatitis or chronic pelvic pain
syndrome (CPPS) mount an immune response to semenogelin (SG), a
protein related to seminal vesicle secretory protein 2 in mice
(SVS2). SG and SVS2 share similarities in gene structure,
three-dimensional structural and functional domains with other
proteins collectively referred to as "rapidly evolving substrates
for transglutaminase," as described in Lundwall, A. et al. (1995)
FEBS Lett. 374:53-56. Identification of these antigens as targets
of an autoimmune response allows the development of a diagnostic
method for determining the etiology of CPPS or asymptomatic
inflammatory prostatitis. Identification of these proteins as
immunogenic antigens also provides for the development of immune
response modulators, where administration of such a composition to
an individual who has prostatitis reduces the symptoms associated
with prostatitis. Furthermore, identification of these target
antigens allows generation of assays to correctly distinguish
prostatitis over other prostate disorders, such as prostate cancer.
In certain embodiments, the diagnosis developed based on
prostatitis-associated antigen can lessen the need for invasive
biopsy.
Prostatitis-Associated Antigens
[0050] The present disclosure provides prostatitis-associated
antigens (PAA), and compositions comprising the antigens. PAA are
useful in various applications, including diagnostic assays and
treatment methods.
[0051] Target antigens that are the target of an autoimmune
response generated in an individual who has prostatitis, e.g.
chronic prostatitis, are also referred herein as "self-antigens,"
"prostatitis-associated antigens," or "prostatitis-associated
target antigens." An individual that has lost immunologic tolerance
for SG, SVS2, or related proteins may develop autoantibodies or
"prostatitis-associated autoantibodies" in response to such
self-antigens.
[0052] In certain embodiments, PAA are useful in diagnostic assays,
as described in more detail below, to detect an antibody response
in a prostatitis patient who has not yet undergone treatment for
the prostatitis, e.g., to correctly diagnose prostatitis from other
prostate disorders, e.g. prostate cancer, and to determine
likelihood of beneficial clinical response to treatment with an
immunomodulatory treatment regimen. PAA are also useful, as
described below, to detect an antibody response in a prostatitis
patient for prognosis during treatment, e.g., to determine
likelihood of beneficial clinical response to treatment with an
immunomodulatory treatment regimen. PAA are also useful in
immunogenic compositions, to induce an immune response to the
target antigen, e.g., in an animal to model human prostatitis. In
other embodiments, PAA are also useful for generating therapeutic
antibodies, which antibodies are useful for treating
prostatitis.
[0053] PAA are targets of an autoimmune response generated in an
individual who has chronic prostatitis or CPPS and who may exhibit
a clinically beneficial response to treatment with an
immunomodulatory treatment regimen. As used herein "PAA" includes,
but is not limited to, human semenogelin (SG) I isoform a; human
semenogelin I isoform b; human semenogelin II; mouse SVS2; active
fragments of a PAA; a polypeptide comprising an amino acid sequence
that is substantially similar to the amino acid sequence of a human
SG or a rodent SVS2 polypeptide; a fusion polypeptide comprising a
PAA and a heterologous fusion partner; a synthetic PAA; and the
like Amino acid sequences of human SG are available at, e.g.,
GenBank Accession Numbers: NP.sub.--002998 (Semenogelin I),
NP.sub.--002999 (Semenogelin II); and UniProtKB/Swiss-Prot
Accession Numbers: P04279 (SEMG1_Human), and Q02328
(SEMG2_Human).
[0054] A polypeptide comprising an amino acid sequence that is
substantially similar to the amino acid sequence of a human SG or a
rodent SVS2 polypeptide includes a polypeptide comprising an amino
acid sequence having at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, at least about 98%, or at least about 99%, amino acid
sequence identity to a contiguous stretch of from about 25 amino
acids (aa) to about 50 aa, from about 50 aa to about 75 aa, from
about 75 aa to about 100 aa, from about 100 aa to about 150 aa,
from about 150 aa to about 200 aa, from about 200 aa to about 250
aa, from about 250 aa to about 300 aa, from about 300 aa to about
350 aa, from about 350 aa to about 400 aa, from about 400 aa to
about 450 aa, from about 450 aa to about 500 aa, from about 500 aa
to about 550 aa, or from about 550 aa to about 580 aa, of a human
SG (e.g., a human SG comprising SEQ ID NO:1; SEQ ID NO:2; or SEQ ID
NO:3) or a rodent SVS2 polypeptide (e.g., a rodent SVS2 comprising
SEQ ID NO:4; SEQ ID NO:5).
[0055] The present disclosure provides an isolated PAA, antigenic
fragments of a PAA, and variants of a PAA. In some embodiments, a
subject PAA is isolated from a natural source, e.g., is in an
environment other than its naturally-occurring environment. In
other embodiments, a subject PAA is recombinantly made, e.g., in a
genetically modified host cell (e.g., bacteria; yeast; Picchia;
insect cells; and the like), where the genetically modified host
cell is genetically modified with a nucleic acid comprising a
nucleotide sequence encoding the PAA. In certain cases, the PAA is
synthetic, e.g., a subject synthetic PAA is synthesized chemically
in a laboratory (e.g., by cell-free chemical synthesis). The PAA
can be a full-length or one or more fragments of SG, SVS2, or
proteins functionally and structurally similar to SVS2.
[0056] In some embodiments, a subject PAA comprises an amino acid
sequence having at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 98%, or at least about 99%, amino acid sequence
identity to a contiguous stretch of from about 25 amino acids (aa)
to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa
to about 100 aa, from about 100 aa to about 150 aa, from about 150
aa to about 200 aa, from about 200 aa to about 250 aa, from about
250 aa to about 300 aa, from about 300 aa to about 350 aa, from
about 350 aa to about 400 aa, from about 400 aa to about 450 aa, or
from about 450 to about 462 aa, of the amino acid sequence depicted
in FIG. 9A (SEQ ID NO:1).
[0057] In some embodiments, a subject PAA comprises an amino acid
sequence having at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 98%, or at least about 99%, amino acid sequence
identity to a contiguous stretch of from about 25 amino acids (aa)
to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa
to about 100 aa, from about 100 aa to about 150 aa, from about 150
aa to about 200 aa, from about 200 aa to about 250 aa, from about
250 aa to about 300 aa, from about 300 aa to about 350 aa, or from
about 350 aa to about 402 aa, of the amino acid sequence depicted
in FIG. 9B (SEQ ID NO:2).
[0058] In some embodiments, a subject PAA comprises an amino acid
sequence having at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 98%, or at least about 99%, amino acid sequence
identity to a contiguous stretch of from about 25 amino acids (aa)
to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa
to about 100 aa, from about 100 aa to about 150 aa, from about 150
aa to about 200 aa, from about 200 aa to about 250 aa, from about
250 aa to about 300 aa, from about 300 aa to about 350 aa, from
about 350 aa to about 400 aa, from about 400 aa to about 450 aa,
from about 450 to about 500 aa, from about 500 aa to about 550 aa,
or from about 550 aa to about 582 aa, of the amino acid sequence
depicted in FIG. 9C (SEQ ID NO:3).
[0059] In some embodiments, a subject PAA comprises an amino acid
sequence having at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 98%, or at least about 99%, amino acid sequence
identity to a contiguous stretch of from about 25 amino acids (aa)
to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa
to about 100 aa, from about 100 aa to about 150 aa, from about 150
aa to about 200 aa, from about 200 aa to about 250 aa, from about
250 aa to about 300 aa, from about 300 aa to about 350 aa, or from
about 350 aa to about 375 aa, of the amino acid sequence depicted
in FIG. 9D (SEQ ID NO:3).
[0060] In some embodiments, a subject PAA comprises an amino acid
sequence having at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, at least about 98%, or at least about 99%, amino acid sequence
identity to a contiguous stretch of from about 25 amino acids (aa)
to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa
to about 100 aa, from about 100 aa to about 150 aa, from about 150
aa to about 200 aa, from about 200 aa to about 250 aa, from about
250 aa to about 300 aa, from about 300 aa to about 350 aa, from
about 350 aa to about 400 aa, or from about 400 aa to about 414 aa,
of the amino acid sequence depicted in FIG. 9E (SEQ ID NO:5).
[0061] In some embodiments, a subject PAA has a length of from
about 15 aa to about 25 aa, from about 25 aa to about 50 aa, from
about 50 aa to about 100 aa, from about 100 aa to about 150 aa,
from about 150 aa to about 200 aa, from about 200 aa to about 250
aa, from about 250 aa to about 300 aa, from about 300 aa to about
350 aa, from about 350 aa to about 400 aa, from about 400 aa to
about 450 aa, from about 450 aa to about 500 aa, from about 500 aa
to about 550 aa, or from about 550 aa to about 580 aa. Where a
subject PAA is a fusion protein comprising a PAA and a heterologous
fusion partner polypeptide, a subject PAA fusion protein can have a
total length that is equal to the sum of the PAA and the
heterologous fusion partner polypeptide.
[0062] In some embodiments, a subject PAA comprises a detectable
label, e.g., a radioisotope, an enzyme which generates a detectable
product, a fluorescent protein, a chromogenic protein, and the
like. A subject PAA can be conjugated to other moieties, such as
members of specific binding pairs, e.g., biotin (member of
biotin-avidin specific binding pair), an antibody, a lectin, and
the like. A subject PAA can also be bound to (e.g., immobilized
onto) a solid support, including, but not limited to, polystyrene
plates or beads, magnetic beads, test strips, membranes, and the
like.
[0063] In some embodiments, a subject PAA is detectably labeled,
either directly or indirectly. Direct labels include radioisotopes
(e.g., .sup.125I; .sup.35S, and the like); enzymes whose products
generate a signal (e.g., luciferase, .beta.-galactosidase, horse
radish peroxidase, alkaline phosphatase, and the like); fluorescent
labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin,
and the like); fluorescence emitting metals, e.g., .sup.152Eu, or
others of the lanthanide series, attached to the antibody through
metal chelating groups such as EDTA; chemiluminescent compounds,
e.g., luminol, isoluminol, acridinium salts, and the like;
bioluminescent compounds, e.g., luciferin; fluorescent proteins;
and the like. Indirect labels include second antibodies specific
for a subject antibody, wherein the second antibody is labeled as
described above; and members of specific binding pairs, e.g.,
biotin-avidin, and the like.
[0064] In some embodiments, a subject PAA is a fusion protein
comprising a PAA and a fusion partner polypeptide such as a
fluorescent or chromogenic polypeptide, or an enzyme that generates
a product that produces a detectable signal. Suitable enzymes
include, but are not limited to, .beta.-galactosidase, luciferase,
horse radish peroxidase, alkaline phosphatase, etc. Suitable
fluorescent proteins include, but are not limited to, a green
fluorescent protein (GFP), including, but not limited to, a GFP
derived from Aequoria victoria or a derivative thereof, a number of
which are commercially available; a GFP from a species such as
Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as
described in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein
Chem. 20:507-519; a yellow fluorescent protein; a red fluorescent
protein; any of a variety of fluorescent and colored proteins from
Anthozoan species, as described in, e.g., Matz et al. (1999) Nature
Biotechnol. 17:969-973, U.S. Patent Publication No. 2002/0197676,
or U.S. Patent Publication No. 2005/0032085; and the like.
Multimerized PAA
[0065] In some embodiments, a subject PAA is multimerized, e.g.,
two or more PAA polypeptides are linked, e.g., in tandem, or as
cross-linked monomeric units. Multimers include dimers, trimers,
tetramers, pentamers, etc. Monomeric PAA polypeptides are linked to
one another directly or via a linker.
[0066] Thus, in some embodiments, a subject PAA polypeptide has the
formula (X.sub.1-(Y).sub.0-40-X.sub.2-(Y).sub.0-40).sub.n, where
X.sub.1 and X.sub.2 are PAA polypeptides, Y is a linker, and n is
an integer from 1 to about 10 (e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10). Where a linker is used, Y is one or more amino acids, or
other linking groups. X.sub.1 and X.sub.2 can be the same or
different, e.g., can have the same amino acid sequence, or can
differ from one another in amino acid sequence. Thus, e.g., a
subject PAA polypeptide can have the formula
X.sub.1-(Y).sub.0-40-X.sub.2, e.g., where the PAA polypeptide is a
dimer.
[0067] In some embodiments, a subject multimerized PAA is a
homodimer, where two monomeric PAA subunits are cross-linked, e.g.,
via internal amino acid residues such as cysteine residues. In
other embodiments, a subject multimerized PAA is a homotrimer,
where three monomeric PAA subunits are cross-linked, e.g., via
internal amino acid residues such as cysteine residues. In other
embodiments, a subject multimerized PAA is a homotetramer, where
four monomeric PAA subunits are cross-linked, e.g., via internal
amino acid residues such as cysteine residues.
[0068] Linkages for homo- or hetero-polymers or for coupling to
carriers can be provided in a variety of ways. For example,
cysteine residues can be added at both the amino- and
carboxyl-termini, where the peptides are covalently bonded via
controlled oxidation of the cysteine residues. Also useful are a
large number of heterobifunctional agents which generate a
disulfide link at one functional group end and a peptide link at
the other, including N-succidimidyl-3-(2-pyridyldithio) proprionate
(SPDP). This reagent creates a disulfide linkage between itself and
a cysteine residue in one protein and an amide linkage through the
amino on a lysine or other free amino group in the other. A variety
of such disulfide/amide forming agents is known. See, for example,
Immun. Rev. 62:185 (1982). Other bifunctional coupling agents form
a thioether rather than a disulfide linkage. Many of these
thioether forming agents are commercially available and include
reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid,
2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic
acid and the like. The carboxyl groups can be activated by
combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic
acid, sodium salt. An exemplary coupling agent is succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Of course,
it will be understood that linkage should not substantially
interfere with either of the linked groups to function for its
intended use.
Carriers
[0069] In some embodiments, a subject PAA polypeptide is linked to
a carrier. The term "linked," as used herein interchangeably with
the term "coupled," refers to proximately associated, e.g., the PAA
polypeptide and the carrier are in close spatial proximity. In some
embodiments, the linkage is a covalent linkage. In other
embodiments, the linkage is a non-covalent linkage. In some
embodiments, the PAA polypeptide is linked directly to the carrier.
In other embodiments, the PAA polypeptide is linked indirectly,
e.g., via a linker molecule.
[0070] Examples of suitable carriers include large, slowly
metabolized macromolecules such as: proteins; polysaccharides, such
as sepharose, agarose, cellulose, cellulose beads and the like;
polymeric amino acids such as polyglutamic acid, polylysine, and
the like; amino acid copolymers; inactivated virus particles;
inactivated bacterial toxins such as toxoid from diphtheria,
tetanus, cholera, leukotoxin molecules; liposomes; inactivated
bacteria; dendritic cells; and the like. Carriers are described in
further detail below.
