U.S. patent application number 13/203831 was filed with the patent office on 2012-01-05 for polypeptide structural motifs associated with cell signaling activity.
This patent application is currently assigned to ATYR PHARMA, INC.. Invention is credited to Leslie Ann Greene.
Application Number | 20120004185 13/203831 |
Document ID | / |
Family ID | 42666252 |
Filed Date | 2012-01-05 |
United States Patent
Application |
20120004185 |
Kind Code |
A1 |
Greene; Leslie Ann |
January 5, 2012 |
POLYPEPTIDE STRUCTURAL MOTIFS ASSOCIATED WITH CELL SIGNALING
ACTIVITY
Abstract
Isolated polypeptides comprising or consisting essentially of
specific structural motifs (e.g., three .beta.-sheets and two
.alpha.-helices) are provided, wherein the polypeptides exhibit at
least one cell signaling and/or other non-canonical activity of
biological relevance. Also provided are polynucleotides encoding
such polypeptides, binding agents that bind such polypeptides,
analogs, variants and fragments of such polypeptides, etc., as well
as compositions and methods of identifying and using any of the
foregoing.
Inventors: |
Greene; Leslie Ann; (San
Diego, CA) |
Assignee: |
ATYR PHARMA, INC.
San Diego
CA
|
Family ID: |
42666252 |
Appl. No.: |
13/203831 |
Filed: |
February 26, 2010 |
PCT Filed: |
February 26, 2010 |
PCT NO: |
PCT/US10/25642 |
371 Date: |
August 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61156370 |
Feb 27, 2009 |
|
|
|
Current U.S.
Class: |
514/21.2 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/348; 435/375;
435/419; 435/6.17; 435/7.1; 436/501; 436/86; 436/94; 514/21.3;
530/324; 530/350; 536/23.1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 2317/24 20130101; C12N 9/0036 20130101; G01N 2500/20 20130101;
Y10T 436/143333 20150115; C12Y 601/01012 20130101; G01N 2333/52
20130101; G01N 2500/04 20130101; C07K 16/40 20130101; A61K 38/00
20130101; G01N 2500/10 20130101; C07K 14/521 20130101; G01N 33/68
20130101; C12Y 601/01021 20130101; C12N 9/93 20130101; G01N
2333/9015 20130101 |
Class at
Publication: |
514/21.2 ;
530/324; 530/350; 514/21.3; 536/23.1; 435/320.1; 435/325; 435/375;
435/7.1; 435/6.17; 436/86; 436/94; 435/348; 435/254.2; 435/419;
435/252.3; 436/501 |
International
Class: |
A61K 38/19 20060101
A61K038/19; C07H 21/00 20060101 C07H021/00; C12N 15/63 20060101
C12N015/63; C12N 5/00 20060101 C12N005/00; G01N 33/53 20060101
G01N033/53; A61P 43/00 20060101 A61P043/00; G01N 33/68 20060101
G01N033/68; G01N 33/50 20060101 G01N033/50; C12N 5/10 20060101
C12N005/10; C12N 1/19 20060101 C12N001/19; C12N 1/21 20060101
C12N001/21; G01N 33/566 20060101 G01N033/566; C07K 14/52 20060101
C07K014/52; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. An isolated polypeptide consisting essentially of three
.beta.-sheets and two .alpha.-helices, wherein the polypeptide
exhibits a cell signaling or other non-canonical activity.
2. The isolated polypeptide of claim 1, wherein the polypeptide
consists essentially of three antiparallel .beta.-sheets flanked at
each end by an .alpha.-helix.
3. The isolated polypeptide of claim 1, wherein the polypeptide
comprises about 60 to 200 amino acid residues.
4. The isolated polypeptide of claim 1, wherein the polypeptide
comprises a contiguous fragment of a human protein, or a sequence
having at least 80% identity to said contiguous fragment.
5. The isolated polypeptide of claim 1, wherein the polypeptide
comprises at least two non-contiguous fragments of a human protein,
or sequences having at least 80% identity to said non-contiguous
fragments.
6. The isolated polypeptide of claim 1, wherein the polypeptide is
a) a GlyRS polypeptide comprising residues 345-438 of SEQ ID NO: 1
or an active fragment or variant thereof; b) an AspRS polypeptide
comprising residues 367-448 of SEQ ID NO: 2 or an active fragment
or variant thereof; c) a HisRS polypeptide comprising residues
294-372 of SEQ ID NO: 3 or an active fragment or variant thereof;
d) a ThrRS polypeptide comprising residues 469-586 of SEQ ID NO: 4
or an active fragment or variant thereof; e) a GluProRS polypeptide
comprising residues 1171-1253 of SEQ ID NO: 5 or an active fragment
or variant thereof; f) a SerRS polypeptide comprising residues
325-410 of SEQ ID NO: 6 or an active fragment or variant thereof;
g) a PheRS polypeptide comprising residues 380-449 of SEQ ID NO: 7
or an active fragment or variant thereof; h) a LysRS polypeptide
comprising residues 425-523 of SEQ ID NO: 8 or an active fragment
or variant thereof; i) a AsnRS polypeptide comprising residues
416-494 of SEQ ID NO: 9 or an active fragment or variant thereof;
j) a AlaRS polypeptide comprising residues 148-258 of SEQ ID NO: 10
or an active fragment or variant thereof; k) a thioredoxin
polypeptide comprising residues 20-105 of SEQ ID NO: 11 or an
active fragment or variant thereof; l) a macrophage inhibitory
factor polypeptide comprising residues 1-90 of SEQ ID NO: 12 or an
active fragment or variant thereof; and/or m) a peroxiredoxin 5
isoform B polypeptide comprising residues 32-68 and 125-161 of SEQ
ID NO: 13 or an active fragment or variant thereof.
7.-18. (canceled)
19. The isolated polypeptide of claim 1, wherein the polypeptide
has chemokine activity.
20. A pharmaceutical composition comprising a polypeptide according
to claim 1.
21. An a) isolated polynucleotide encoding a polypeptide according
to claim 1, b) vector comprising an isolated polynucleotide of a),
or c) host cell comprising a vector of b).
22-23. (canceled)
24. A method for identifying a polypeptide fragment having a cell
signaling activity, comprising the steps of identifying a protein
sequence containing a structural motif comprised of two
.alpha.-helices and three .beta.-sheets, determining the amino acid
residue boundaries of the structural motif within said protein, and
thereby identifying a polypeptide fragment having cell signaling
activity.
25. The method of claim 24, wherein the structural motif consists
essentially of at least three antiparallel .beta.-sheets flanked at
each end by an .alpha.-helix.
26. The method of claim 24, wherein the polypeptide fragment
comprises about 60 to 200 amino acids of the protein.
27. The method of claim 24, wherein the step of identifying a
protein sequence containing a structural motif comprised of two
.alpha.-helices and three anti-parallel .beta.-sheets is performed
using a secondary structure prediction method.