[0071] Suitable carriers are well known in the art, and include,
e.g., thyroglobulin, albumins such as human serum albumin, tetanus
toxoid; Diphtheria toxoid; polyamino acids such as
poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;
influenza virus hemagglutinin, influenza virus nucleoprotein;
hepatitis B virus core protein, hepatitis B virus surface antigen;
purified protein derivative (PPD) of tuberculin from Mycobacterium
tuberculosis; inactivated Pseudomonas aeruginosa exotoxin A (toxin
A); Keyhole Limpet Hemocyanin (KLH); filamentous hemagglutinin
(FHA) of Bordetella pertussis; T helper cell (Th) epitopes of
tetanus toxoid (TT) and Bacillus Calmette-Guerin (BCG) cell wall;
recombinant 10 kDa, 19 kDa and 30-32 kDa proteins from M. leprae or
from M. tuberculosis, or any combination of these proteins; and the
like. See, e.g., U.S. Pat. No. 6,447,778 for a discussion of
carriers, and for methods of conjugating peptides to carriers.
[0072] Pseudomonas aeruginosa exotoxin A (toxin A) has been used
effectively as a carrier in conjugate vaccines. Pseudomonas
aeruginosa exotoxin A may be purified from the supernatant of
fermentor-grown cultures of Pseudomonas aeruginosa PA 103. Toxin A
has been classified as a superantigen based upon results in
animals. Toxin A can be completely and irreversibly detoxified by
covalent coupling to adipic acid dihydrazide (ADH), a 4 carbon
spacer molecule. This step destroys the ADPR-transferase activity
of the toxin molecule, hence rendering it nontoxic. The non-reacted
hydrazide group can be used to covalently couple a polypeptide to
toxin A. Toxin A may also be coupled to a polypeptide using a
carbodiimide reagent.
[0073] PPD-peptide conjugates are conveniently prepared with
glutaraldehyde as coupling agent. See, e.g., Rubinstein et al.
(1995) AIDS 9:243-51.
[0074] The methods by which a subject polypeptide is conjugated
with a carrier include disulfide linkages through a C terminal
peptide cysteine linkage, coupling with glutaraldehyde solution for
two hours, coupling with tyrosine, or coupling with water soluble
carbodiimide.
[0075] In some embodiments, a subject PAA polypeptide is lipidated.
Lipidation increases a cytotoxic T cell (CTL) response to the
peptide that is linked to the lipid. The lipid residue, such as
palmitic acid or the like, is attached to the amino terminus of the
peptide. The lipid can be attached directly to the peptide, or,
indirectly via a linkage, such as a Ser-Ser, Gly, Gly-Gly, Ser
linkage or the like. As another example, E. coli lipoprotein, such
as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P.sub.3 CSS), can
be used to prime specific CTL when covalently attached to the
peptide. See, Deres et al., Nature 342:561-564 (1989). A subject
PAA polypeptide can be conjugated with uncharged fatty acid
residues of different chain lengths and degrees of unsaturation,
ranging from acetic to stearic acid as well as to negatively
charged succinyl residues via the appropriate carboxylic acid
anhydrides. See, e.g., U.S. Pat. No. 6,419,931.
[0076] A subject PAA polypeptide may be conjugated directly or
indirectly, e.g., via a linker molecule, to a carrier. A wide
variety of linker molecules are known in the art and can be used in
the conjugates. The linkage from the peptide to the carrier may be
through a peptide reactive side chain, or the N- or C-terminus of
the peptide. A linker may be an organic, inorganic, or semi-organic
molecule, and may be a polymer of an organic molecule, an inorganic
molecule, or a co-polymer comprising both inorganic and organic
molecules.
[0077] If present, the linker molecules are generally of sufficient
length to permit the PAA polypeptide and a linked carrier to allow
some flexible movement between the PAA polypeptide and the carrier.
The linker molecules are generally about 6-50 atoms long. The
linker molecules may also be, for example, aryl acetylene, ethylene
glycol oligomers containing 2-10 monomer units, diamines, diacids,
amino acids, or combinations thereof. Other linker molecules which
can bind to polypeptides may be used in light of this
disclosure.
Compositions
[0078] The present disclosure provides compositions comprising a
subject PAA, which in some embodiments are immunogenic
compositions. Compositions comprising a subject PAA may include a
buffer, which is selected according to the desired use of the PAA,
and may also include other substances appropriate to the intended
use. Those skilled in the art can readily select an appropriate
buffer, a wide variety of which are known in the art, suitable for
an intended use. In some instances, the composition can comprise a
pharmaceutically acceptable excipient, a variety of which are known
in the art and need not be discussed in detail herein.
Pharmaceutically acceptable excipients have been amply described in
a variety of publications, including, for example, "Remington: The
Science and Practice of Pharmacy", 19.sup.th Ed. (1995), or latest
edition, Mack Publishing Co; A. Gennaro (2000) "Remington: The
Science and Practice of Pharmacy", 20th edition, Lippincott,
Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug
Delivery Systems (1999) H. C. Ansel et al., eds 7.sup.th ed.,
Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical
Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer.
Pharmaceutical Assoc.
[0079] A subject PAA composition can comprise, in addition to a
subject PAA, one or more of: a salt, e.g., NaCl, MgCl, KCl,
MgSO.sub.4, etc.; a buffering agent, e.g., a Tris buffer,
N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES),
2-(N-Morpholino)ethanesulfonic acid (MES),
2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),
3-(N-Morpholino)propanesulfonic acid (MOPS),
N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS),
etc.; a solubilizing agent; a detergent, e.g., a non-ionic
detergent such as Tween-20, etc.; a protease inhibitor; glycerol;
and the like.
[0080] Pharmaceutical compositions can be prepared in various
forms, such as granules, tablets, pills, suppositories, capsules,
suspensions, sprays, suppositories, transdermal applications (e.g.,
patches, etc.), salves, lotions and the like. Pharmaceutical grade
organic or inorganic carriers and/or diluents suitable for oral and
topical use can be used to make up compositions containing the
therapeutically active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents.
Immunogenic Compositions
[0081] The present disclosure provides an immunogenic composition
comprising a subject PAA. A subject immunogenic composition is
useful for inducing in an individual an immune response to a
PAA.
[0082] In some embodiments, a subject immunogenic composition
comprises a PAA and an adjuvant. Suitable adjuvants include those
suitable for use in humans. Examples of known suitable adjuvants
that can be used in humans include, but are not necessarily limited
to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v
squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan
trioleate (Span 85)), a CpG-containing nucleic acid (where the
cytosine is unmethylated), QS21 (saponin adjuvant), MPL
(Monophosphoryl Lipid A), 3DMPL (3-O-deacylated MPL), extracts from
Aquilla, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte
Biol. 64:713), LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG)
microparticles, Quil A, interleukins, and the like. For veterinary
applications including but not limited to animal experimentation,
one can use Freund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,
referred to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE), and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion.
[0083] Further exemplary adjuvants to enhance effectiveness of the
composition include, but are not limited to: (1) oil-in-water
emulsion formulations (with or without other specific
immunostimulating agents such as muramyl peptides (see below) or
bacterial cell wall components), such as for example (a) MF59.TM.
(WO90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant
approach, eds. Powell & Newman, Plenum Press 1995), containing
5% Squalene, 0.5% Tween 80 (polyoxyethylene sorbitan mono-oleate),
and 0.5% Span 85 (sorbitan trioleate) (optionally containing
muramyl tri-peptide covalently linked to dipalmitoyl
phosphatidylethanolamine (MTP-PE)) formulated into submicron
particles using a microfluidizer, (b) SAF, containing 10% Squalane,
0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion, and (c) RIBI.TM. adjuvant system
(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components such
as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), e.g., MPL+CWS (DETOX.TM.); (2) saponin
adjuvants, such as QS21 or STIMULON.TM. (Cambridge Bioscience,
Worcester, Mass.) may be used or particles generated therefrom such
as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid
of additional detergent e.g. WO 00/07621; (3) Complete Freund's
Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4)
cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma
interferon), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), other TNF superfamily molecules (e.g.,
CH40L, OX40L, and the like), etc.; (5) monophosphoryl lipid A (MPL)
or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454,
optionally in the substantial absence of alum when used with
pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL
with, for example, QS21 and/or oil-in-water emulsions e.g.
EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides
comprising CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr
Opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3,
849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et
al, J. Immunol, 1998, 160, 870-876; Chu et al., J. Exp. Med, 1997,
186, 1623-1631; Lipford et al, Eur. J. Immunol., 1997, 27,
2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg
et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996,
93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840-1845;
Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al,
Cell Immunol, 1996, 167, 72-78; Yamamoto et al, Jpn. J. Cancer
Res., 1988, 79, 866-873; Stacey et al, J. Immunol., 1996, 157,
2116-2122; Messina et al, J. Immunol, 1991, 147, 1759-1764; Yi et
al, J. Immunol, 1996, 157, 4918-4925; Yi et al, J. Immunol, 1996,
157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761; and Yi
et al, J. Immunol, 1998, 160, 5898-5906; International patent
applications WO96/02555, WO98/16247, WO98/18810, WO98/40100,
WO98/55495, WO98/37919 and WO98/52581) e.g., containing at least
one CG dinucleotide, where the cytosine is unmethylated; (8) a
polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549;
(9) a polyoxyethylene sorbitan ester surfactant in combination with
an octoxynol (WO01/21207) or a polyoxyethylene alkyl ether or ester
surfactant in combination with at least one additional non-ionic
surfactant such as an octoxynol (WO01/21152); (10) a saponin and an
immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)
(WO00/62800); (11) an immunostimulant and a particle of metal salt
e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g.
WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a
sterol) e.g. WO98/57659; (14) other substances that act as
immunostimulating agents to enhance the efficacy of the
composition. Muramyl peptides include
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25
acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl--
sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.
[0084] A subject immunogenic composition can include a conventional
pharmaceutically acceptable excipient, such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, magnesium,
carbonate, and the like. A subject immunogenic composition can
include one or more pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents and
the like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of antigen (e.g., a PAA) in these formulations can
vary widely, and can be selected based on various factors such as
fluid volumes, viscosities, body weight and the like in accordance
with the particular mode of administration selected and the
patient's needs. The resulting compositions may be in the form of a
solution, suspension, tablet, pill, capsule, powder, gel, cream,
lotion, ointment, aerosol or the like.
[0085] The PAA concentration of a subject immunogenic composition
in the pharmaceutical formulations can vary widely, e.g., less than
about 0.1%, from about 0.1% to about 2%, from about 2% to 20%, or
from about 20% to about 50%, or more, by weight, and will be
selected on the basis of various factors such as fluid volumes,
viscosities, etc., in accordance with the particular mode of
administration selected.
Diagnostic Methods
[0086] The present disclosure provides methods of diagnosing
autoimmune-mediated prostatitis (e.g., non-bacterial prostatitis);
methods for monitoring patient response to treatment for chronic
(non-bacterial) prostatitis; methods of distinguishing prostate
cancer from prostatitis; and methods of evaluating the cause of
male sterility. The methods generally involve detecting a level of
antibody to a PAA in a male individual.
Diagnosing Non-Bacterial Prostatitis or CPPS
[0087] In certain embodiments, the present disclosure provides
methods of determining whether a patient has chronic prostatitis or
CPPS caused by an autoimmune response against PAA. A subject
diagnostic method generally involves detecting the level of
PAA-specific antibody and/or PAA-specific T cells in a biological
sample obtained from a male individual.
[0088] In some cases, a subject diagnostic method can entail
determining a baseline value of an immune response in a normal
control, and comparing this with a value for the test immune
response. A significantly high (i.e., greater than the typical
margin of experimental error in repeat measurements of the same
sample, expressed as one standard deviation from the mean of such
measurements) level of the immune response relative to a normal
control signals the presence of an autoimmune response against the
PAA in the sample. If the value for immune response is not
significantly different, this signals the lack of an autoimmune
response against the prostatitis-associated antigen. In other
methods, a normal control value (i.e., a mean and standard
deviation) of immune response is determined for a control
population. Typically the individuals in the control population are
free of chronic (non-bacterial) prostatitis. Measured values of
immune response in a patient may be compared with the normal
control value.
[0089] Thus, for example, a test level of an indicator of an immune
response to a PAA (e.g., a level of PAA-specific antibody; a level
of PAA-specific T cells) in a test individual is compared to a
reference level of the indicator. The term "a reference level"
refers to a level of PAA-specific antibody or a level of
PAA-specific T cell that is present in a subject(s) that is/are
asymptomatic for CP/CPPS. The "reference level" can be an average
level of an indicator of an immune response to PAA (e.g. an average
level of PAA-specific antibody or an average level of PAA-specific
T cell) obtained from data from multiple subjects that are
asymptomatic for CP/CPPS. Alternatively, a test sample can be
compared directly to the level present in a control sample, e.g.
when testing pre-treatment versus post-treatment (see below).
[0090] For example, a normal control level (or reference control
level) of an immune response can be readily determined by
determining the level of antibodies against PAA in a statistically
significant number of individuals who do not have prostatitis or
who do not have an autoimmune response against PAA. In certain
cases, a positive control level is a range of level of autoantibody
generated in an autoimmune response detected in a statistically
significant number of individuals who have chronic prostatitis or
CPPS. Similarly, a normal control (or reference control) level of
PAA-specific T cells can readily be determined by determining the
level of PAA-specific T cells in a statistically significant number
of individuals who do not have CP/CPPS or who do not have an
autoimmune response against PAA.
[0091] Various methods known in the art may be used to determine
the level of an immune response, e.g., to determine the level of an
antigen-specific antibody, to determine the level of
antigen-specific T cells, etc. A biological sample obtained from a
patient is analyzed. The biological sample for analysis can be
seminal fluid, semen, blood, plasma, serum, or tissue biopsy sample
(e.g., prostate biopsy sample). The sample is analyzed for
indication of an immune response to a PAA. The immune response can
be determined from the presence of, e.g., antibodies or T-cells
that specifically bind to the PAA. The level of immune response may
also be a measurement of inflammation, prostatitis symptoms,
prostate specific antigens, antibodies against PAA, and anti-PAA
T-cell response, e.g. CD4.sup.+ response, CD8.sup.+ response.
[0092] Where T cell responses are of interest, the sample is a
sample comprising lymphocytes, e.g. seminal fluid, a blood sample,
the cellular portion of a blood sample, etc. T cells can be stained
with a peptide/MHC complex, for example using detectably labeled
MHC reagents (i.cndot.TAg.TM. MHC Tetramers, Beckman Coulter;
BD.TM. DimerX reagents; ProImmune Pro5.RTM. MHC class I Pentamers
etc.) to determine the presence of T cells having specificity for a
PAA. Alternatively, T cells may be assayed in vitro for reactivity
to a PAA, using methods known in the art. For example, a sample
comprising T cells may be contacted with a PAA presented by an
antigen presenting cell (APC); or provided as a stable MHC complex;
and the response of the cells quantitated, for example by
proliferation, cytokine synthesis, cytotoxicity and the like.
Measured values may thus include quantitation of PAA-specific T
cells, quantitation of T cell proliferation in response to a PAA,
quantitation of cytokine release, e.g. IFN-.gamma., IL-2, etc. in
response to presented PAA, percentage of specific cell lysis of an
APC presenting a PAA, and the like.