28. The method of claim 24, wherein the step of identifying a
protein sequence containing a structural motif comprised of two
.alpha.-helices and three anti-parallel .beta.-sheets is performed
using a secondary structure prediction method selected from the
group consisting of PHDsec, NSSP, SOPM, DSC, SSPRED, MultiPredict,
PSA, NNPREDICT, APSSP, GOR, HNN, HTMSRAP, Jpred, JUFO, nnPredict,
Porter, PredictProtein, Prof, PSIpred, SOPMA, SSpro and
DLP-SVM.
29. A method for modulating cell signaling comprising contacting a
cell with an effective amount of a composition comprising: a) a
polypeptide according to claim 1; b) a polynucleotide according to
claim 21; and/or c) an antibody or antigen-binding fragment thereof
that specifically binds a polypeptide according to claim 1.
30. The method of claim 29, wherein the method is a method of: a)
modulating chemokine activity; b) modulating TNF-.alpha. secretion;
c) inducing TNF-.alpha. secretion; d) modulating immune cell
chemotaxis; and/or e) inducing monocyte chemotaxis.
31.-34. (canceled)
35. A screening method for identifying a modulator of cell
signaling, comprising the steps of: (a) forming a reaction mixture
including: (i) a component selected from the group consisting of a
polypeptide according to claim 1; a polynucleotide according to
claim 21; and/or an antibody or antigen-binding fragment thereof
that specifically binds a polypeptide according to claim 1; (ii) a
binding partner, cellular effector and/or cell type known to be
bound and/or modulated by said component; and (iii) a test
compound; and (b) detecting an interaction of said component with
the binding partner, cellular effector and/or cell type, wherein a
change in the interaction in the presence of test compound,
relative to the interaction in the absence of the test compound,
thereby identifies a modulator of cell signaling.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/156,370,
filed Feb. 27, 2009, which is incorporated by reference in its
entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120161.sub.--411_PC_SEQUENCE_LISTING.txt. The text file is 61 KB,
was created on Feb. 26, 2010, and is being submitted electronically
via EFS-Web, concurrent with the filing of the specification.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to polypeptides
containing structural motifs associated with cell signaling and
other biologically relevant activities, compositions comprising
such polypeptides, and methods of identifying and using same.
[0005] 2. Description of the Related Art
[0006] Aminoacyl-tRNA synthetase (AARS) proteins, which catalyze
the aminoacylation of tRNA molecules, are essential for decoding
genetic information during the process of translation. Each of the
eukaryotic tRNA synthetases consists of a core enzyme, which is
closely related to the prokaryotic tRNA synthetase, as well as
additional domains that are appended to the amino-terminal end,
carboxyl-terminal end or inserted into a region internal to the
core enzyme. Human tyrosyl-tRNA synthetase (TyrRS), for example,
has a carboxyl-terminal domain that is not part of prokaryotic and
lower eukaryotic TyrRS molecules. Several aminoacyl-tRNA
synthetases have been demonstrated to have non-canonical functions
distinct from their involvement in translation. For example,
mini-tyrosyl tRNA synthetase (mini-TyrRS), the N-terminal domain of
TyrRS which corresponds to amino acid residues 1-364 and is cleaved
by polymorphonuclear cell elastase and plasmin, exhibits
non-canonical activities not found in the full-length protein. In
vitro, mini-TyrRS has been shown to stimulate neutrophil activation
and chemotaxis, endothelial cell proliferation and migration, and
is pro-angiogenic in chick chorioallantoic membrane (CAM) and mouse
matrigel assays. Mini-TyrRS has an ELR motif that, like
CXC-chemokines such as IL-8, is involved in many of its chemokine
and angiogenic activities. As in other ELR-containing cytokines,
mutation of this motif inhibits mini-TyrRS binding to and
stimulation of leukocytes and angiogenesis.
[0007] In addition, truncated forms of TrpRS have been demonstrated
to have anti-angiogenic properties. In normal human cells, there
are two forms of TrpRS that can be detected: a major form
consisting of the full-length molecule (amino acid residues 1-471)
and a minor truncated form. The minor form is generated by the
deletion of an amino-terminal domain through alternative splicing
of the pre-mRNA and is termed mini-TrpRS. The amino-terminus of
miniTrpRS has been determined to be the methionine residue at
position 48 of the full-length TrpRS molecule. Alternatively,
truncated TrpRS can be generated by proteolysis. For example,
bovine TrpRS is highly expressed in the pancreas and is secreted
into the pancreatic juice, thus resulting in the production of a
truncated TrpRS molecule. Additional studies indicate that
miniTrpRS inhibits VEGF-induced cell proliferation and migration
(Wakasugi et al., Proc. Natl. Acad. Sci. 99: 173-177 (2002)). In
particular, a chick CAM assay shows that miniTrpRS blocks
angiogenic activity of VEGF. In contrast, the full-length TrpRS
does not inhibit angiogenesis. Thus, removal of the first 48 amino
acid residues exposes the anti-angiogenic activity of TrpRS.
Therefore, as with TyrRS, certain forms of TrpRS possess activities
other than the aminoacylation of tRNA.
[0008] Other cytoplasmic proteins have also been shown to have
isoforms with extracellular biological activities. For example,
human thioredoxin (Hu-Trx) serves a key role in redox activities
within the cytoplasm of cells, but has a truncated secreted form
that is a mitogenic cytokine (Arner, 2000; Eur J biochem
267:6102-6109).
[0009] Unfortunately, there are no reliable methods available for
predicting or identifying which cytoplasmic proteins or polypeptide
fragments derived from AARS proteins and/or other proteins may have
novel and previously unappreciated activities. The present
invention addresses these needs and offers other related
advantages.
SUMMARY OF THE INVENTION
[0010] The present invention relates to isolated polypeptides
comprising or consisting essentially of particular structural
motifs, as described herein, which exhibit at least one cell
signaling and/or other non-canonical activity of biological
relevance. The present invention further relates to polynucleotides
encoding such polypeptides, binding agents that bind such
polypeptides, analogs, variants and fragments of such polypeptides,
etc., as well as compositions and methods of identifying and using
any of the foregoing.
[0011] Therefore, according to one aspect of the invention, there
are provided isolated polypeptides comprising or consisting
essentially of three .beta.-sheets and two .alpha.-helices, wherein
the polypeptides exhibit a cell signaling and/or other
non-canonical activity relative to the protein from which they were
derived. The precise order and orientation of the required
.beta.-sheets and .alpha.-helices within a polypeptide of the
invention may vary while still giving rise to the desired cell
signaling and/or other non-canonical activities. However, in a
particularly illustrative embodiment, a polypeptide of the
invention consists essentially of three antiparallel .beta.-sheets
flanked at each end by an .alpha.-helix.