[0093] In some embodiments, diagnostic methods involve detecting
the number of PAA-specific CD4.sup.+ T cells in a biological sample
obtained from an individual. The number of PAA-specific CD4.sup.+ T
cells can be determined using, e.g., a .sup.51Cr release assay,
where target cells pulsed with a PAA and labeled with .sup.51Cr are
contacted with a test sample that may contain PAA-specific
CD4.sup.+ T cells. The number of PAA-specific CD4.sup.+ T cells is
determined by measuring release of .sup.51Cr from the target
cells.
[0094] In certain embodiments, the level of an autoantibody,
present in a biological sample from an individual who has
prostatitis or from a test individual suspected of having
prostatitis, to a PAA, is a measured value of an autoimmune
response. If the level of PAA-specific autoantibody is
substantially higher than a control level, this indicates that the
prostatitis is caused by an autoimmune response against the PAA and
also predicts an increased likelihood that the individual may
exhibit a clinically beneficial response to treatment with an
immunomodulatory treatment regimen. Autoantibodies to be detected
that are specific for a PAA can include antibodies of any isotype;
single-chain Fv; Fab; Fab; Fv; F(ab').sub.2; and the like. A
biological sample obtained from an individual is contacted with
PAA. If a significantly high level of autoantibodies from an
individual form a complex with the PAA, it is an indication that an
autoimmune response has been mounted in the individual and
prostatitis may be diagnosed.
[0095] In other embodiments, an antibody profile is detected in a
biological sample (e.g., seminal fluid, blood, or a blood product
such as serum, plasma, etc.), and the antibody profile either
correlates directly with or is inversely correlated with, the
severity of the symptoms associated with prostatitis. It may also
predict the level of a clinically beneficial response to treatment
with an immunomodulatory treatment regimen.
[0096] In some embodiments, the level of autoantibody to two or
more PAA is determined. For example, in some embodiments, the level
of autoantibody to different isoforms or fragments of SG is
determined. Where the level of autoantibody to two or more PAA is
determined, the two or more antibody levels are collectively
referred to as an "antibody profile." Antibody profile of an
individual who has prostatitis may be compared to the control
antibody profile, e.g., profile obtained from an individual who
does not have prostatitis in order to determine whether there is an
autoimmune response that caused the prostatitis. The antibody
profile may also help determine whether an immunomodulatory
treatment may be beneficial to the individual with prostatitis.
[0097] In some embodiments, the level of an autoantibody is
expressed as a "normalized level." For example, either Quantile
Normalization or Robust Linear Normalization can be utilized to
obtain a "normalized level". In one embodiment, Quantile
Normalization is utilized to obtain a normalized response level. In
the context of protein arrays, this method forces the arrays to
have identical intensity distribution to allow comparison between
arrays that may have systematic measurement errors.
[0098] In another embodiment, Robust Linear Normalization is
utilized to obtain a normalized response level. This method uses a
statistical linear model and positive control proteins, e.g., IgG
and V5 to fit the model and also removes systematic measurement
errors.
[0099] The level of an antibody to a PAA can be determined using
any of a number of immunological assays. For example, a detectably
labeled PAA, or a panel of detectably labeled PAA, can be employed,
where the level of signal produced in such an assay is proportional
to the amount of antibody in a biological sample. Suitable assays
include, e.g., enzyme-linked immunosorbent assays (ELISA),
radioimmunoassay (RIA), western blot, surface plasmon resonance,
and the like.
[0100] In some embodiments, the assay is a sandwich assay, in which
an antibody specific for the Fc portion of human antibody is
immobilized on an insoluble support; a biological sample from a
test individual (e.g., an individual having prostatitis; an
individual having prostatitis who has been treated with an
immunomodulatory treatment regimen; an individual suspected of
having prostatitis) is contacted with the immobilized antibody,
forming a complex between antibodies present in the biological
sample and the immobilized antibody; and the complex is contacted
with a detectably labeled PAS. The level of signal produced by the
detectably labeled PAA indicates the level of antibody in the
biological sample that is specific for a PAA. Suitable insoluble
support materials include, e.g., agarose, sepharose,
nitrocellulose, silica, polystyrene, and the like. The insoluble
support can be in any of a variety of forms, including, e.g.,
beads, magnetic beads, films, strips, chips, multi-well plates, and
the like.
[0101] In some embodiments, the outcome of a subject diagnostic
method (e.g., whether an individual is considered to have CP/CPPS)
is provided in a report. Thus, in some embodiments, a subject
method further includes a step of preparing or generating a report
that includes information regarding the results of the determining
(e.g., assaying) step. For example, a subject method can further
include a step of generating or outputting a report providing the
results of the level of anti-PAA antibodies in the individual
and/or the number of anti-PAA T cells in the individual, which
report can be provided in the form of an electronic medium (e.g.,
an electronic display on a computer monitor), or in the form of a
tangible medium (e.g., a report printed on paper or other tangible
medium).
Monitoring Patient Response to Treatment for Chronic
Prostatitis
[0102] The present disclosure provides methods of monitoring
patient response to a treatment for CP/CPPS. The methods generally
involve assaying the level of an immune response in an individual
to a PAA at a first time point and at at least a second time, where
the second time point is later than the first time point, and where
the difference (if any) in the level of immune response to a PAA
between the first time point and the second time point provides an
indication as to whether the individual is responding to the
treatment for CP/CPPS.
[0103] The first time point can be before treatment begins; and the
at least second time point can be a time point after the beginning
of treatment. For example, the first time point can be from one
month to two weeks, from about two weeks to about one week, from
about one week to about one day, from about one day to about 12
hours, from about 12 hours to about 1 hour, or less than one hour
(e.g., 5 minutes to 15 minutes, 15 minutes to 30 minutes, or 30
minutes to about 60 minutes) before treatment for CP/CPPS begins.
The second time point can be from about 12 hours to about 1 day,
from about 1 day to about 1 week, from about 1 week to about 1
month, or more than 1 month, after treatment begins. The level of
an immune response to a PAA can be assayed at additional time
points, e.g., third, fourth, fifth, and further time points. The
level of an immune response to a PAA can be assayed at
substantially regular time intervals, e.g., once per week, one
every two weeks, once a month, once every three months, etc.,
following the beginning of treatment for CP/CPPS.
[0104] In the context of disease prognosis, the level of autoimmune
response against a PAA present in a biological sample from an
individual who has prostatitis and who has undergone treatment with
an immunomodulatory treatment regimen may be substantially lower
than the level of the immune response in an individual before
treatment with the immunomodulatory treatment regimen.
[0105] In other methods, a control value of immune response (e.g.,
a mean and standard deviation) is determined from a control
population of individuals who have undergone treatment with a
therapeutic agent. Measured values of immune response in a patient
are compared with the control value. If the measured level in a
patient is not significantly different (e.g., more than one
standard deviation) from the control value, treatment can be
discontinued. If the level in a patient is significantly above the
control value, continued administration of the treatment regimen is
warranted. If the level in the patient persists above the control
value, then a change in treatment regimen can be indicated.
[0106] In other methods, a patient who is not presently receiving
treatment but has undergone a previous course of treatment is
monitored for immune response to determine whether a resumption of
treatment is required. The measured value of immune response in the
patient can be compared with a value of immune response previously
achieved in the patient after a previous course of treatment. A
significant decrease relative to the previous measurement (i.e.,
greater than a typical margin of error in repeat measurements of
the same sample) is an indication that treatment is effective.
Alternatively, the value measured in a patient can be compared with
a control value (mean plus standard deviation) determined in a
population of patients after undergoing a course of treatment.
Alternatively, the measured value in a patient can be compared with
a control value in populations of prophylactically treated patients
who remain free of symptoms of disease, or populations of
therapeutically treated patients who show amelioration of disease
characteristics. In all of these cases, a significant increase
relative to the control level (i.e., more than a standard
deviation) is an indicator that treatment should be resumed in a
patient.
Differential Diagnosis
[0107] The present disclosure further provides a method for
differential diagnosis to distinguish between chronic
(non-bacterial) prostatitis and prostate cancer (or benign prostate
hyperplasia; BPH). A subject method of differential diagnosis
generally involves assaying a level of an immune response to a PAA
in an individual. In some embodiments, a subject method further
involves assaying a level of prostate-specific antigen (PSA) in a
biological sample (e.g., serum, plasma, blood, etc.) obtained from
the individual. Where the level of PSA is higher than normal, and
the level of anti-PAA immune response is normal, at least a
provisional diagnosis of prostate cancer or BPH can be made, and
additional test(s) for prostate cancer and/or BPH may be ordered.
Where the level of PSA is higher than normal, and the level of
anti-PAA immune response is higher than normal, a diagnosis of
CP/CPPS can be made. Where the level of PSA is higher than normal,
the level of anti-PAA immune response is higher than normal, and a
diagnosis of CP/CPPS is made, more invasive diagnostic methods such
as biopsy can be avoided. Where the level of PSA is higher than
normal, the level of anti-PAA immune response is higher than
normal, and a diagnosis of CP/CPPS is made, treatment for CP/CPPS
may be indicated.
[0108] Levels of PSA less than 4 ng/mL are considered normal;
levels of PSA between 4 ng/mL and 10 ng/mL are associated with a
20-30% risk of prostate cancer; levels of PSA between 10 ng/mL and
20 ng/mL are associated with a 50-75% risk of prostate cancer; and
levels of PSA greater than 20 ng/mL are associated with a 90% risk
of prostate cancer.
[0109] Methods of determining the level of PSA in a biological
sample (e.g., blood; serum; plasma; etc.) are well established in
the art. Any known method can be used in conjunction with a subject
method of differential diagnosis. Typically, the assay is an
immunological assay employing antibody specific for PSA. See, e.g.,
U.S. Pat. No. 7,045,605; U.S. Pat. No. 6,107,049; U.S. Pat. No.
6,361,955.
[0110] In some embodiments, the outcome of a subject differential
diagnostic method (e.g., whether an individual is considered to
have CP/CPPS or whether an individual is considered to have
prostate cancer or BPH) is provided in a report. Thus, in some
embodiments, a subject method further includes a step of preparing
or generating a report that includes information regarding the
results of the determining (e.g., assaying) step. For example, a
subject method can further include a step of generating or
outputting a report providing the results of the level of anti-PAA
antibodies in the individual and/or the number of anti-PAA T cells
in the individual and/or the level of PSA in the individual, which
report can be provided in the form of an electronic medium (e.g.,
an electronic display on a computer monitor), or in the form of a
tangible medium (e.g., a report printed on paper or other tangible
medium).
Evaluating Basis of Male Sterility
[0111] The present disclosure further provides a method for
evaluating the basis for male sterility in a male. The method
generally involves assaying a level of an immune response to a PAA
in a male individual. Where the results of the assay indicate a
higher than normal (or reference) level of immune response to PAA,
the cause of male sterility can be attributed to CP/CPPS. Treatment
for CP/CPPS can then be prescribed.
Assessing Likelihood of Response to Treatment
[0112] The present disclosure provides methods of assessing the
likelihood that a patient having prostatitis will exhibit a
beneficial response to treatment with an immunomodulatory treatment
regimen. Patients subject to such an assessment include: patients
who have symptoms of chronic prostatits, CPPS, non-infectious
chronic prostatitis, or who have an elevated levels of PSA.
Patients may also include those who have been treated with an
immunomodulatory treatment regimen or who may be subjected to an
immunomodulatory treatment.
[0113] For example, the level in an individual who has prostatitis
(and who has not yet been treated for the prostatitis) of an
antibody against one or more PAA that is at least about 25%, at
least about 50%, at least about 75%, at least about 100% (or
two-fold), at least about 2.5-fold, at least about 5-fold, at least
about 10-fold, at least about 25-fold, at least about 50-fold, or
at least about 100-fold higher than a normal control level of
antibody (e.g., in an individual without prostatitis or without an
autoimmune response), indicates that the cause of prostatitis may
be due to an autoimmune response against semenogelin proteins. It
also indicates an increased likelihood that the individual will
exhibit a clinically beneficial response to treatment with an
immunomodulatory treatment regimen.
[0114] For example, the level in an individual who has prostatitis
(and who has not yet been treated for the prostatitis) of an
antibody against one or more target antigens that is at least about
25%, at least about 50%, at least about 75%, at least about 100%
(or two-fold), at least about 2.5-fold, at least about 5-fold, at
least about 10-fold, at least about 25-fold, at least about
50-fold, or at least about 100-fold higher than a normal control
level of antibody may predict an at least about 10%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, or greater than
80%, increased likelihood that the individual will exhibit a
clinically beneficial response to treatment with an
immunomodulatory treatment regimen.
Diagnostic Kits
[0115] The present disclosure further provides kits for use in any
of the above-mentioned diagnostic methods. Such kits typically
comprise two or more components necessary for performing a
diagnostic assay for the detection of immune responses specific to
a prostatitis-associated target antigen. Components can be
compounds, reagents, containers and/or equipment. Kits also
typically contain labeling providing directions for use of the kit.
For example, one container within a kit can contain one or more
fragments or a full-length prostatitis-associated target antigen.
Alternatively, the target antigens may be provided attached to a
support material. One or more additional containers can enclose
elements, such as reagents or buffers, to be used in the assay.
Such kits can also, or alternatively, contain a detection reagent
that contains a reporter group suitable for direct or indirect
detection of antibody binding. For example, secondary antibodies to
recognize antibodies, e.g. human IgG, may be provided. The term
labeling refers to any written or recorded material that is
attached to, or otherwise accompanies a kit at any time during its
manufacture, transport, sale or use. For example, the term labeling
encompasses advertising leaflets and brochures, packaging
materials, instructions, audio or video cassettes, computer discs,
as well as writing imprinted directly on kits.
[0116] In certain cases, a subject kit comprises a subject target
antigen immobilized on a solid support and a labeled reagent
capable of binding to an antibody specific for the subject target
antigen.
PAA-Specific Antibodies
[0117] The present disclosure provides antibodies specific for a
subject PAA. In certain embodiments, a subject PAA-specific
antibody is isolated, e.g., is in an environment other than its
naturally-occurring environment. Suitable antibodies specific for a
PAA include antibodies of any isotype; single-chain Fv; Fab; Fab;
Fv; F(ab').sub.2; artificial antibodies; humanized antibodies; and
the like. In some embodiments, a subject antibody is specific for a
mutant prostate-associated target antigen.
[0118] Suitable antibodies are obtained by immunizing a host animal
with peptides comprising all or a portion of a subject prostate
cancer-associated target antigen. Suitable host animals include
mouse, rat sheep, goat, hamster, rabbit, etc. The host animal will
generally be from a different species than the immunogen where the
immunogen is from a naturally occurring source, e.g., a human
sample, where representative host animals include, but are not
limited to, e.g., rabbits, goats, mice, etc.