[0012] The size of a polypeptide of the invention can vary while
still having the desired structural and functional features
described herein. However, in certain illustrative embodiments, an
isolated polypeptide of the invention will have a size in the range
of about 40-400, 50-300 or 60-200 amino acid residues.
[0013] In many embodiments, a polypeptide of the invention will be
a contiguous fragment of a mammalian protein (e.g., a human
protein), or a sequence sharing substantial structural identity
(e.g., at least 70%, 80% or 90%) with said contiguous fragment.
However, in other embodiments, it will be understood that the
polypeptide may be comprised of two or more non-contiguous
fragments of a protein, or sequences sharing substantial sequence
identity with said non-contiguous fragments.
[0014] In a specific embodiment of the invention, an isolated
polypeptide of the invention is a GlyRS (GRS) polypeptide
comprising or consisting essentially of residues 345-438 of SEQ ID
NO: 1, which fragment has been identified herein as containing a
structural motif of the invention and having cell signaling
activity. In a related embodiment, the isolated polypeptide is an
active fragment or variant of a polypeptide comprising residues
345-438 of SEQ ID NO: 1, e.g., a fragment of the polypeptide which
retains the same or similar cell signaling activity or a variant
which shares substantial sequence identity thereto (e.g., at least
80% or 90%) and which retains the same or similar cell signaling
activity.
[0015] In another specific embodiment of the invention, the
polypeptide is an AspRS (DRS) polypeptide comprising or consisting
essentially of residues 367-448 of SEQ ID NO: 2 or an active
fragment or variant thereof.
[0016] In another specific embodiment, the polypeptide is a HisRS
(HRS) polypeptide comprising or consisting essentially of residues
294-372 of SEQ ID NO: 3 or an active fragment or variant
thereof.
[0017] In yet another specific embodiment, the polypeptide is a
ThrRS (TRS) polypeptide comprising or consisting essentially of
residues 469-586 of SEQ ID NO: 4 or an active fragment or variant
thereof.
[0018] In another specific embodiment, the polypeptide is a
GluProRS (EPRS) polypeptide comprising or consisting essentially of
residues 1171-1253 of SEQ ID NO: 5 or an active fragment or variant
thereof.
[0019] In still another specific embodiment of the invention, the
polypeptide is a SerRS (SRS) polypeptide comprising or consisting
essentially of residues 325-410 of SEQ ID NO: 6 or an active
fragment or variant thereof.
[0020] In still another specific embodiment of the invention, the
polypeptide is a PheRS (FRS) polypeptide comprising or consisting
essentially of residues 380-449 of SEQ ID NO: 7 or an active
fragment or variant thereof.
[0021] In still another specific embodiment of the invention, the
polypeptide is a LysRS (KRS) polypeptide comprising or consisting
essentially of residues 425-523 of SEQ ID NO: 8 or an active
fragment or variant thereof.
[0022] In still another specific embodiment of the invention, the
polypeptide is a AspRS (NRS) polypeptide comprising or consisting
essentially of residues 416-494 of SEQ ID NO: 9 or an active
fragment or variant thereof.
[0023] In still another specific embodiment of the invention, the
polypeptide is a AlaRS (ARS) polypeptide comprising or consisting
essentially of residues 148-258 of SEQ ID NO: 10 or an active
fragment or variant thereof.
[0024] In another specific embodiment of the invention, the
polypeptide is a thioredoxin polypeptide comprising or consisting
essentially of residues 20-105 of SEQ ID NO: 11 or an active
fragment or variant thereof. In another embodiment, the polypeptide
is a Trx80 polypeptide comprising or consisting essentially of
residues 20-84 of Trx80, which is a secreted form of thioredoxin
containing the N-terminal 84 amino acid residues of
thioredoxin.
[0025] In another specific embodiment, the polypeptide is a
macrophage inhibitory factor polypeptide comprising or consisting
essentially of residues 1-90 of SEQ ID NO: 12 or an active fragment
or variant thereof.
[0026] In yet another specific embodiment, the polypeptide is a
human peroxiredoxin 5 isoform B polypeptide comprising or
consisting essentially of residues 32-68 and 125-161 of SEQ ID NO:
13 or an active fragment or variant thereof.
[0027] In certain embodiments, the cell signaling activity of a
polypeptide of the invention is a chemokine and/or cytokine
activity and may be determined and/or confirmed using any of a
variety of illustrative assays known and established in the art. In
specific embodiments, cell signaling activity may be determined
using essentially any assay that measures cytokine activity,
chemokine activity, chemotaxis, cell migration, cytokine release,
cell differentiation and/or cell toxicity. In more specific
embodiments, cytokine activity may be determined, for example,
using an assay which measures GPCR-dependent chemotaxis of
monocytes, release of interleukins, and/or apoptosis.
[0028] Certain embodiments include methods of modulating chemokine
activity, such as TNF-.alpha. secretion. Specific embodiments
include methods of inducing TNF-.alpha. secretion. Certain
embodiments include methods of modulating immune cell chemotaxis.
Specific embodiments include methods of inducing monocyte
chemotaxis. Certain embodiments include methods of modulating
Toll-like receptor signaling. Specific embodiments include methods
of inducing Toll-like receptor 2 signaling.
[0029] According to another aspect of the invention, there is
provided an isolated polynucleotide encoding a polypeptide as
described herein.
[0030] According to yet another aspect, the invention provides a
pharmaceutical composition comprising a polypeptide as described
herein, or a polynucleotide encoding a polypeptide as described
herein.
[0031] According to another aspect, the invention provides a vector
comprising an isolated polynucleotide as described herein, as well
as a host cell comprising such a vector.
[0032] According to yet another aspect, the invention provides
screening methods for identifying proteins containing a structural
motif of the invention and thereby identifying novel polypeptide
fragments within said proteins that possess cell signaling and/or
other non-canonical activities. For example, in one embodiment, the
invention provides a method for identifying a polypeptide fragment
having a cytokine activity by identifying a protein sequence
containing a structural motif as described herein, e.g., comprised
of two .alpha.-helices and three .beta.-sheets, determining the
amino acid residue boundaries of the structural motif within said
protein, and thereby identifying a polypeptide fragment of the
protein which has a cytokine activity.
[0033] In certain embodiments of the described screening methods,
the step of identifying a protein sequence containing a structural
motif comprised of two .alpha.-helices and three anti-parallel
.beta.-sheets is performed using a secondary structure prediction
method known and available in the art, which may illustratively
include, but are not limited to, PHDsec, NSSP, SOPM, DSC, SSPRED,
MultiPredict, PSA, NNPREDICT, APSSP, GOR, HNN, HTMSRAP, Jpred,
JUFO, nnPredict, Porter, PredictProtein, Prof, PSIpred, SOPMA,
SSpro and DLP-SVM.
[0034] In still other aspects, the polypeptides, antibodies and/or
other compositions of the present invention may be used in
essentially any type of screening assay known and available in the
art. For example, compositions of the invention (e.g.,
polypeptides, polynucleotides and/or antibodies) may be used in
conjunction with essentially any known screening methodology in
order to identify suitable cell types and/or disease conditions
amenable to treatment according to the present invention. In other
examples, compositions of the invention (e.g., polypeptides,
polynucleotides and/or antibodies) may be used in conjunction with
known screening methodologies in order to identify binding
partners, competitive inhibitors, cellular effectors, and the like,
that mediate or modulate, either directly or indirectly, the cell
signaling and/or non-canonical activities of the compositions
herein. For example, in a particular embodiment, a screening method
is provided for identifying test compounds as inhibitors, or
alternatively, potentiators, of an interaction between a
composition of the invention and one or more of its binding
partners, cellular effectors and/or cell types subject to
modulation. This may include, for example, steps of forming a
reaction mixture including: (i) a composition of the invention,
(ii) a binding partner, cellular effector and/or cell type known to
be modulated by said composition, and (iii) a test compound; and
detecting interaction of the test compound with the binding
partner, cellular effector and/or cell type. A statistically
significant change (potentiation or inhibition) in activity or
modulation in the presence of the test compound, relative to the
interaction in the absence of the test compound, indicates a
potential agonist (mimetic or potentiator) or antagonist
(inhibitor) of activity.
BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS
[0035] SEQ ID NO: 1 is the full length amino acid sequence of human
glycyl-tRNA synthetase (GlyRS).
[0036] SEQ ID NO: 2 is the full length amino acid sequence of human
aspartyl-tRNA synthetase (AspRS).
[0037] SEQ ID NO: 3 is the full length amino acid sequence of human
histidyl-tRNA synthetase (HisRS).
[0038] SEQ ID NO: 4 is the full length amino acid sequence of human
threonyl-tRNA synthetase (ThrRS).
[0039] SEQ ID NO: 5 is the full length amino acid sequence of human
glutamyl-/prolyl-tRNA synthetase (GluProRS).
[0040] SEQ ID NO: 6 is the full length amino acid sequence of human
seryl-tRNA synthetase (SerRS).
[0041] SEQ ID NO: 7 is the full length amino acid sequence of human
phenylalanyl-tRNA synthetase (FRSa).
[0042] SEQ ID NO: 8 is the full length amino acid sequence of human
lysyl-tRNA synthetase (KRS).
[0043] SEQ ID NO: 9 is the full length amino acid sequence of human
asparaginyl-tRNA synthetase (NRS).
[0044] SEQ ID NO: 10 is the full length amino acid sequence of
human alanyl-tRNA synthetase (ARS).
[0045] SEQ ID NO: 11 is the full length amino acid sequence of
human thioredoxin.
[0046] SEQ ID NO: 12 is the full length amino acid sequence of
human macrophage inhibitory factor.
[0047] SEQ ID NO: 13 is the full length amino acid sequence of
human peroxiredoxin 5 isoform B.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows an illustrative structural motif of the
invention comprising three antiparallel .beta.-sheets flanked by an
.alpha.-helix at each end.
[0049] FIGS. 2A-2C show the identification of a fragment of human
GlyRS containing a structural motif of the invention and
demonstration that the fragment has chemokine activity. FIG. 2A
shows the location of the G6 fragment and structural motif within
full-length GlyRS. FIG. 2B shows a structural model of GlyRS
residues 344-420. FIG. 2C shows that the G6 fragment induces
GPCR-dependent migration of THP-1 monocyte cells.
[0050] FIG. 3 shows that the identified structural motif is found
in other class II AARS proteins, including GlyRS (GRS), ThrRS
(TRS), HisRS (HRS), AspRS (DRS), GluProRS (EPRS), SerRS (SRS),
AsnRS (NRS), AlaRS (ARS), PheRS (FRS) and LysRS (KRS).
[0051] FIG. 4 shows the structural conservation of a motif of the
invention across multiple class II AARS proteins, despite its
relatively low sequence conservation.
[0052] FIG. 5 shows that a structural motif of the invention is
also found in non-AARS proteins, including (A) human thioredoxin,
(B) macrophage inhibitory protein (residues 1-90), and (C)
peroxiredoxin 5 isoform B (residues 32-68 and 125-161). One feature
of the proteins that share this structural motif is that they are
unconventionally secreted.
[0053] FIG. 6 shows that a structural motif of the invention is
found within a fragment of human seryl tRNA synthetase (SerRS) that
exhibits cell-signaling activity. This figure shows the location of
the S3 fragment (residues 253-484 of full-length SerRS) and the
structural motif (residues 325-410) within the full-length SerRS
polypeptide sequence.
[0054] FIGS. 7A-7C show that the S3 fragment (residues 253-484 of
full-length SerRS) containing a structural motif of the invention
binds to human monocytes and B-cells and stimulates secretion of
TNF-.alpha. from a human monocytic cell line. FIG. 7A shows S3
binding to human monocytes, FIG. 7B shows S3 binding to human
B-cells, and FIG. 7C shows induction of TNF-.alpha. secretion from
human monocytic cell line (THP-1) as compared to LPS control.
[0055] FIG. 8 shows that the S3 fragment signaled through Toll-like
receptor 2 to elicit a strong secretion of alkaline phoshpatase in
a dose-dependent manner (x-axis=dosage in nM;
y-axis=OD.sub.600).
[0056] FIG. 9 shows that S3-mediated signaling through Toll-like
receptor 2 (TLR2) was inhibited by pre-treatment with a monoclonal
antibody that blocks binding to mTLR2.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The practice of the present invention will employ, unless
indicated specifically to the contrary, conventional methods of
molecular biology and recombinant DNA techniques within the skill
of the art, many of which are described below for the purpose of
illustration. Such techniques are explained fully in the
literature. See, e.g., Sambrook, et al., Molecular Cloning: A
Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical
Approach, vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B.
Hames & S. Higgins, eds., 1985); Transcription and Translation
(B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.
Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B.
Perbal, ed., 1984).
[0058] All publications, patents and patent applications cited
herein are hereby incorporated by reference in their entirety.
[0059] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0060] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises," and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0061] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they materially
affect the activity or action of the listed elements.
[0062] An "agonist" refers to a molecule that intensifies or mimics
the non-canonical biological activity of an AARS. Agonists may
include proteins, nucleic acids, carbohydrates, small molecules, or
any other compound or composition that modulates the activity of an
AARS either by directly interacting with the AARS or its binding
partner, or by acting on components of the biological pathway in
which the ATRS participates. Included are partial and full
agonists.
[0063] The term "antagonist" refers to a molecule that inhibits or
attenuates the non-canonical biological activity of an AARS.
Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small molecules, or any other compound or
composition that modulates the activity of an AARS or its binding
partner, either by directly interacting with the AARS or its
binding partner or by acting on components of the biological
pathway in which the AARS participates. Included are partial and
full antagonists.