[0119] In certain embodiments, the antibodies specific for target
antigens may be useful in determining the essential epitopes on the
target antigens. Antibody profiles to different fragments of SG or
other SVS2-related proteins may help pinpoint the portion of the SG
or SVS2-related protein that is immunogenic. The immunogenic
portion of the SG or SVS2-related protein may also contain an
epitope for autoantibodies developed in an individual with an
autoimmune response. If the autoantibody-specific epitopes of SG or
SVS2-related proteins are known, the immunogenic composition and
the method of the present disclosure may employ such immunogenic
fragments containing such epitopes instead of a larger or
full-length SG.
[0120] A subject anti-PAA antibody will in some embodiments
comprise an anti-cancer agent for delivery to a site of a cancer
cell to further facilitate tumor killing or clearance. The
anti-cancer agent can be attached covalently or non-covalently,
directly or via a linker, to the anti-PAA antibody. Suitable
anti-cancer agents that can be attached to a subject anti-PAA
antibody include, but are not limited to, an anti-proliferation
moiety (e.g., vascular endothelial growth factor (VEGF)
antagonist); a toxin (e.g., an anti-cancer toxin, e.g., ricin,
Pseudomonas exotoxin A, and the like); a radionuclide (e.g.
.sup.90Y, .sup.131I, .sup.177L, .sup.10B and the like), anti-cancer
drugs (e.g. doxorubicin, 5-fluorouracil, leucovorin, a taxane
(e.g., docetaxel, paclitaxel), mitozantrone, vinblastine,
estramustine phosphate, etoposide, etc.), and/or can optionally be
modified to provide for improved pharmacokinetic profile (e.g., by
PEGylation (e.g., attachment of one or more poly(ethylene glycol)
moieties), hyperglycosylation, and the like). A subject anti-PAA
antibody can comprise, attached thereto, a ribosome inactivating
protein (RIP), a photo-activatable cytotoxin, a vinca alkaloid, an
anthracycline, an invertebrate toxin, or a bacterial toxin.
Methods of Treating Prostatitis
[0121] The present disclosure provides methods of treating CP/CPPS
in an individual having CP/CPPS, the methods generally involving
administering to the individual an agent in an amount effective to
decrease the level of an anti-PAA immune response.
[0122] In some embodiments, an effective amount of an agent is an
amount that is effective, when administered in one or more doses,
to reduce the level of autoantibodies against PAA and/or the amount
of T cell response against PAA in an individual by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, or more than 80%, compared to the
level of anti-PAA antibody or PAA-specific T cells in the
individual not treated with the agent.
[0123] A subject treatment regimen includes an "immunomodulatory
treatment regimen," involving administration to an individual of an
effective amount of an agent that reduces an autoimmune response to
a PAA in an individual Immunomodulatory treatment regimens for the
treatment of prostatitis include any treatment that modulates an
immune response in the individual such that an autoimmune response
to a PAA is reduced.
[0124] In some embodiments, immunomodulatory agents include
inhibitors of T cells, such as small molecule inhibitors,
oligonucleotide inhibitors, or peptide inhibitors. The agent may be
immunosuppressants directed against T cells. In certain cases, the
immunosuppressant may be directed specifically against CD4.sup.+ T
cells. In certain embodiments, the immunosuppressive effect may be
initiated by inhibiting cyclin-dependent kinases or Src kinases.
See Singh R P et al. (2008) Inflamm Allergy Drug Targets 7:253-9,
Lopez-Diego (2008) Nat Rev Drug Discov. 7: 909-25, and Javeed et
al. (2008) Mol Diagn Ther. 12: 171-81 for more details on
immunomodulatory agents.
[0125] In some embodiments, "an immunomodulatory treatment regimen"
includes treatment with two or more agents, one or more of which is
an immunomodulatory agent.
[0126] In some embodiments, an immunomodulatory treatment regimen
comprises administration of an agent that increases the activity
and/or level of PAA-specific Tregs in an individual. For example,
in some embodiments, an agent is administered that increases the
level of PAA-specific CD4.sup.+Foxp3.sup.+ regulatory T cells in an
individual.
[0127] In some embodiments, an effective amount of an agent is an
amount that, when administered in one or more doses, results in an
at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, or more than 90%, reduction in
the level of anti-PAA immune response in the individual before
treatment with the agent.
[0128] In some embodiments, an effective amount of an agent is an
amount that, when administered in one or more doses, results in an
at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, or more than 90%, reduction in
the severity of one or more symptoms of CP/CPPS in the individual,
compared to the severity of the one or more symptoms in the absence
of treatment with the agent.
Methods of Treating Prostate Cancer and/or Benign Prostatic
Hyperplasia
[0129] The present disclosure provides methods of treating prostate
cancer, and methods of treating benign prostatic hyperplasia (BPH).
The methods generally involve administering to an individual in
need thereof an effective amount of a PAA. In some embodiments, the
methods involve administering to an individual in need thereof an
effective amount of a subject immunogenic composition. In some
embodiments, the methods involve administering to an individual in
need thereof an effective amount of a subject PAA-specific
antibody, e.g., a subject PAA-specific antibody that comprises an
anti-cancer agent attached thereto.
[0130] Whether a subject method is effective to treat prostate
cancer can be determined using any known method, e.g., measuring
levels of PSA in a biological sample from the individual; analyzing
a biopsy sample from a prostate tumor to determine the number of
cancer cells; measuring the size of the prostate tumor; carrying
out an imaging method (e.g., ultrasound, computed tomography,
magnetic resonance imaging) to assess the size of the tumor;
etc.
Administering a PAA
[0131] In some embodiments, an effective amount of a PAA (or an
immunogenic composition comprising a PAA) is an amount that induces
an immune response to a PAA in the individual, where the immune
response is effective to achieve one or more of: reducing the
growth rate of prostate cancer in an individual, reducing the size
of a prostate tumor in an individual, and reducing the number of
prostate cancer cells in the individual. In some embodiments, an
effective amount of a PAA (or an immunogenic composition comprising
a PAA) is an amount that is effective to induce a T cell response
to a prostate cancer cell, e.g., to induce a T cell response to a
cell surface antigen present on a prostate cancer cell. T cell
responses include cytotoxic T cell responses (e.g., CD8.sup.+ T
cell responses), CD4.sup.+ T cell responses, etc.
[0132] For example, in some embodiments, an effective amount of a
PAA (or an immunogenic composition comprising a PAA) is an amount
that is effective, when administered in one or more doses, to
reduce the number of prostate cancer cells in an individual by at
least about 10%, at least about 20%, at least about 25%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, or more than 80%,
compared to the number of prostate cancer cells in the untreated
individual.
[0133] In some embodiments, a PAA, or an immunogenic composition
comprising a PAA, is administered as a single dose. In other
embodiments, a PAA, or an immunogenic composition comprising a PAA,
is administered in multiple doses, e.g., a first dose, followed by
one or more booster doses.
[0134] A PAA, or an immunogenic composition comprising a PAA, can
be administered via various enteral and parenteral routes of
administration, where parenteral routes of administration include
intramuscular, intravenous, subcutaneous, and the like, and where
enteral routes of administration include oral, buccal, rectal, etc.
A PAA can be administered as a bolus or by continuous infusion over
a period of time, by intramuscular, subcutaneous, intravenous,
oral, intratumoral, peritumoral, intralesional, or perilesional
routes, to exert local as well as systemic therapeutic effects.
[0135] A PAA, or a subject immunogenic composition comprising a
PAA, is administered to an individual in one or more doses.
Suitable amounts of a PAA per dose range from about 100 .mu.g to
about 100 mg, e.g., from about 100 .mu.g to about 250 .mu.g, from
about 250 .mu.g to about 500 .mu.g, from about 500 .mu.g to about 1
mg, from about 1 mg to about 10 mg, from about 10 mg to about 25
mg, from about 25 mg to about 50 mg, from about 50 mg to about 75
mg, or from about 75 mg to about 100 mg.
[0136] Suitable dosage forms encompass pharmaceutically acceptable
carriers that are inherently nontoxic and nontherapeutic. Examples
of such carriers include ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride-mixtures of saturated
vegetable fatty acids, water, salts, or electrolytes such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and
polyethylene glycol. Other carriers include polysaccharides such as
sodium carboxymethylcellulose or methylcellulose,
polyvinylpyrrolidone, polyacrylates,
polyoxyethylene-polyoxypropylene-block polymers, polyethylene
glycol, and wood wax alcohols. Conventional depot forms are
suitably used. Such forms include, for example, microcapsules,
nano-capsules, liposomes, plasters, tablet, capsules, etc.
Administering a PAA-Specific Antibody
[0137] In some embodiments, the methods involve administering to an
individual in need thereof an effective amount of a subject
PAA-specific antibody. The PAA-specific antibody is administered in
one or more doses, as described above for administration of PAA. In
some embodiments, a subject method involves administering a subject
PAA-specific antibody that comprises an anti-cancer agent attached
thereto. A PAA-specific antibody can be administered in a
composition together with one or more pharmaceutically acceptable
carriers, as described above for administration of PAA.
[0138] In some embodiments, an effective amount of an anti-PAA
antibody is an amount that is effective, when administered in one
or more doses, to reduce the number of prostate cancer cells in an
individual by at least about 10%, at least about 20%, at least
about 25%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, or
more than 80%, compared to the number of prostate cancer cells in
the untreated individual.
Combination Therapy
[0139] In some embodiments, a subject method of treating prostate
cancer or BPH involves combination therapy, e.g., administering an
effective amount of a PAA (or an effective amount of a subject
immunogenic composition comprising a PAA) and administering at
least one additional therapy to treat the prostate cancer or BPH,
where suitable additional therapies include, e.g., surgical
treatment, radiation therapy, hormone therapy, and
chemotherapy.
[0140] Surgical treatments that can be carried out in conjunction
with a subject combination therapy include, but are not limited to,
prostatectomy and orchiectomy. Radiation therapy that can be
carried out in conjunction with a subject combination therapy
includes, but is not limited to, external beam radiation therapy,
intensity-modulated radiation therapy, and brachytherapy. Hormone
therapy that can be carried out in conjunction with a subject
combination therapy includes, but is not limited to,
androgen-deprivation therapy; administration of a leuteinizing
hormone releasing hormone (LHRH) agonist (e.g., leupropride,
goserelin, triptorelin); administration of an anti-androgen (e.g.,
bicalutamide, flutamide, nilutamide); and the like. Chemotherapy
that can be carried out in conjunction with a subject combination
therapy includes, but is not limited to, administration of one or
more of a taxane (e.g., docetaxel, paclitaxel), mitozantrone,
doxorubicin, vinblastine, estramustine phosphate, and
etoposide.
Subjects Suitable for Diagnosis and/or Treatment
[0141] Individuals who are to be diagnosed using a subject
diagnostic method include male individuals (e.g., mammals,
including humans). Such individuals include males who are suspected
of having CP/CPPS (e.g., non-bacterial prostatitis). Such
individuals also include individuals who have undergone a test for
PSA levels, and who have exhibited higher than normal levels of
PSA, where such individuals include males who have serum PSA levels
higher than 4 ng/mL, e.g., from about 4 ng/mL to about 10 ng/mL,
from about 10 ng/mL to about 20 ng/mL, or greater than 20 ng/mL.
Individuals who are to be diagnosed using a subject diagnostic
method include males from 10 years to 12 years, from 13 years to 18
years, from 18 years to 25 years, from 25 years to 30 years, from
30 years to 40 years, from 40 years to 50 years, from 50 years to
60 years, or from 60 years to 70 years, in age, or older than 70
years.
[0142] Individuals who are to be diagnosed using a subject
differential diagnostic assay include male individuals who have had
a rectal digital examination and who are considered to possibly
have prostate cancer; male individuals who have undergone a test
for PSA levels, and who have exhibited higher than normal levels of
PSA, where such individuals include males who have serum PSA levels
higher than 4 ng/mL, e.g., from about 4 ng/mL to about 10 ng/mL,
from about 10 ng/mL to about 20 ng/mL, or greater than 20 ng/mL.
Individuals who are to be diagnosed using a subject differential
diagnostic method include males from 10 years to 12 years, from 13
years to 18 years, from 18 years to 25 years, from 25 years to 30
years, from 30 years to 40 years, from 40 years to 50 years, from
50 years to 60 years, or from 60 years to 70 years, in age, or
older than 70 years.
[0143] Individuals who are suitable for treatment with a subject
method of treating prostatitis include male individuals who have a
higher than normal level of anti-PAA antibody and/or T cell
response, e.g., who have a level of anti-PAA antibody and/or a
level of anti-PAA T cells that is at least about 10%, at least
about 15%, at least about 20%, at least about 25%, at least about
30%, at least about 40%, at least about 50%, at least about 75%, at
least about 100% (or 2-fold), at least about 2.5-fold, at least
about 3-fold, at least about 4-fold, at least about 5-fold, at
least about 10-fold, at least about 25-fold, or at least about
50-fold, or more than 50-fold, higher than a normal control level
of anti-PAA antibody and/or anti-PAA T cells. Individuals, who
exhibit higher than normal levels of anti-PAA antibody and/or
higher than normal levels of anti-PAA T cells, that are suitable
for treatment with a subject method include males from 10 years to
12 years, from 13 years to 18 years, from 18 years to 25 years,
from 25 years to 30 years, from 30 years to 40 years, from 40 years
to 50 years, from 50 years to 60 years, or from 60 years to 70
years, in age, or older than 70 years.
[0144] Individuals who are suitable for treatment with a subject
method of treating prostate cancer or BPH include male individuals
(e.g., human males) who have been diagnosed with prostate cancer or
BPH; male individuals (e.g., human males) who are considered at
greater risk of developing prostate cancer or BPH compared to
normal control individuals or compared to the general male
population; male individuals (e.g., human males) who have been
treated for prostate cancer and who failed to respond to such
treatment; and male individuals (e.g., human males) who have been
treated for prostate cancer, who responded to the treatment, but in
whom the prostate cancer recurred.
EXAMPLES
[0145] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
Role of Prostate Antigen-Specific Immune Response in
Prostatitis
Materials and Methods
Mice and Reagents
[0146] Aire-KO mice were generated as previously described in
Penna, G. et al. (2007) Eur Urol 51:524-533. Aire-KO male mice used
in these experiments were backcrossed into the C57BL/6 (B6) and NOD
Lt/J backgrounds >10 generations and generated using
heterozygous breeders. WT B6 mice used in the experiments were
littermates of the Aire-KO mice. C57BL/6.times.NOD Lta F2
Aire-deficient mice were derived from intercrosses of C57BL/6 and
NOD Lt/J Aire-KO mice. All mice were housed in a pathogen-free
barrier facility at the UCSF. Experiments complied with the Animal
Welfare Act and NIH guidelines for the ethical care and use of
animals in biomedical research and were approved by the UCSF Animal
Care and Use Committee. Tissue culture experiments were performed
in RPMI 1640 media (Cambrex, Walkersville, N.J.) supplemented with
10% fetal calf serum (Cambrex, Walkersville, N.J.),
penicillin/streptomycin (Sigma, St. Louis, Mo.) and non-essential
amino acids (Cambrex, Walkersville, N.J.).