[0064] The term "modulating" includes "increasing" or
"stimulating," as well as "decreasing" or "reducing," typically in
a statistically significant or a physiologically significant amount
as compared to a control. An "increased" or "enhanced" amount is
typically a "statistically significant" amount, and may include an
increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30
or more times (e.g., 500, 1000 times) (including all integers and
decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8,
etc.) the amount produced by no composition (the absence of an
agent or compound) or a control composition. A "decreased" or
reduced amount is typically a "statistically significant" amount,
and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
decrease in the amount produced by no composition (the absence of
an agent or compound) or a control composition, including all
integers in between.
[0065] A "subject," as used herein, includes any animal that
exhibits a symptom, or is at risk for exhibiting a symptom, which
can be treated or diagnosed with a composition of the invention.
Suitable subjects (patients) include laboratory animals (such as
mouse, rat, rabbit, or guinea pig), farm animals, and domestic
animals or pets (such as a cat or dog). Non-human primates and,
preferably, human patients, are included.
[0066] "Treatment" or "treating," as used herein, includes any
desirable effect on the symptoms or pathology of a disease or
condition that can be effected by the cell-modulatory activities of
a composition as described herein, and may include even minimal
changes or improvements in one or more measurable markers of the
disease or condition being treated. "Treatment" or "treating" does
not necessarily indicate complete eradication or cure of the
disease or condition, or associated symptoms thereof. The subject
receiving this treatment is any subject in need thereof. Exemplary
markers of clinical improvement will be apparent to persons skilled
in the art.
[0067] As used herein, the terms "polypeptide" and "protein" are
used according to conventional meaning, i.e., as a sequence of
amino acids. Polypeptides are not limited to a specific length,
but, in the context of the present invention, typically represent a
fragment of a full length protein, and may include
post-translational modifications, for example, glycosylations,
acetylations, phosphorylations and the like, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring. Polypeptides and proteins of the invention
may be prepared using any of a variety of well known recombinant
and/or synthetic techniques, illustrative examples of which are
further discussed below.
[0068] The present invention relates generally to the
identification of polypeptide structural motifs associated with
unexpected biological activities of therapeutic relevance. For
example, as further described herein, a protein structural motif
was identified within an isolated fragment of the human glycyl-tRNA
synthetase (GRS) protein, and this structural motif is believed to
be responsible for the unexpected cell signaling activity found to
be induced by the GlyRS fragment. Further analysis of the
identified structural motif revealed its presence not only within
other human AARS proteins, but also within other proteins unrelated
to tRNA synthesis. Thus, based upon this discovery, the present
invention provides a means for identifying polypeptide fragments
having previously unrecognized cell signaling and/or other
biological activities, and further provides the polypeptide
fragments so identified. For example, in one illustrative
embodiment, by identifying the presence of a structural motif of
the invention within cytoplasmic proteins being screened, it is
possible to delineate novel secreted polypeptide fragments of those
cytoplasmic proteins having previously unappreciated activities
unrelated to their canonical cytoplasmic activities.
[0069] Accordingly, the present invention broadly provides isolated
polypeptides containing a structural motif as described herein,
wherein the polypeptides exhibit at least one cell signaling or
other non-canonical activity (e.g., an activity that is either not
observed for the full length protein from which the polypeptide was
derived or an activity that is significantly different (e.g.,
increased or decreased) relative to the full length protein from
which it was derived).
[0070] In a particular embodiment of the invention, isolated
polypeptides are provided that contain a structural motif comprised
of two .alpha.-helices and three .beta.-sheets, wherein the
polypeptides exhibit at least one cell signaling or other
non-canonical activity. In a more specific embodiment, isolated
polypeptides are provided that contain a structural motif comprised
of two .alpha.-helices and three antiparallel .beta.-sheets,
wherein the polypeptides exhibit at least one cell signaling or
other non-canonical activity. In an even more specific embodiment,
isolated polypeptides are provided that contain a structural motif
comprised of three antiparallel .beta.-sheets flanked by an
.alpha.-helix at each end (e.g., as depicted illustratively in FIG.
1), wherein the polypeptides exhibit at least one cell signaling or
other non-canonical activity.
[0071] A structural motif of the present invention, e.g., having
three .beta.-sheets and two .alpha.-helices, may be identified
within a protein using essentially any of a variety of well known
and established methodologies available in the art for the
prediction of protein secondary structure. Secondary structure
prediction is a set of techniques that can predict with high
accuracy the local secondary structures of proteins based on
knowledge of their primary amino acid sequence and/or other
parameters. For proteins, a prediction generally consists of
assigning regions of the amino acid sequence as likely to adopt a
particular secondary structure, e.g., .alpha.-helices,
.beta.-strands, .beta.-sheets, etc. The success of a prediction can
be evaluated, for example, by comparing it to the results of the
DSSP algorithm applied to the crystal structure of the protein (if
available). Specialized algorithms have been developed for the
detection of these and other well-defined patterns in proteins.
[0072] Accordingly, using such methodologies, the presence of
structural motifs of the present invention within proteins being
screened can be predicted with high accuracy using any of a number
of well-known and publicly available programs and/or modeling tools
for secondary structure prediction, several illustrative examples
of which are set forth below: [0073] ESyPred3D
(http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/),
Lambert C, Leonard N, De Bolle X, Depiereux E. ESyPred3D:
Prediction of proteins 3D structures. Bioinformatics. 2002
September; 18(9):1250-1256, which is an automated homology modeling
program; [0074] I_Tasser
(http://zhang.bioinformatics.ku.edu/I-TASSER/), which builds models
based on multiple threading alignments; [0075] Bhageerath
(http://www.scfbio-iitd.res.in/bhageerath/index.jsp) an energy
based protein structure prediction server' [0076] PHDsec
(http://www.embl-heidelberg.de/predictprotein/), which is a
multiple alignment-based neural network system; [0077] NSSP
(http://dot.imgen.bcm.tmc.edu:9331/pssprediction/pssp.html), which
is a multiple alignment-based nearest-neighbor method; [0078] SOPM
(http://www.ibcp.fr/predict.html), which is a multiple
alignment-based method combining various prediction programs;
[0079] DSC
(http://bonsai.lif.icnet.uk/bmm/dsc/dsc_read_align.html), which is
a multiple alignment-based program using statistics; [0080] SSPRED:
(http://www.embl-heidelberg.de/sspred/ssp_mul.html), which is a
multiple alignment-based program using statistics; [0081]
MultiPredict (http://kestrel.ludwig.ucl.ac.uk/zpred.html), which is
a multiple alignment-based method using physicochemical information
from a set of aligned sequences and statistical secondary structure
decision constants; [0082] PSA (http://bmerc-www.bu.edu/psa/),
which analyzes amino acid sequences to predict secondary structures
and folding classes; [0083] NNPREDICT
(http://www.cmpharm.ucsf.edu/.about.nomi/nnpredict.html), which is
a single-sequence based neural network prediction; [0084] APSSP
(http://imtech.res.in/raghava/apssp/), which is known as the
Advanced Protein Secondary Structure Prediction Server; [0085] GOR
(http://npsa-pbiljbcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html),
which relies on Information theory/Bayesian inference (Garnier et
al, 1996); [0086] HNN
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html)
which relies on a Hierarchical Neural Network method (Guermeur,
1997); [0087] HTMSRAP
(http://biotechnology.tbzmed.ac.ir/htmsrap/index.htm), which
employs Helical TransMembrane Segment Rotational Angle Prediction;
[0088] Jpred (http://www.compbio.dundee.ac.uk/.about.www-jpred/),
which involves neural network-based secondary structure prediction;
[0089] JUFO
(http://www.meilerlab.org/view.php?section=0&page=6), which
involves neural network-based protein secondary structure
prediction; [0090] nnPredict
(http://www.cmpharm.ucsf.edu/.about.nomi/nnpredict.html); [0091]
Porter (http://distill.ucd.ie/porter/); [0092] PredictProtein
(http://www.predictprotein.org/), which includes PHDsec, PHDacc,
PHDhtm, PHDtopology, PHDthreader, MaxHom and EvalSec; [0093] Prof
(http://www.aber.ac.uk/.about.phiwww/prof/), which uses cascaded
multiple classifiers for secondary structure prediction; [0094]
PSIpred (http://bioinf.cs.ucl.ac.uk/psipred/), which includes
various protein structure prediction methods; [0095] SOPMA
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)
(Geourjon and Deleage, 1995) [0096] SSpro
(http://www.igb.uci.edu/?page=tools&subPage=psss), which
includes secondary structure prediction using bidirectional
recurrent neural networks; and, [0097] DLP-SVM
(http://www.tuat.ac.jp/.about.domserv/cgi-bin/DLP-SVM.cgi), which
involves domain linker prediction using SVM.
[0098] Additional information regarding certain of these and other
secondary structure prediction methodologies for single sequences
can be found, for example, in Chou et al. (1974) Biochemistry, 13,
211-222; Lim (1974) Journal of Molecular Biology, 88, 857-872;
Garnier et al. (1978) Journal of Molecular Biology, 120, 97-120;
Kabsch et al. (1983) FEBS Letters, 155, 179-182; Deleage et al.
(1987) Protein Engineering, 1, 289-294 (DPM;
http://www.ibcp.fr/serv_pred.html); Presnell et al. (1992)
Biochemistry, 31, 983-993; Holley et al. (1989) Proceedings of the
National Academy of Science, 86, 152-156; King et al. (1990).
Journal of Molecular Biology, 216, 441-457; and Kneller et al.
(1990) Journal of Molecular Biology, 214, 171-182 (NNPRED;
http://www.cmpharm.ucsf.edu/.about.nomi/nnpredict.html).
[0099] Furthermore, additional information regarding certain of
these and other automated methods for predicting secondary
structure from multiply aligned protein sequences can be found, for
example, in Zvelebil et al. (1987) Journal of Molecular Biology,
195, 957-961 (ZPRED); Rost et al. (1993) Journal of Molecular
Biology,
232,584-599(PHD;http://www.embl-heidelberg.de/predictprotein/predictprote-
in.html); Salamov et al. (1995) Journal of Molecular Biology, 247,
1 (NNSSP;
http://dot.imgen.bcm.tmc.edu:9331/seq-search/struc-predict.html);
Geourjon et al. (1994), Protein Engineering, 7, 157-16 (SOPMA;
http://www.ibcp.fr/serv_pred.html); Solovyev et al. (1994) Computer
Applications in the Biosciences, 10, 661-669. (SSP;
http://dot.imgen.bcm.tmc.edu:9331/seq-search/struc-predict.html);
Wako et al. (1994) Journal of Molecular Biology, 238, 693-708;
Mehta et al. (1995) Protein Science 4, 2517-25 (SSPRED;
http://www.embl-heidelberg.de/sspred/sspred_info.html); and King et
al. (1996) Protein Sci 5, 2298-2310. (DSC;
http://www.bmm.icnetuk/dsc/dsc_form_align.html).
[0100] Structure visualization software can also be used, if
desired, in conjunction with publicly available crystal structures
for AARS or non-AARS proteins (e.g., via Swiss Prot database on the
NIH Entrez server) to visualize a protein of interest and identify
structural motifs (e.g., Accelrys Software Inc., Discovery Studio
Modeling Environment, Release 2.0, San Diego: Accelrys Software
Inc., 2007). Still other illustrative software tools available for
identifying secondary structural motifs include ESyPred3d (Lambert
2002; ESyPred3D: Prediction of proteins 3D structures.
Bioinformatics 18:1250-1256). Structure predictions can then be
visually inspected (e.g., in Accelrys Discovery Visualizer) for
peptide domains that have the desired structural motif.
[0101] After identifying a protein or polypeptide containing a
desired structural motif of the invention (e.g., containing three
.beta.-sheets flanked on each end by .alpha.-helices), using one or
more suitable techniques such as those discussed above, the amino
acid boundaries of the motif are determined and isolated
polypeptide fragments containing the motif can be produced (e.g.,
synthetically or recombinantly) and tested for confirmation of cell
signaling activity.
[0102] A polypeptide of the invention is said to have a "cell
signaling activity" when the polypeptide exhibits one or more
activities commonly associated with and/or induced by cell
signaling proteins, either directly or indirectly. In certain
embodiments, the cell signaling activity is a cytokine and/or
chemokine activity. Cytokines are a category of cell-signaling
molecules and chemokines are a family of small cytokines, or
proteins secreted by cells.
[0103] Proteins are generally classified as chemokines according to
shared structural characteristics such as small size (they are all
approximately 8-10 kilodaltons in size), and the presence of four
cysteine residues in conserved locations that are key to forming
their 3-dimensional shape. A hallmark activity of chemokines is
their ability to act as chemoattractants to induce and/or control
the migration of cells. Cells that are attracted by chemokines
generally follow a signal of increasing chemokine concentration
towards the source of the chemokine.
[0104] Chemokines serve various biological roles. Some chemokines
control cells of the immune system during processes of immune
surveillance, such as directing lymphocytes to the lymph nodes so
they can screen for invasion of pathogens by interacting with
antigen-presenting cells residing in these tissues. These are known
as homeostatic chemokines and are produced and secreted without any
need to stimulate their source cells. Some chemokines have roles in
development, such as promoting angiogenesis (the growth of new
blood vessels), or guiding cells to tissues that provide specific
signals critical for cellular maturation. Other chemokines are
inflammatory and are released from a wide variety of cells in
response to bacterial infection, viruses and agents that cause
physical damage. Their release is often stimulated by
pro-inflammatory cytokines such as interleukin 1. Inflammatory
chemokines function mainly as chemoattractants for leukocytes,
recruiting monocytes, neutrophils and other effector cells from the
blood to sites of infection or tissue damage. Certain inflammatory
chemokines activate cells to initiate an immune response or promote
wound healing. They are released by many different cell types and
serve to guide cells of both innate immune system and adaptive
immune system. Chemokines exert many of their biological effects by
interacting with G protein-linked transmembrane receptors called
chemokine receptors, which are selectively found on the surfaces of
their target cells.