Antigens
[0147] The natural mouse SVS2 and human SG were purified from mouse
and human semen plasma, respectively, based on the method described
previously (Malm, J. et al., (1996) Eur J Biochem 238:48-53). In
brief, fresh seminal plasma were collected in a 40 mmol/1 Tris/HCL
buffer pH9.7 with 4M urea, 25 mmol/1 EDTA, 30 mmol/l DTT, 3 mmol/l
benzamidine and 0.5 mmol/1 Pefabloc (Boehringer, Ridgefield, Conn.)
at 4.degree. C., and was applied on a heparin-sepharose column
(0.7.times.2.5 cm, GE Healthcare, Pittsburgh, Pa.) that was
equilibrated with 0.1 mol/l Tris pH8.5, 2M urea, 5 mmol/1 EDTA, 3
mmol/l DTT, 3 mmol/l benzamidine and 0.2 mmol/1 Pefabloc buffer.
Following application of the sample, the column was washed with
5.times.2 ml of equilibration buffer then 4 ml of 0.1M NaCl. The
column was eluted with 4 ml of 0.25M NaCl buffer. The eluant was
desalted and concentrated with 30 Kd cut-off Centricons (Millipore,
Billerica, Mass.). The recombinant MBP-SVS2 and MBP-SG1 proteins
were made as described in pMal Protein Fusion and Purification
System's protocol from New England Biolabs Inc. with the following
modifications. SVS2 gene that was cloned from mouse prostate and
SG1 gene that was cloned from human prostate biopsy were subcloned
in frame with MBP and transformed into Top Ten competent cells.
Plasmid DNA isolated was transformed into BL21 (DE3) pLysS bacteria
to express the fusion protein. Eluted proteins were concentrated
with the Amicon Centricon columns with a 10-kD cutoff and washed 2
times with PBS. For ELISPOT assays, purified MBP, MBP-SVS2 and
MBP-SG1 proteins were passed through a ProteoSpin endotoxin removal
kit (Norgen Biotek Corp. Thorold, Canada). Protein concentrations
were measured with BioRad Dc assay (BioRad, Hercules, Calif.) using
BSA as the standard.
Immunoprecipitation
[0148] Immunoprecipitation of autoantigens was performed using
protein G agarose coupled to Aire-sufficient or Aire-KO sera as
described previously (Harlow, E., and Lane, D. 1999. Using
antibodies: A lab manual. 495). In brief, whole prostate tissues
were homogenized in 0.15 M NaCl, 0.05 M Tris (pH 8), and 0.1% CHAPS
(Sigma, St. Louis, Mo.). Protein agarose G-coupled columns were
washed in 30 mL PBS, and prostate extracts from RAG-/- mice
prepared in CHAPS buffer were passed through the matrix. Columns
were washed with 30 mL PBS and washed again with 30 mL of 10 mM
phosphate, pH 6.8. Eluates were collected by passing 0.5 ml of 100
mM glycine, pH 2.5, over the column and collecting the flow
through. Eluates from multiple runs were pooled and concentrated in
a centrifugal protein concentrator (Vivaspin; Sartorius, UK).
In-Gel Digestion and PMF
[0149] SVS2 was identified by provisional peptide mass
fingerprinting (PMF) as previously described in DeVoss, J. et al.
(2006) J Exp Med 203:2727-2735. In brief, gel bands were excised,
destained (stain-stripped) three times in 50% acetonitrile and 25
mM ammonium bicarbonate (pH 8), dehydrated with 100% acetonitrile,
and dried in a Speed-Vac (Savant). Gel pieces were digested for 16
h at 37.degree. C. Peptides were extracted, and PMF was used for
preliminary protein identification. Mass spectra were produced by
MALDI-TOF representing protonated molecular ions (MH.sup.+) of
tryptic peptides from the proteins present in each gel spot. The
mass spectra were internally mass calibrated using two trypsin
autolysis products present in the digest mixture. Preliminary
protein identities were established by matching the experimentally
determined peptide masses to those produced by an in silico tryptic
digestion of the Swiss-Prot protein database (available at the
ExPASy proteomics server of the Swiss institute of Bioinformatics)
within the window of experimental mass measurement accuracy. The
PMF data-searching algorithm (available through MS-Fit at the
website: prospector.ucsf.edu) was used to perform the database
searches.
Immunoblotting
[0150] Mouse or human sera were screened for the presence of
autoantibodies by Western blotting. Rabbit anti-human SG antibody
(Santa Cruz Biotechnology, Santa Cruz, Calif.) was used as a
positive control. Secondary Goat anti-mouse, Goat anti-rabbit HRP
antibody (Upstate, Billerica, Mass.) and Goat anti-human IgG HRP
antibody (Invitrogen, Carlsbad, Calif.) were used in the respective
assays. The resulting films developed with either Upstate
Visualizer or ECL chemiluminescent substrate. For competition
studies, C57BL/6.times.NOD Lt/J F2 Aire-KO sera was preincubated
with serial dilutions of either recombinant MBP-SVS2 or control MBP
protein in TBS-T with 1% nonfat dry milk for 2 h at room
temperature before use as the primary reagent to blot the
membranes. The concentrations of protein used in these experiments
were 30, 7.5, 1.88, 0.47 mcg.
Real-Time PCR
[0151] Real-time PCR was performed on cDNA prepared from
DNase-treated RNA that were derived from the tested tissues or the
CD45 negative thymic stroma cells that were purified by FACS
sorting according to the previous method (Gray, D. H. et al.,
(2002) J Immunol Methods 260:15-28). Aire and cyclophilin
specific-primers and probes were used as previously described
(DeVoss, J. et al., (2006) J Exp Med 203:2727-2735). SVS2 taqman
primers/Fam-labeled MGB probes (Mn01251795_g1) were purchased from
Applied Biosystems (Foster City, Calif.). Reactions were run on the
HT7900 sequence detection system (Applied Biosystems).
ELISPOT Analysis
[0152] Mouse splenocytes were harvested from Aire-KO, WT B6 mice,
or immunized WT B6 mice. Human PBMC was generated from a CPPS
individual and a normal donor. The release of IFN-.gamma. was
measured by ELISPOT assay. Briefly, Multiscreen HTS IP plates
(Millipore, Billerica, Mass.) were coated overnight with 2 .mu.g/ml
of anti-mouse IFN-.gamma. mAb or anti-human IFN-.gamma. mAb (BD
Biosciences, San Diego, Calif.) at 4.degree. C. The plates were
washed with PBS and blocked with RPMI medium containing 10% FCS for
2 h at 37.degree. C. Mouse splenocytes or human PBMC were added to
each well with the different antigens and incubated for 18 hour in
RPMI complete medium. The plates were washed with PBS before adding
2 .mu.g/ml of biotin-labeled anti-mouse or anti-human IFN-.gamma.
mAb (2 .mu.g/ml; BD Biosciences) respectively and incubated
overnight at 4.degree. C. After washing and further incubation with
avidin--horseradish peroxidase (1:100 dilutions; BD Biosciences)
for 1 h at room temperature, the plates were washed and developed
using substrate solution (AEC; BD Biosciences, San Diego, Calif.).
Positive spots displayed in the plate membranes were examined using
an automated ELISPOT reader (AID; Autoimmun Diagnostika GmbH,
Strassburg, Germany). The number of spot-forming cells was the
average number of spots in duplicate or triplicate wells.
Generation of Antigen-Specific T Cell Lines
[0153] Aire-KO B6 male mice were immunized with the purified SVS2
(200 .mu.g/mouse) emulsified in an equal volume of CFA (Difco). The
antigens were injected s.c. into the foot pads. Mice were boosted
twice with the same amount of antigen emulsified in IFA at 10 days
intervals. 10 days following the second boost, single-cell
suspensions from draining lymph nodes of SVS2-immunized mice were
incubated with purified SVS2 (50 .mu.g/ml). Twenty-four hours
later, mouse IL-2 (50 u/ml), IL-7 (10 ng/ml) and IL 15 (10 ng/ml)
(Peprotech) were supplemented into the media. Subsequent
restimulations occurred at weekly intervals by the addition of
purified SVS2-pulsed, irradiated B6 splenocytes to the cultures
followed by the same cytokine supplementation 24 hours later. The
OVA-specific T cell lines were generated in parallel in separate
mice through the same procedure. After two cycles of restimulation,
the dead cells were removed by centrifugation with ficoll
(Histopaque-1083, Sigma), and the cultured cells were harvested for
further analysis and adoptive transfer experiments.
Intracellular Cytokine Staining of Antigen-Specific T Cells
[0154] Antigen-specific T cell lines were incubated with the
irradiated B6 splenocytes pulsed with MBP-SVS2 (50 .mu.g/ml) for 24
hours. GolgiStop buffer (BD Biosciences) was added at a final
concentration of 1 .mu.g/ml for the last 4 hours of incubation. The
cells were then harvested for intracellular cytokine staining.
Briefly, the cells were first stained with anti-mouse CD4-APC
antibody (BD Biosciences) and fixed with 4% formaldehyde in PBS for
10 min Followed by being permeabilized with 1.times.BD perm/wash
buffer (BD Biosciences) for 15 min, the cells were stained with
anti-mouse IFN-.gamma.-PE (eBioscience), anti-mouse IL 10-PE
(Biolegend), or anti-mouse IL17a-FITC (BD Biosciences) antibodies.
The cells were assessed by flow cytometry and analyzed with Flowjo
(TreeStar).
Adoptive Transfer Experiment
[0155] Splenocytes were harvested from either B6 Aire-KO mice or
immunized B6 mice. CD4+ or CD8+ T cells or CD19+ B cells were
depleted using FACS sorting. Briefly, cells were stained with
anti-CD4-PE, anti-CD8-PE or anti-CD19-PE antibodies (BD Pharmingen)
for 30 min on ice, and then the PE negative cells (CD4+ depleted
cells, CD8+ depleted cells and CD19+ depleted cells) were collected
(FACSAria, BD, San Diego, Calif.). 5.times.106 sorted cells (CD4+
depleted, CD8+ depleted or CD19+ depleted) were injected i.v. into
B6-RAG-/- mice. Animals were aged 40 days after the transfer,
sacrificed, and analyzed.
SVS2 Immunization Experiments
[0156] B6 Male mice at 25-weeks of age were immunized s.c. with the
recombinant MBP-SVS2 fusion protein (200 .mu.g/mouse) or MBP (200
.mu.g/mouse) as the control. Both proteins were emulsified in an
equal volume of CFA (Difco, Lawrence, Kans.) supplemented with
mycobacterium tuberculosis H37Ra. The antigens were injected S.C.
on the back. Mice were boosted twice with the same amount of
antigen emulsified in IFA at 2-week intervals. Two weeks following
the second boost, tissues and sera were harvested for further
analysis or adoptive transfer experiments.
Histopathology
[0157] For histology staining, prostate tissues were either fixed
in 10% neutral buffered formalin and embedded in paraffin or frozen
in OCT (Tissue Tek) tissue embedding compound at -80 C. After
sectioning, tissues were stained with hematoxylin and eosin (Sigma,
St. Louis, Mo.). For immunohistochemical staining, prostates were
snap-frozen in OCT (Tissue Tek), sectioned at 6 um thick, and
processed for staining using Vectastain Elite ABC Kit (Vector,
Burlingame, Calif.) with biotinylated anti-rat AB against rat
anti-mouse CD3 (clone CT-CD3; Caltag Laboratories, Carlsbad,
Calif.), CD4 (clone H129.19; BD Pharmingen), CD8a (clone 53-6.7; BD
Pharmingen), CD19 (clone 1D3; BD Pharmingen), followed by
streptavidin-peroxidase conjugate. Colorimetric detection was
visualized by using DAB substrate (Pierce, Rockford, Ill.).
Histology was assessed by a pathologist, who scored lymphocytic
infiltrates while blinded to mouse phenotype or treatment. The
presence of inflammatory cells was scored by a pathologist
according to the following categories: no infiltration, rare
scattered inflammatory cells, obvious scattered cells without
confluence, confluent sheets of cells and/or epithelial injury by
inflammatory cells.
Human Subjects
[0158] Sera were obtained from consenting patients with clinical
histories of CPPS as part of an IRB-approved protocol at the
University of Washington Medical Center. Clinical assessment of
prostatitis in the prostate biopsies was performed by the pathology
department. Control sera were obtained from male volunteer blood
donors without histories of prostatitis symptoms. Sera were stored
in aliquots at -80.degree. C. until used.
Statistical Analysis
[0159] For the mouse study, differences between groups were
evaluated using two-tailed Student's T test. Differences were
considered to be statistically significant at p<0.05. For human
studies, differences between groups were evaluated by Fishers exact
test. Differences were considered to be statistically significant
at p<0.05.
Results
Role of Prostate-Associated Antigen in Prostatitis
[0160] Aire-KO mice spontaneously develop multi-organ autoimmunity,
including those of the eye, salivary glands, ovaries and stomach.
Inflammation in the prostate was also observed in most Aire-KO
mice, but the nature of immune recognition for autoimmune
prostatitis in Aire-KO mice had not been reported. Histological
experiments were carried as detailed below in order to confirm the
pathology of prostate in Aire-KO mice.
[0161] Histological changes in the prostates of Aire-KO mice were
examined in the staining of representative prostate sections from
Aire-KO and Aire-sufficient mice (WT). In Aire-KO mice, the
inflammation seen included cellular extravasation into the
interstitial tissue between the glands as well as infiltration into
glandular epithelium of both the dorsal and ventral prostate glands
leading to effacement of the usual glandular architecture in severe
cases (FIG. 1A; the urethra (u) and ductus deferens (dd), two ducts
that course through the prostate glands are denoted; upper panels:
4.times. magnification; lower panels: 40.times. magnification).
Moderate to severe prostate inflammation, including intraepithelial
infiltration of prostate glands by lymphocytes, was observed in 5
of 7 examined male C57BL/6 Aire-KO mice at 10-20 weeks of age, but
completely absent in all of the 5 Aire-wild-type (WT) littermate
controls (18-20 wk old) (FIG. 1B) (Table 2). Scoring was performed
blindly by a pathologist using a scoring system adapted from
grading used in evaluating human prostate tissues (p<0.001,
unpaired Student's T-test) Immunohistochemistry performed on
Aire-KO prostate glands demonstrated a predominance of CD4+ T cells
among the infiltrating cells in the prostates (FIG. 1C, upper
panels). The staining was performed for CD4, CD8, CD19 and anti-rat
IgG in cryostatic sections of prostates from a representative 20
week-old Aire-KO (upper panel) and WT (lower panels) mice. Staining
is representative of 6 Aire-KO mice and 5 WT mice. CD8+ T cells
were observed but in fewer numbers than CD4+ T cells. Nevertheless
both CD4+ and CD8+ T cells could be seen infiltrating the glandular
epithelia of these prostates. CD19+ B cells were seen at low
frequency, and if present, resided within the stroma of the
prostate tissue. In contrast, wild-type mice lacked lymphocytes
within either the glandular cell layer or the stroma (FIG. 1C,
lower panels).
TABLE-US-00001 TABLE 1 Histologic score of inflammatory infiltrates
Pathology Score 0 1 2 3 4 Mononuclear Infiltration in Glands - - +
++ +++ Mononuclear Infiltration in Stroma - + any any +++ "-", no
infiltration; "+", rare scattered inflammatory cells; "++", obvious
scattered cells without confluence; "+++", confluent sheets of
cells and/or epithelial injury by inflammatory cells (replacing or
destroying mucosal cells).
[0162] FIGS. 1A-C. Spontaneous Development of Autoimmune
Prostatitis in the Aire-KO Mouse.
[0163] (A) H&E staining of representative prostate sections
from Aire-KO and Aire-sufficient mice (WT). The urethra (u) and
ductus deferens (dd), two ducts that course through the prostate
glands are denoted. Upper panels: 4.times. magnification; lower
panels: 40.times. magnification. (B) Mononuclear infiltration in
H&E stained prostate sections from 7 Aire-KO and 5
Aire-sufficient (WT) littermate mice on C57BL/6 background (18-20
wk old) was graded blindly by a pathologist using a scoring system
adapted from grading used in evaluating human prostate tissues
(Suppl. Table 1). p<0.001, unpaired Student's T-test. (C)
Immunohistochemical staining for CD4, CD8, CD19 and anti-rat IgG in
cryostatic sections of prostates from a representative 20 week-old
Aire-KO (upper panel) and WT (lower panels) mice. Staining is
representative of 6 Aire-KO mice and 5 WT mice.
Aire-KO Mouse Develop Spontaneous Immune Responses to SVS2.
[0164] In order to define the relevant prostate autoantigens that
mediate this prostatitis, sera of Aire-KO mice were screened for
autoantibodies to proteins present in the protein lysates derived
from mouse prostate tissue.
[0165] Sera of Aire-deficient and WT mice were used in western
blots to determine whether autoantibodies were present
Immunoblotting of whole prostate extracts was performed with the
sera from male Aire-deficient and WT mice on the B6/NOD F2
background (individual mice listed by number). Each lane of the
multi-screen immunoblot represents the reactivity of sera from
individual mice. Gel bands stained by sera from multiple Aire-KO
mice were excised and analyzed by mass spectroscopy.
[0166] Next, immunoblotting of prostate, eye, lacrimal, and
salivary gland extracts were performed with the sera from male
Aire-KO and WT mice on the B6/NOD F2 background (individual mice
listed by number) (FIG. 8). Each lane of the multi-screen
immunoblot represents the reactivity of sera from individual mice.
Autoantibodies against prostate-derived proteins could be detected
in most Aire-KO sera (FIG. 2A). Some sera showed broad reactivity,
whereas others showed more limited and/or weaker reactivity,
consistent with heterogeneity in the immune response. Nevertheless,
common bands were also evident across multiple mice, indicating
that there were immune responses to proteins shared by the majority
of the Aire-KO mice. These common bands within the prostate lysate
were not evident in immunoblots to lysates from other tissues, such
as the eye, salivary, or lacrimal glands, in which Aire-KO mice
also develop spontaneous inflammation (FIG. 8).
[0167] In an effort to purify and identify the targeted antigens,
sera from Aire-KO mice with strong autoreactivity to the lysates
and from WT mice were used to make immunoprecipitation columns.
These columns were used to enrich for antibody-binding proteins
from pooled prostate tissue extracts. Enriched proteins from both
Aire-KO and WT sera-coupled columns were concentrated and subjected
to gel electrophoresis. Multiple distinct bands that were
visualized by silver staining and that were unique to the Aire-KO
sera column were excised and analyzed by mass spectroscopy. Seminal
vesicle secretory protein 2 (SVS2) was successfully identified with
29% sequence coverage of the protein through peptide mass
fingerprinting (PMF) from both the 26 and 38-kD bands. The peptide
sequences identified through PMF are denoted in underline and lie
predominantly within the N terminus of the SVS2 protein (GenBank
Accession No. AAI07277; accession no. 78070545) (FIG. 2B). SVS2 is
an androgen-regulated protein expressed by the prostate and
secreted into seminal fluid, where it serves as a major clotting
protein involved in the formation of the copulatory plug.
[0168] To confirm that SVS2 was the protein recognized by
autoantibodies in the Aire-KO mice, purified SVS2 was generated
through two approaches. Mouse-derived SVS2 protein was purified
from the seminal vesicle fluid according to the protocol that was
utilized to purify human SG, a protein that has structural
similarities to mouse SVS2 protein. This purification procedure
yielded the expected 38-kD protein. As an alternative approach,
recombinant SVS2 was also produced as an 80-kD fusion protein with
maltose binding protein (MBP) expressed in E. coli. Recombinant
MBP-SVS2 could be purified from bacteria lysate with an amylose
column. Recombinant MBP was also expressed and purified from E.
coli and used as a control in subsequent experiments.
[0169] Sera from Aire-KO mice contained autoantibodies that could
recognize both the purified, semen-derived protein and the
recombinant protein by western blot (FIG. 2C). Immunoblots of
recombinant MBP-SVS2 and purified SVS2 from semen plasma with sera
from Aire-KO mice (right) and Aire-sufficient mice (left) on
B6.times.NOD F2 background. MBP and OVA are the negative controls.
Aire-KO sera did not bind to the control MBP or another irrelevant
protein ovalbumin (OVA). The absence of SVS2 reactivity in the
Aire-sufficient sera further supports the importance of Aire
regulation in autoreactivity to this antigen.
[0170] To confirm that the prostate extract-reactive autoantibodies
were specific to SVS2, Aire-KO sera was pre-incubated with varying
concentrations MBP-SVS2 fusion protein before immunoblotting the
prostate extracts. Anti-SVS2 positive sera from Aire-KO mice were
preincubated with either recombinant MBP-SVS2 or MBP control before
being used in the immunoblots against the prostate extract. Wild
type and B6.times.NOD F2 Aire-heterozygous sera were used as
controls. Increasing concentrations of MBP-SVS2 abrogated
immunoreactivity to the bands while pre-incubation with MBP did not
alter this immunoreactivity (FIG. 2D). These results indicate that
SVS2 is a prostate autoantigen that is targeted by autoantibodies
in the Aire-KO mice. Moreover, these results also indicate that
some of the recognized lower molecular weight proteins represent
fragments of SVS2.
[0171] FIGS. 2A-D. Aire-KO Mice Possess Spontaneous Autoantibodies
to SVS2.
[0172] (A) Immunoblotting of whole prostate extracts was performed
with the sera from male Aire-KO and WT mice on the B6/NOD F2
background (individual mice listed by number). Each lane of the
multi-screen immunoblot represents the reactivity of sera from
individual mice. Gel bands stained by sera from multiple Aire-KO
mice were excised and analyzed by mass spectroscopy. (B) Multiple
microsequenced peptides were found to be derived from SVS2
(accession no. 78070545). The peptide sequences identified through
peptide mass fingerprinting (PMF) are denoted in underline and lie
predominantly within the N terminus of the protein. (C) Immunoblots
of recombinant MBP-SVS2 and purified SVS2 from semen plasma with
sera from Aire-KO mice (right) and -sufficient mice (left) on
B6.times.NOD F2 background. MBP and OVA are the negative controls.
(D) Anti-SVS2 positive sera from Aire-KO mice were preincubated
with either recombinant MBP-SVS2 or MBP control before being used
in the immunoblots against the prostate extract. Wild type and
B6.times.NOD F2 Aire-heterozygous sera were used as controls.
[0173] FIG. 8. Aire-Deficient Male Mice Possess Prostate-Specific
Autoantibodies.
[0174] Immunoblotting of prostate, eye, lacrimal, and salivary
gland extracts were performed with the sera from male Aire-KO and
WT mice on the B6/NOD F2 background (individual mice listed by
number). Each lane of the multi-screen immunoblot represents the
reactivity of sera from individual mice.
SVS2 Expression in the Thymus is Aire-Dependent.
[0175] To elucidate the role Aire plays in central tolerance of
self-antigens derived from SVS2, distribution of SVS2 expression
was examined as detailed below.
[0176] To determine the distribution of SVS2 expression, RNA was
isolated from different tissues and assessed by real-time PCR to
quantify the expression of SVS2 mRNA. Values are normalized to
cyclophilin expression for each tissue and are plotted in arbitrary
units as denoted. Consistent with prior reports, SVS2 was
specifically expressed in the prostate gland but was essentially
undetectable in other organs tested, including tissues where
spontaneous inflammation can develop in Aire-KO mice (FIG. 3A). As
a next step, it is important to determine whether or not SVS2 is
expressed in the thymus and whether its thymic expression is
Aire-dependent. CD45-negative thymic stromal cells were purified
from WT and Aire-KO mice and analyzed the expression of SVS2 by
quantitative real-time PCR. Values are normalized to cyclophilin
Results presented are representative of 2 experiments performed.
Error bars represent mean.+-.SD. As seen in FIG. 3B, SVS2 was
expressed at significant levels in the WT thymus compared to the
Aire-KO thymus. This result demonstrates that thymic expression of
the SVS2 gene is indeed Aire-dependent.
[0177] FIGS. 3A and 3B. SVS2 is Highly Expressed in the Prostate
and is an Aire-Dependent Antigen Expressed in the Thymus.
[0178] (A). The expression levels of SVS2 were quantitated by
RT-PCR on cDNA prepared from diverse tissues. Values are normalized
to cyclophilin expression for each tissue and are plotted in
arbitrary units as denoted. (B). Quantitative real-time PCR assay
for the SVS2 expression in the CD45.sup.- thymic stroma cells of
Aire-KO and Aire-WT mice. Values are normalized to cyclophilin
Results presented are representative of 2 experiments performed.
Error bars represent mean.+-.SD.
Autoimmune Prostatitis is Mediated by CD4+ T Cells.
[0179] Identifying the immune effectors that participate in the
cascade of events leading to prostatitis elucidates the mechanism
by which autoimmunity towards prostate proteins is developed.
[0180] Adoptive transfer experiments were performed to determine
the immune effectors that could mediate prostatitis. Splenocytes
from Aire-KO B6 mice were pooled, depleted of either CD4+ T cells,
CD8+ T cells or CD19+ B cells by FACS, and adoptively transferred
into immunodeficient B6/RAG-KO mice. Mice that received CD8+ T
cell-depleted (middle panel), or B cell-depleted (right panel)
splenocytes developed prostate inflammation with similar
pathological characteristics to the Aire-KO mice (FIG. 4A, middle
and right panels). Adoptive transfer of CD4+ T cell-depleted
splenocytes (left panel), however, failed to induce this level of
prostatitis (FIG. 4A, left panel). Results presented are
representative of two separate adoptive transfer experiments, each
with two mice per group, with magnification of 100.times.. These
results indicate that autoimmune T cells mediate the spontaneous
prostatitis seen in Aire-KO mice, of which CD4+ T cells play a
dominant role.
[0181] Based on these results, it is important to determine whether
Aire-KO mice possess SVS2-reactive T cells spontaneously. 200,000
splenocytes per well from Aire-KO mice and WT mice were assessed
for T cell responses against MBP-SVS2 (10 mcg/ml) or MBP (10
mcg/ml) by IFN-.gamma. ELISPOT. Through this approach, specific
responses could be detected to the MBP-SVS2 fusion protein. While
there was heterogeneity in the immune responses to MBP-SVS2 in
Aire-KO mice, the responses seen were significantly different from
WT mice where no responses to MBP-SVS2 could be detected (FIG. 4B).
Aire-KO mice also had higher background reactivity to the control
antigen MBP compared to WT mice, so additional mice were assessed
for immune responses to non-recombinant, purified proteins. Results
from Aire-KO and WT mice are shown (n=7 per group) in FIG. 4C. Mean
SFU/group are indicated with dashes with significance assessed by
paired Students T-test. Again, spontaneous T cell responses to
semen-derived SVS2 were detected only in the Aire-KO mice (FIG.
4C).
[0182] IFN-.gamma. ELISPOT assays were also performed with purified
semen-derived SVS2 (10 mcg/ml) and OVA (10 mcg/ml) (n=5 per group).
Mean SFU/group are again indicated with dashes with significance
assessed by two-sided Students T-test (FIG. 4D).
[0183] To confirm that SVS2-reactive T cells in fact mediate
prostatitis, an SVS2-reactive CD4.sup.+ T cell line was generated
by stimulating splenocytes from male Aire-KO mice in vitro with
purified SVS2. Upon restimulation with SVS2-MBP, the SVS2-reactive
T cell line produced IFN-.gamma., but not IL-17 or IL-10. An
ovalbumin (OVA)-reactive T cell line was generated in parallel as a
control. Adoptive transfer of the SVS2-reactive T cell line into
B6/RAG-KO mice induced prostatitis (pathology score 4) in 3 of 3
treated mice, whereas adoptive transfer of the OVA-specific T cell
line resulted in no inflammation.
[0184] FIGS. 4A-D. Spontaneous T Cells Responses to SVS2 in Aire-KO
Mice.
[0185] (A) Splenocytes derived from B6 Aire-KO were depleted of
CD4, CD8 T cells or B cells by FACS. 5.times.10.sup.6 sorted cells
were adoptively transferred into RAG-deficient B6 mice. H&E
staining of representative frozen prostate sections indicated that
cells depleted of CD8+ T cells (middle), CD19+ B cells (right), but
not the CD4+ T cells (left) were capable of transferring disease
into RAG-deficient mice. Results presented are representative of
two separate adoptive transfer experiments, each with two mice per
group. Magnification 100.times.. (B) 200,000 splenocytes per well
from Aire-KO mice and WT mice were assessed for T cell responses
against MBP-SVS2 (10 mcg/ml) or MBP (10 mcg/ml) by IFN-.gamma.
ELISPOT. (C) Results from Aire-KO and WT mice are shown (n=7 per
group). Mean SFU/group are indicated with dashes with significance
assessed by paired Students T-test. (D) IFN-.gamma. ELISPOT assays
were also performed with purified semen-derived SVS2 (10 mcg/ml)
and OVA (10 mcg/ml) (n=5 per group). Mean SFU/group are again
indicated with dashes with significance assessed by two-sided
Students T-test.
Tolerance to SVS2 can be Broken and Prostatitis can be Induced in
Wild-Type B6 Mice.
[0186] Based on the results presented above, central tolerance
mediated by Aire seems to play a major role in preventing
autoimmune prostatitis. In order to further identify the mechanism
that resembles human prostatitis, it would be important to
determine whether central tolerance to SVS2 is complete in a
wild-type setting.
[0187] SVS2 was examined for immunogenicity in WT B6 mice by
immunizing these mice with recombinant MBP-SVS2. In doing so,
whether inducing an immune response to SVS2 is sufficient for
triggering prostatitis could also be determined. WT mice were
challenged with either MBP-SVS2 (FIG. 5A) or MBP (FIG. 5B)
emulsified in complete Freund's adjuvant (CFA). After two booster
immunizations with the corresponding protein in incomplete Freund's
adjuvant (IFA), sera, splenocytes, and tissues were harvested for
analysis. Sera from individual immunized B6 mice were used to
immunoblot semen-purified SVS2 or OVA. Each blot represents an
individual animal. One group of 4 mice is shown and represents a
total of 14 mice that were immunized with MBP-SVS2 or with MBP. To
minimize cross-reactivity to potential contaminants in the
recombinant antigens, semen-derived SVS2 was used to perform the in
vitro assays. Autoantibodies against the semen-derived SVS2 could
be detected in the sera of 7 out of 14 mice (FIG. 5A).