[0105] Given these various biological roles and activities, the
chemokine, cytokine or cell signaling activities of a polypeptide
of the invention can be determined using any of a number of routine
and art-recognized assays. In certain embodiment, cell signaling
activity is determined using essentially any assay that measures
chemotaxis, cell migration, cytokine release, cell differentiation
and/or cell viability. In more specific embodiments, chemokine
activity is determined, for example, using an assay which measures
GPCR-dependent chemotaxis of monocytes release of interleukins
and/or apoptosis.
[0106] The isolated polypeptides of the invention can be
essentially any length and can be of essentially any origin as long
as they provide the necessary elements to constitute a structural
motif possessing the desired cell signaling or other non-canonical
activity.
[0107] In certain illustrative embodiments, a polypeptide of the
invention will range in size from about 30-100, 30-200, 30-300,
30-400, 30-500, 40-100, 40-200, 40-300, 40-400, 40-500, 50-100,
50-200, 50-300, 50-400, 50-500, 60-100, 60-200, 60-300, 60-400 or
60-500 amino acids.
[0108] In certain embodiments, a polypeptide of the invention is a
truncated mammalian (e.g., human) protein or an active variant
thereof. A truncated protein refers to a polypeptide which is
shorter than its corresponding full length protein, for example,
due to removal of amino acids from its N- and/or C-terminal ends.
The extent of the truncation, that is, the number of N- and/or
C-terminal amino acid residues removed from a full length protein
can vary considerably while still providing desired cellular
effects when administered to a cell, tissue or subject, as
described herein. In certain embodiments, at least about 5, 10, 15,
20, 25, 50, 75, 100, 150, 200, 250, 300, 350 amino acids, or more,
including all intermediate lengths, are truncated from the N-
and/or C-terminus of a full length protein. Intermediate lengths
are intended to include all integers there between, for example, 6,
7, 8, etc., 51, 52, 53, etc., 201, 202, 203, etc.
[0109] In other embodiments, a polypeptide of the invention is
comprised of one or more fragments of a mammalian protein, or
active variants thereof. For example, in one illustrative
embodiment, a polypeptide of the invention is comprised of a linear
stretch of contiguous amino acid (e.g., having a length within the
ranges noted above) derived from a mammalian protein, such as a
human protein. Alternatively, a polypeptide of the invention may be
comprised of non-contiguous fragments of a mammalian protein,
wherein the non-contiguous fragments are sufficient to constitute a
structural motif as described herein.
[0110] In a specific embodiment of the invention, the polypeptide
is a fragment of a GlyRS protein, wherein the fragment has a cell
signaling or other non-canonical activity. In a more specific
embodiment, the polypeptide is a truncated form of a GlyRS protein
comprising amino acid residues 345-420 of the human GlyRS protein
set forth in SEQ ID NO: 1, or an active fragment or variant
thereof.
[0111] In another embodiment of the invention, the polypeptide is a
fragment of an AspRS protein, wherein the fragment has a cell
signaling or other non-canonical activity. In a more specific
embodiment, the polypeptide is a truncated form of an AspRS protein
comprising amino acid residues 367-448 of the human AspRS protein
set forth in SEQ ID NO: 2, or an active fragment or variant
thereof.
[0112] In another embodiment, the polypeptide is a fragment of a
HisRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment, the
polypeptide is a truncated form of a HisRS protein comprising amino
acid residues 294-372 of the human HisRS protein set forth in SEQ
ID NO: 3, or an active fragment or variant thereof.
[0113] In yet another embodiment of the invention, the polypeptide
is a fragment of a ThrRS protein, wherein the fragment has a cell
signaling or other non-canonical activity. In a more specific
embodiment, the polypeptide is a truncated form of a ThrRS protein
comprising amino acid residues 469-586 of the human ThrRS protein
set forth in SEQ ID NO: 4, or an active fragment or variant
thereof.
[0114] In another embodiment, the polypeptide is a fragment of a
GluProRS protein, wherein the fragment has a cell signaling or
other non-canonical activity. In a more specific embodiment, the
polypeptide is a truncated form of a GluProRS protein comprising
amino acid residues 1171-1253 of the human GluProRS protein set
forth in SEQ ID NO: 5, or an active fragment or variant
thereof.
[0115] In another embodiment, the polypeptide is a fragment of a
SerRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment, the
polypeptide is a truncated form of a SerRS protein comprising amino
acid residues 325-410 of the human SerRS protein set forth in SEQ
ID NO: 6, or an active fragment or variant thereof.
[0116] In another embodiment, the polypeptide is a fragment of a
PheRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment of the
invention, the polypeptide is a PheRS (FRS) polypeptide comprising
residues 380-449 of SEQ ID NO: 7 or an active fragment or variant
thereof.
[0117] In another embodiment, the polypeptide is a fragment of a
LysRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment of the
invention, the polypeptide is a LysRS (KRS) polypeptide comprising
residues 425-523 of SEQ ID NO: 8 or an active fragment or variant
thereof.
[0118] In another embodiment, the polypeptide is a fragment of a
AsnRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment of the
invention, the polypeptide is a AsnRS (NRS) polypeptide comprising
residues 416-494 of SEQ ID NO: 9 or an active fragment or variant
thereof.
[0119] In another embodiment, the polypeptide is a fragment of a
AlaRS protein, wherein the fragment has a cell signaling or other
non-canonical activity. In a more specific embodiment of the
invention, the polypeptide is a AlaRS (ARS) polypeptide comprising
residues 148-258 of SEQ ID NO: 10 or an active fragment or variant
thereof.
[0120] In still another embodiment, the polypeptide is a fragment
of a thioredoxin protein, wherein the fragment has a cell signaling
or other non-canonical activity. In a more specific embodiment, the
polypeptide is a truncated form of a thioredoxin protein comprising
amino acid residues 20-105 of the human thioredoxin protein set
forth in SEQ ID NO: 11, or an active fragment or variant
thereof.
[0121] In another embodiment, the polypeptide is a fragment of a
macrophage inhibitory factor protein, wherein the fragment has a
cell signaling or other non-canonical activity. In a more specific
embodiment, the polypeptide is a truncated form of a macrophage
inhibitory factor protein comprising amino acid residues 1-90 of
the human macrophage inhibitory factor protein set forth in SEQ ID
NO: 12, or an active fragment or variant thereof.