[0188] None of the 14 MBP-immunized mice had any autoantibody
activity against SVS2 (FIG. 5B). The pooled splenocytes from 4 mice
that were immunized with either the recombinant MBP-SVS2 (left) or
MBP (right) were also assessed by IFN-.gamma. ELISPOT for
reactivity to purified SVS2 (10 mcg/ml), OVA (10 mcg/ml), or PBS.
The splenocytes depleted of CD8+ or CD4+ T cells were also
assessed. The splenocytes from MBP-SVS2 immunized mice demonstrated
specific reactivity to the semen-derived SVS2, while the
splenocytes of MBP immunized mice had no specific immune response
against SVS2 (FIG. 5C). Error bars represent mean+SD. Results are
representative of two experiments.
[0189] Depletion of CD4+ T cells from the MBP-SVS2 immunized
splenocytes by FACS sorting eliminated the SVS2-specific response,
while depletion of CD8+ T cells had essentially no effect. These
results demonstrate that tolerance to this self-antigen can be
broken and that both antibody and CD4+ T cell responses can be
induced in Aire-sufficient mice.
[0190] FIGS. 5A-C. Induction of SVS2-Specific Antibody and T Cell
Response by Immunizing Aire-Sufficient B6 Mice with MBP-SVS2.
[0191] WT B6 mice were immunized with either (A) MBP-SVS2 or (B)
MBP emulsified in CFA. Sera from individual immunized B6 mice were
used to immunoblot semen-purified SVS2 or OVA. Each blot represents
an individual animal. One group of 4 mice is shown and represents a
total of 14 mice that were immunized with MBP-SVS2 or with MBP. (C)
The pooled splenocytes from 4 mice that were immunized with either
the recombinant MBP-SVS2 (left) or MBP (right) were assessed by
IFN-.gamma. ELISPOT for reactivity to purified SVS2 (10 mcg/ml),
OVA (10 mcg/ml), or PBS. The splenocytes depleted of CD8+ or CD4+ T
cells were also assessed. Error bars represent mean+SD. Results are
representative of two experiments.
[0192] Next, the prostates of immunized mice were examined for the
induction of prostatitis. Male B6 mice were immunized s.c. with the
recombinant MBP-SVS2 fusion protein (upper row) or MBP control
protein (lower row) emulsified in CFA (FIG. 6A). After two booster
immunizations with the respective proteins in IFA, the prostates
were analyzed by immunohistochemical staining for CD3+ cells in
frozen sections of prostate from the immunized mice with MBP-SVS2
(upper) or MBP (lower). While the severity of inflammation was less
than that seen in Aire-KO mice, nevertheless 10 out of 14
MBP-SVS2-immunized mice had intraprostatic CD3+ T cell infiltration
(FIG. 6A). These results demonstrate that tolerance to SVS2 can be
broken in WT mice with pathologic consequences.
[0193] To confirm that the induced prostatitis is T cell mediated,
adoptive transfer experiments were performed as follows and the
results shown in FIG. 6. Pooled splenocytes from the mice that were
immunized with MBP-SVS2 (upper row) were adoptively transferred
into B6/RAG-KO mice. MBP immunized splenocytes were adoptively
transferred into B6/RAG-KO mice as controls (lower row). 4 weeks
following transfer, the prostates of recipient mice were assessed
for prostatitis. Lymphocyte infiltration in the prostates of each
immunized mouse was scored blindly by a pathologist (p<0.005,
Students T-test) (FIG. 6B). The intraprostatic CD3+ T cell
infiltrates into the glandular epithelium were observed in all mice
that received the SVS2-MBP immunized splenocytes (FIG. 6B). None of
the mice that received MBP-immunized splenocytes displayed any
prostatitis (FIG. 6C). These results further support the role of
the T cell response induced by SVS2 in mediating disease.
[0194] FIGS. 6A-D. Induction of Prostatitis in Wild-Type B6 Mice
with SVS2 Immunization.
[0195] (A) Male B6 mice were immunized s.c. with the recombinant
MBP-SVS2 fusion protein (upper row) or MBP control protein (lower
row) emulsified in CFA. After two booster immunizations with the
respective proteins in IFA, the prostates were analyzed by
immunohistochemical staining for CD3+ cells in frozen sections of
prostate from the immunized mice with MBP-SVS2 (upper) or MBP
(lower). (B) Lymphocyte infiltration in the prostates of each
immunized mouse was scored blindly by a pathologist. p<0.005,
Students T-test. (C) Pooled splenocytes from the mice immunized
with the recombinant MBP-SVS2 fusion protein (upper row) or MBP
control protein (lower row) were adoptively transferred (AT) i.v.
into RAG-deficient mice. 4 weeks later, the prostates were analyzed
by immunohistochemical staining for CD3+ cells. (D) Lymphocyte
infiltration in the prostates of each mouse was again scored by a
pathologist. p<0.005, Students T-test.
Chronic Prostatitis Patients Develop Autoimmune Response to Human
Semenogelin.
[0196] Central tolerance to SVS2 in mice has proven to be crucial
in the prevention of autoimmunity leading to prostatitis. As for
human prostatitis, the results accumulated from mouse studies
presented above may also be applicable. The following experiment
demonstrated that T cell immunity against a human semen protein
semenogelin is involved in human prostatitis.
[0197] While no clear human homologue for SVS2 exists, SVS2 is a
member of a family of prostate-specific proteins collective known
as "rapidly evolving substrates for transglutaminase" (Lundwall,
A., and Lazure, C. (1995) FEBS Lett 374:53-56). Similar
prostate-specific proteins also exist in humans. Using the same
method that was used to purify SVS2 from mouse semen, we purified
one such protein, semenogelin (SG), from human semen plasma and
examined whether CPPS patients can possess antibodies to this
autoantigen Immunoblotting of purified semenogelin was performed
with the sera from CPPS patients (upper panel of FIG. 7A) and
age-matched male blood donors (lower panel of FIG. 7A). Each lane
of the multi-screen immunoblot represents the reactivity of sera
from individual sample. The 52-kDa band represents SG1 and the
71-kDa band represents SG2. Rabbit anti-human semenogelin as a
positive control (the 1.sup.st lane) and human nonspecific IgG was
used as a negative control (the last lane). 15 representative sera
were shown from each group.
[0198] As expected, the SG preparations contained both isoforms:
the 52-kDa SG1 and the 71-kDa SG2. 26 of 39 sera from patients with
the clinical diagnosis of CPPS possessed autoantibodies to these
proteins (FIG. 7A, top), while only 8 of 39 sera from age-matched
normal males possessed these autoantibodies (FIG. 7A, bottom)
(Fisher's exact test, two-sided p<0.0001). 16 of the 39 assessed
CPPS patients had undergone prostate biopsies as part of their
clinical management. 9 of the 16 CPPS patients had prostatitis on
biopsy. Interestingly, all patients with biopsy-proven prostatitis
had autoantibodies to SG (Table 2).
TABLE-US-00002 TABLE 2 Presence of prostatitis on biopsy Patient ID
Antibody Prostatitis 1109 + + 1196 + + 1197 + + 1229 + + 1241 + +
1248 + + 1261 + + 1274 + + 1277 + + 1218 + - 1235 + - 1237 + - 1259
+ - 1262 + - 1250 - - 1256 - -
[0199] The specificity of these autoantibodies was confirmed
against recombinant human MBP-SG1- and MBP-SG2 fusion proteins
(FIG. 7B) Immunoblots of recombinant MBP-SG1, MBP-SG1 and
semenogelin purified from semen plasma are used to detect IgG
antibodies within sera from a patient with CPPS (middle) and an
age-matched normal male donor (right). Polyclonal rabbit-anti-human
semenogelin Ab was used as positive control (left). MBP and BSA
represent irrelevant protein controls.
[0200] T cell immunity against this antigen could also be detected
in the peripheral blood mononuclear cell (PBMC) from a CPPS patient
by IFN-.gamma. ELISPOT (FIG. 7C). PBMC from a CPPS patient (opened
bar) and an age-matched normal male donor (filled bar) were
assessed for T cell responses against the purified semenogelin and
MBP-SG1 fusion protein by IFN-.gamma. ELISPOT. 100,000 cells per
well were cultured overnight with antigens at 10 .mu.g/ml. The
number of spot forming units (SFU) was shown as the mean of
triplicate wells.
[0201] FIGS. 7A-C. Chronic Prostatitis Patients Possess Autoimmune
Response to Human Semenogelin.
[0202] (A) Immunoblotting of purified semenogelin was performed
with the sera from CPPS patients (upper panel) and age-matched male
blood donors (lower panel). Each lane of the multi-screen
immunoblot represents the reactivity of sera from individual
sample. The 52-kDa band represents Sg1 and the 71-kDa band
represents Sg2. Rabbit anti-human semenogelin as a positive control
(the 1.sup.st lane) and human nonspecific IgG was used as a
negative control (the last lane). 15 representative sera were shown
from each group. (B) Immunoblots of recombinant MBP-Sg1, MBP-Sg1,
and semenogelin purified from semen plasma were used to detect IgG
antibodies within sera from a patient with CPPS (middle) and an
age-matched normal male donor (right). Polyclonal rabbit-anti-human
semenogelin Ab was used as positive control (left). MBP and BSA
represent irrelevant protein controls. (C) PBMC from a CPPS patient
(opened bar) and an age-matched normal male donor (filled bar) were
assessed for T cell responses against the purified semenogelin and
MBP-Sg1 fusion protein by IFN-.gamma. ELISPOT. 100,000 cells per
well were cultured overnight with antigens at 10 ug/ml. The number
of spot forming units (SFU) was shown as the mean of triplicate
wells.
Example 2
Growth of Prostate Cancer Cells in Aire.sup.-/- (Knockout) and
Wild-Type Mice
[0203] Five male Aire-KO mice and five wild-type male mice were
injected s.c. with Tramp-C2 cells (ATCC number CRL-2731; mouse
prostate cancer cell line; 2.times.10.sup.5 cells per mouse) on day
0. Tumor growth was monitored every 3 days; the size of the tumor
was measured by caliper. When the diameter of the tumor in a given
mouse reached 2 cm, mice were sacrificed. All remaining mice were
sacrificed on day 67.
[0204] The results are shown in FIG. 10. As shown in FIG. 10, the
survival rate for Aire-KO mice was much higher than that for
wild-type mice. Thus, prostate cancer cells grow more slowly in
Aire-KO mice than in wild-type mice.
Example 3
Expression of PAA in BPH and Prostate Cancer
[0205] Reverse transcription-polymerase chain reaction (RT-PCR) was
performed on RNA obtained from prostate tissue of 2 human male
prostate cancer patients undergoing radical prostatectomy (patient
identifiers 2134 and 2136). Prostate tissues were dissected into
normal prostate (N), BPH (B), and tumors (T). RNA was also prepared
from 2 prostate cancer cell lines (LNCap; and DU-145). RT-PCR was
performed using primers specific for Sg1 (upper panel) and primers
specific for Sg2 (lower panel). The results, shown in FIG. 11,
indicate that BPH and prostate cancer cells express Sg11 and
Sg12.
[0206] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
51462PRTHomo sapiens 1Met Lys Pro Asn Ile Ile Phe Val Leu Ser Leu
Leu Leu Ile Leu Glu1 5 10 15 Lys Gln Ala Ala Val Met Gly Gln Lys
Gly Gly Ser Lys Gly Arg Leu 20 25 30 Pro Ser Glu Phe Ser Gln Phe
Pro His Gly Gln Lys Gly Gln His Tyr 35 40 45 Ser Gly Gln Lys Gly
Lys Gln Gln Thr Glu Ser Lys Gly Ser Phe Ser 50 55 60 Ile Gln Tyr
Thr Tyr His Val Asp Ala Asn Asp His Asp Gln Ser Arg65 70 75 80 Lys
Ser Gln Gln Tyr Asp Leu Asn Ala Leu His Lys Thr Thr Lys Ser 85 90
95 Gln Arg His Leu Gly Gly Ser Gln Gln Leu Leu His Asn Lys Gln Glu
100 105 110 Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val
Val Ile 115 120 125 His His Lys Gly Gly Lys Ala His Arg Gly Thr Gln
Asn Pro Ser Gln 130 135 140 Asp Gln Gly Asn Ser Pro Ser Gly Lys Gly
Ile Ser Ser Gln Tyr Ser145 150 155 160 Asn Thr Glu Glu Arg Leu Trp
Val His Gly Leu Ser Lys Glu Gln Thr 165 170 175 Ser Val Ser Gly Ala
Gln Lys Gly Arg Lys Gln Gly Gly Ser Gln Ser 180 185 190 Ser Tyr Val
Leu Gln Thr Glu Glu Leu Val Ala Asn Lys Gln Gln Arg 195 200 205 Glu
Thr Lys Asn Ser His Gln Asn Lys Gly His Tyr Gln Asn Val Val 210 215
220 Glu Val Arg Glu Glu His Ser Ser Lys Val Gln Thr Ser Leu Cys
Pro225 230 235 240 Ala His Gln Asp Lys Leu Gln His Gly Ser Lys Asp
Ile Phe Ser Thr 245 250 255 Gln Asp Glu Leu Leu Val Tyr Asn Lys Asn
Gln His Gln Thr Lys Asn 260 265 270 Leu Asn Gln Asp Gln Gln His Gly
Arg Lys Ala Asn Lys Ile Ser Tyr 275 280 285 Gln Ser Ser Ser Thr Glu
Glu Arg Arg Leu His Tyr Gly Glu Asn Gly 290 295 300 Val Gln Lys Asp
Val Ser Gln Ser Ser Ile Tyr Ser Gln Thr Glu Glu305 310 315 320 Lys
Ala Gln Gly Lys Ser Gln Lys Gln Ile Thr Ile Pro Ser Gln Glu 325 330
335 Gln Glu His Ser Gln Lys Ala Asn Lys Ile Ser Tyr Gln Ser Ser Ser
340 345 350 Thr Glu Glu Arg Arg Leu His Tyr Gly Glu Asn Gly Val Gln
Lys Asp 355 360 365 Val Ser Gln Arg Ser Ile Tyr Ser Gln Thr Glu Lys
Leu Val Ala Gly 370 375 380 Lys Ser Gln Ile Gln Ala Pro Asn Pro Lys
Gln Glu Pro Trp His Gly385 390 395 400 Glu Asn Ala Lys Gly Glu Ser
Gly Gln Ser Thr Asn Arg Glu Gln Asp 405 410 415 Leu Leu Ser His Glu
Gln Lys Gly Arg His Gln His Gly Ser His Gly 420 425 430 Gly Leu Asp
Ile Val Ile Ile Glu Gln Glu Asp Asp Ser Asp Arg His 435 440 445 Leu
Ala Gln His Leu Asn Asn Asp Arg Asn Pro Leu Phe Thr 450 455 460
2402PRTHomo sapiens 2Met Lys Pro Asn Ile Ile Phe Val Leu Ser Leu
Leu Leu Ile Leu Glu1 5 10 15 Lys Gln Ala Ala Val Met Gly Gln Lys
Gly Gly Ser Lys Gly Arg Leu 20 25 30 Pro Ser Glu Phe Ser Gln Phe
Pro His Gly Gln Lys Gly Gln His Tyr 35 40 45 Ser Gly Gln Lys Gly
Lys Gln Gln Thr Glu Ser Lys Gly Ser Phe Ser 50 55 60 Ile Gln Tyr
Thr Tyr His Val Asp Ala Asn Asp His Asp Gln Ser Arg65 70 75 80 Lys
Ser Gln Gln Tyr Asp Leu Asn Ala Leu His Lys Thr Thr Lys Ser 85 90
95 Gln Arg His Leu Gly Gly Ser Gln Gln Leu Leu His Asn Lys Gln Glu
100 105 110 Gly Arg Asp His Asp Lys Ser Lys Gly His Phe His Arg Val
Val Ile 115 120 125 His His Lys Gly Gly Lys Ala His Arg Gly Thr Gln
Asn Pro Ser Gln 130 135 140 Asp Gln Gly Asn Ser Pro Ser Gly Lys Gly
Ile Ser Ser Gln Tyr Ser145 150 155 160 Asn Thr Glu Glu Arg Leu Trp
Val His Gly Leu Ser Lys Glu Gln Thr 165 170 175 Ser Val Ser Gly Ala
Gln Lys Gly Arg Lys Gln Gly Gly Ser Gln Ser 180 185 190 Ser Tyr Val
Leu Gln Thr Glu Glu Leu Val Ala Asn Lys Gln Gln Arg 195 200 205 Glu
Thr Lys Asn Ser His Gln Asn Lys Gly His Tyr Gln Asn Val Val 210 215
220 Glu Val Arg Glu Glu His Ser Ser Lys Val Gln Thr Ser Leu Cys
Pro225 230 235 240 Ala His Gln Asp Lys Leu Gln His Gly Ser Lys Asp
Ile Phe Ser Thr 245 250 255 Gln Asp Glu Leu Leu Val Tyr Asn Lys Asn
Gln His Gln Thr Lys Asn 260 265 270 Leu Asn Gln Asp Gln Gln His Gly
Arg Lys Ala Asn Lys Ile Ser Tyr 275 280 285 Gln Ser Ser Ser Thr Glu
Glu Arg Arg Leu His Tyr Gly Glu Asn Gly 290 295 300 Val Gln Lys Asp
Val Ser Gln Arg Ser Ile Tyr Ser Gln Thr Glu Lys305 310 315 320 Leu
Val Ala Gly Lys Ser Gln Ile Gln Ala Pro Asn Pro Lys Gln Glu 325 330
335 Pro Trp His Gly Glu Asn Ala Lys Gly Glu Ser Gly Gln Ser Thr Asn
340 345 350 Arg Glu Gln Asp Leu Leu Ser His Glu Gln Lys Gly Arg His
Gln His 355 360 365 Gly Ser His Gly Gly Leu Asp Ile Val Ile Ile Glu
Gln Glu Asp Asp 370 375 380 Ser Asp Arg His Leu Ala Gln His Leu Asn
Asn Asp Arg Asn Pro Leu385 390 395 400 Phe Thr3582PRTHomo sapiens
3Met Lys Ser Ile Ile Leu Phe Val Leu Ser Leu Leu Leu Ile Leu Glu1 5
10 15 Lys Gln Ala Ala Val Met Gly Gln Lys Gly Gly Ser Lys Gly Gln
Leu 20 25 30 Pro Ser Gly Ser Ser Gln Phe Pro His Gly Gln Lys Gly
Gln His Tyr 35 40 45 Phe Gly Gln Lys Asp Gln Gln His Thr Lys Ser
Lys Gly Ser Phe Ser 50 55 60 Ile Gln His Thr Tyr His Val Asp Ile
Asn Asp His Asp Trp Thr Arg65 70 75 80 Lys Ser Gln Gln Tyr Asp Leu
Asn Ala Leu His Lys Ala Thr Lys Ser 85 90 95 Lys Gln His Leu Gly
Gly Ser Gln Gln Leu Leu Asn Tyr Lys Gln Glu 100 105 110 Gly Arg Asp
His Asp Lys Ser Lys Gly His Phe His Met Ile Val Ile 115 120 125 His
His Lys Gly Gly Gln Ala His His Gly Thr Gln Asn Pro Ser Gln 130 135
140 Asp Gln Gly Asn Ser Pro Ser Gly Lys Gly Leu Ser Ser Gln Cys
Ser145 150 155 160 Asn Thr Glu Lys Arg Leu Trp Val His Gly Leu Ser
Lys Glu Gln Ala 165 170 175 Ser Ala Ser Gly Ala Gln Lys Gly Arg Thr
Gln Gly Gly Ser Gln Ser 180 185 190 Ser Tyr Val Leu Gln Thr Glu Glu
Leu Val Val Asn Lys Gln Gln Arg 195 200 205 Glu Thr Lys Asn Ser His
Gln Asn Lys Gly His Tyr Gln Asn Val Val 210 215 220 Asp Val Arg Glu
Glu His Ser Ser Lys Leu Gln Thr Ser Leu His Pro225 230 235 240 Ala
His Gln Asp Arg Leu Gln His Gly Pro Lys Asp Ile Phe Thr Thr 245 250
255 Gln Asp Glu Leu Leu Val Tyr Asn Lys Asn Gln His Gln Thr Lys Asn
260 265 270 Leu Ser Gln Asp Gln Glu His Gly Arg Lys Ala His Lys Ile
Ser Tyr 275 280 285 Pro Ser Ser Arg Thr Glu Glu Arg Gln Leu His His
Gly Glu Lys Ser 290 295 300 Val Gln Lys Asp Val Ser Lys Gly Ser Ile
Ser Ile Gln Thr Glu Glu305 310 315 320 Lys Ile His Gly Lys Ser Gln
Asn Gln Val Thr Ile His Ser Gln Asp 325 330 335 Gln Glu His Gly His
Lys Glu Asn Lys Ile Ser Tyr Gln Ser Ser Ser 340 345 350 Thr Glu Glu
Arg His Leu Asn Cys Gly Glu Lys Gly Ile Gln Lys Gly 355 360 365 Val
Ser Lys Gly Ser Ile Ser Ile Gln Thr Glu Glu Gln Ile His Gly 370 375
380 Lys Ser Gln Asn Gln Val Arg Ile Pro Ser Gln Ala Gln Glu Tyr
Gly385 390 395 400 His Lys Glu Asn Lys Ile Ser Tyr Gln Ser Ser Ser
Thr Glu Glu Arg 405 410 415 Arg Leu Asn Ser Gly Glu Lys Asp Val Gln
Lys Gly Val Ser Lys Gly 420 425 430 Ser Ile Ser Ile Gln Thr Glu Glu
Lys Ile His Gly Lys Ser Gln Asn 435 440 445 Gln Val Thr Ile Pro Ser
Gln Asp Gln Glu His Gly His Lys Glu Asn 450 455 460 Lys Met Ser Tyr
Gln Ser Ser Ser Thr Glu Glu Arg Arg Leu Asn Tyr465 470 475 480 Gly
Gly Lys Ser Thr Gln Lys Asp Val Ser Gln Ser Ser Ile Ser Phe 485 490
495 Gln Ile Glu Lys Leu Val Glu Gly Lys Ser Gln Ile Gln Thr Pro Asn
500 505 510 Pro Asn Gln Asp Gln Trp Ser Gly Gln Asn Ala Lys Gly Lys
Ser Gly 515 520 525 Gln Ser Ala Asp Ser Lys Gln Asp Leu Leu Ser His
Glu Gln Lys Gly 530 535 540 Arg Tyr Lys Gln Glu Ser Ser Glu Ser His
Asn Ile Val Ile Thr Glu545 550 555 560 His Glu Val Ala Gln Asp Asp
His Leu Thr Gln Gln Tyr Asn Glu Asp 565 570 575 Arg Asn Pro Ile Ser
Thr 580 4375PRTHomo sapiens 4Met Lys Ser Ser Val Phe Val Leu Ser
Leu Leu Leu Ile Leu Glu Arg1 5 10 15 Gln Ser Ala Val Phe Val Gln
Tyr Gly Ala Thr Lys Gly His Phe Gln 20 25 30 Ser Ser Ser Ser Glu
Gly Phe Met Leu Gly Gln Lys Gly Arg Leu Ser 35 40 45 Phe Gly Ile
Lys Gly Gly Ser Asp Glu Ala Ala Glu Glu Ser Leu Phe 50 55 60 Met
Gln Ser Gln Arg Arg Val Tyr Gly Gln Gly Gly Gly Asp Met Thr65 70 75
80 Gln Thr Arg Val Ser Gln Glu His Thr Ser Val Lys Gly Ala Ala Leu
85 90 95 Cys Arg Asn Gly Gln Val Ser Gln Leu Lys Ser Gln Glu Ser
Gln Ile 100 105 110 Lys Ser Tyr Gly Gln Val Lys Ser Ser Gly Gln Leu
Lys Ser Gly Gly 115 120 125 Ser Ala Phe Gly Gln Val Lys Ser Ser Val
Ser Gln Ile Lys Ser Tyr 130 135 140 Gly Gln Leu Lys Ser Gly Gly Gln
Leu Lys Ser Gly Gly Pro Ala Phe145 150 155 160 Gly Gln Val Lys Ser
Gln Glu Ser Gln Ile Lys Ser Tyr Gly Gln Leu 165 170 175 Lys Ser Ser
Gly Gln Leu Lys Ser Gly Gly Ser Ala Phe Gly Gln Val 180 185 190 Lys
Ser Ser Val Ser Gln Ile Lys Ser Tyr Gly Gln Leu Lys Ser Gly 195 200
205 Gly Ser Gln Val Lys Ser Tyr Gly Gln Thr Lys Ser Tyr Gly Glu Glu
210 215 220 Gly Gln Leu Asn Ser Phe Ser Gln Leu Lys Ser Gln Gly Ala
Gln Leu225 230 235 240 Lys Ser Tyr Gly Gln Gln Lys Ser Gln Gln Gln
Ser Ser Phe Ser Gln 245 250 255 Val Lys Ser Gln Ser Ser Gln Leu Lys
Ser Tyr Gly Gln Gln Lys Ser 260 265 270 Leu Lys Gly Phe Ser Gln Gln
Thr Gln His Lys Gly Phe Ala Met Asp 275 280 285 Glu Gly Met Ser Gln
Val Arg Lys Gln Phe Ser Asp Asp Asp Leu Ser 290 295 300 Val Gln Gln
Lys Ser Thr Gln Gln Met Lys Thr Glu Glu Asp Leu Ser305 310 315 320
Gln Phe Gly Gln Gln Arg Gln Tyr Gly Gln Glu Arg Ser Gln Ser Tyr 325
330 335 Lys Gly Tyr Leu Glu Gln Tyr Arg Lys Lys Val Gln Glu Gln Gln
Arg 340 345 350 Lys Asn Phe Asn Pro Gly Asn Tyr Phe Thr Lys Gly Gly
Ala Asp Leu 355 360 365 Tyr Gln Ala Gln Leu Lys Gly 370 375
5414PRTHomo sapiens 5Met Lys Ser Ser Val Phe Ile Leu Ser Leu Phe
Leu Leu Leu Glu Arg1 5 10 15 Gln Ala Ala Val Val Gly Gln Tyr Gly
Gly Thr Lys Gly His Phe Gln 20 25 30 Ser Ser Ser Ser Gly Phe Met
Leu Gly Gln Lys Gly His Leu Asn Phe 35 40 45 Gly Leu Lys Gly Gly
Ser Glu Glu Ala Ala Glu Glu Ser Ile Phe Met 50 55 60 Gln Ser Gln
His Gln Met Phe Gly Gln Asp Gly Gly Asp Met Ala Gln65 70 75 80 Thr
Ser Val Ser Gln Glu His Thr Gly Val Lys Gly Ala Ala Ile Cys 85 90
95 Arg Lys Gly Gln Val Ser Gln Leu Lys Ser Gln Glu Ser Gln Ile Lys
100 105 110 Ser Phe Arg Gln Val Lys Ser Ser Gly Gln Leu Lys Ser Gly
Gly Ser 115 120 125 Gln Leu Lys Ser Phe Gly Gln Val Lys Ser Ser Glu
Ser Gln Leu Lys 130 135 140 Ser Phe Gly Gln Val Lys Ala Ser Gly Ser
Gln Leu Lys Ser Phe Gly145 150 155 160 Gln Val Lys Ala Ser Gly Ser
Gln Leu Lys Ser Tyr Gly Gln Met Lys 165 170 175 Ser Ser Gly Ser Gln
Val Lys Ser Phe Gly Gln Met Lys Ser Ser Gly 180 185 190 Ser Gln Val
Lys Ser Phe Gly Gln Met Lys Ala Ser Glu Ser Gln Ile 195 200 205 Lys
Ser Phe Gly Gln Arg Lys Ser Gln Gly Gly Gln Leu Gln Ser Tyr 210 215
220 Gly Gln Met Lys Ser Tyr Gly Gln Thr Lys Ser Leu Glu Ser Gln
Ala225 230 235 240 Lys Ser Phe Gly Gln Val Lys Ser Gln Ser Gly Gln
Met Lys Ser Ser 245 250 255 Tyr Gly Gln Arg Lys Ser Tyr Gly Glu Glu
Thr Gln Leu Lys Ser Phe 260 265 270 Asp Gln Asp Ala Gln Leu Lys Ser
Tyr Gly Gln Gln Lys Ser Gln Lys 275 280 285 Gln Ser Ser Phe Ser Gln
Val Lys Ser Gln Ser Ala Gln Leu Lys Ser 290 295 300 Phe Gly Gln Gln
Lys Ser Leu Lys Gly Phe Ser Gln Gln Thr Gln Gln305 310 315 320 Lys
Gly Phe Ala Met Asp Glu Asp Leu Ser Gln Val Arg Lys Gln Phe 325 330
335 Asp Asp Asp Asp Leu Ser Val Gln Gln Lys Ser Thr Gln Gln Met Lys
340 345 350 Thr Glu Glu Asp Leu Ser Gln Phe Gly Gln Gln Arg Gln Phe
Gly Gln 355 360 365 Glu Arg Ser Gln Ser Tyr Lys Gly Tyr Leu Ala Gln
Tyr Arg Lys Lys 370 375 380 Leu Gln Glu Gln Gln Gln Gln Lys Asn Phe
Asn Gln Asp Asn Phe Phe385 390 395 400 Thr Lys Gly Gly Ala Gly Leu
Tyr Gln Ala Gln Leu Lys Gly 405 410
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