[0122] In yet another embodiment, the polypeptide is a fragment of
a human peroxiredoxin 5 isoform B protein, wherein the fragment has
a cell signaling or other non-canonical activity. In a more
specific embodiment, the polypeptide is a truncated form of a human
peroxiredoxin 5 isoform B comprising amino acid residues 32-68 and
125-161 of the sequence set forth in SEQ ID NO: 13, or an active
fragment or variant thereof.
[0123] In another specific embodiment, the polypeptide if the
invention is not a fragment of a TrpRS or TyrRS protein.
[0124] In another embodiment, the polypeptide is not a HisRS
fragment consisting of the first 48 amino acids of the HisRS
protein.
[0125] In another embodiment, the polypeptide is not a thioredoxin
fragment consisting of the first 80 or 84 N-terminal amino acids of
the thioredoxin protein.
[0126] The present invention further provides variants of the
polypeptides described herein. Polypeptide variants encompassed by
the present invention will typically exhibit at least about 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity (determined, for example, as described below), along their
lengths, to the corresponding region of a wild-type or reference
sequence from which it is derived.
[0127] A polypeptide variant may differ from a naturally occurring
polypeptide of the invention in one or more substitutions,
deletions, additions and/or insertions. Such variants may be
naturally occurring or may be synthetically generated, for example,
by modifying one or more of the above polypeptide sequences of the
invention and evaluating their biological activity as described
herein using any of a number of techniques well known in the
art.
[0128] In other illustrative embodiments, the variant may be a
splice variant, whether naturally or non-naturally occurring,
wherein the splice variant possesses at least one non-canonical
activity, e.g., as described herein.
[0129] In other illustrative embodiments, the variant contains one
or more point mutations relative to a wild type or reference
polypeptide sequence, whether naturally or non-naturally occurring,
wherein the variant polypeptide possesses at least one
non-canonical activity, e.g., as described herein.
[0130] In certain embodiments, a variant will contain conservative
substitutions. A "conservative substitution" is one in which an
amino acid is substituted for another amino acid that has similar
properties, such that one skilled in the art would expect the
secondary structure and hydropathic nature of the polypeptide to be
substantially unchanged. Modifications may be made in the structure
of the polynucleotides and polypeptides of the present invention
and still obtain a functional molecule that encodes a variant or
derivative polypeptide with desirable characteristics. When it is
desired to alter the amino acid sequence of a polypeptide to create
an equivalent, or even an improved, variant of a polypeptide of the
invention, one skilled in the art, for example, can change one or
more of the codons of the encoding DNA sequence according to Table
1.
[0131] For example, certain amino acids may be substituted for
other amino acids in a protein structure without appreciable loss
of interactive binding capacity with structures such as, for
example, receptors, antigen-binding regions of antibodies or
binding sites on a substrate molecule. Since it is the interactive
capacity and nature of a protein that generally defines that
protein's biological functional activity, certain amino acid
sequence substitutions can be made in a protein sequence, and, of
course, its underlying DNA coding sequence, and nevertheless obtain
a protein with like properties. It is thus contemplated that
various changes may be made in the polypeptide sequences of the
disclosed compositions, or corresponding DNA sequences which encode
said polypeptides without appreciable loss of their desired utility
or activity.
TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0132] In making such changes, the hydropathic index of amino acids
may also be considered. The importance of the hydropathic amino
acid index in conferring interactive biologic function on a protein
is generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). For example, it is known that
the relative hydropathic character of the amino acid contributes to
the secondary structure of the resultant protein, which in turn
defines the interaction of the protein with other molecules, for
example, enzymes, substrates, receptors, DNA, antibodies, antigens,
and the like. Each amino acid has been assigned a hydropathic index
on the basis of its hydrophobicity and charge characteristics (Kyte
and Doolittle, 1982). These values are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0133] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biological functionally equivalent
protein. In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0134] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. As detailed in U.S. Pat. No. 4,554,101, the
following hydrophilicity values have been assigned to amino acid
residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1);
glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein. In such changes, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those within
.+-.1 are particularly preferred, and those within .+-.0.5 are even
more particularly preferred.
[0135] As outlined above, amino acid substitutions may be based on
the relative similarity of the amino acid side-chain substituents,
for example, their hydrophobicity, hydrophilicity, charge, size,
and the like. Exemplary substitutions that take various of the
foregoing characteristics into consideration are well known to
those of skill in the art and include: arginine and lysine;
glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine.
[0136] Amino acid substitutions may further be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; and amino acids with uncharged polar head groups having
similar hydrophilicity values include leucine, isoleucine and
valine; glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
A variant may also, or alternatively, contain non-conservative
changes. In a preferred embodiment, variant polypeptides differ
from a native sequence by substitution, deletion or addition of
five amino acids or fewer. Variants may also (or alternatively) be
modified by, for example, the deletion or addition of amino acids
that have minimal influence on secondary structure and hydropathic
nature of the polypeptide.
[0137] Polypeptides may comprise a signal (or leader) sequence at
the N-terminal end of the protein, which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to a linker or other sequence
for ease of synthesis, purification or identification of the
polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fc region.
[0138] When comparing polypeptide sequences, two sequences are said
to be "identical" if the sequence of amino acids in the two
sequences is the same when aligned for maximum correspondence, as
described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0139] Optimal alignment of sequences for comparison may be
conducted, for example, using the Megalign program in the Lasergene
suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.),
using default parameters. This program embodies several alignment
schemes described in the following references: Dayhoff, M. O.
(1978) A model of evolutionary change in proteins--Matrices for
detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of
Protein Sequence and Structure, National Biomedical Research
Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645
Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,
Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153;
Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D.
(1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol.
Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D.
J. (1983) Proc. Nat'l Acad., Sci. USA 80:726-730.
[0140] Alternatively, optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman (1981) Add. APL. Math 2:482, by the identity alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity methods of Pearson and Lipman (1988)
Proc. Nat'l Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by
inspection.
[0141] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used,
for example with the parameters described herein, to determine
percent sequence identity for the polynucleotides and polypeptides
of the invention. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. For amino acid sequences, a scoring matrix can be used
to calculate the cumulative score. Extension of the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment.
[0142] In one illustrative approach, the "percentage of sequence
identity" is determined by comparing two optimally aligned
sequences over a window of comparison of at least 20 positions,
wherein the portion of the polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical amino acid residue occurs in both sequences
to yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the reference
sequence (i.e., the window size) and multiplying the results by 100
to yield the percentage of sequence identity.
[0143] In certain embodiments of the invention, there are provided
fusion polypeptides, and polynucleotides encoding fusion
polypeptides. Fusion polypeptides refer to polypeptides of the
invention that have been covalently linked, either directly or
indirectly via an amino acid linker, to one or more heterologous
polypeptide sequences (fusion partners). The polypeptides forming
the fusion protein are typically linked C-terminus to N-terminus,
although they can also be linked C-terminus to C-terminus,
N-terminus to N-terminus, or N-terminus to C-terminus. The
polypeptides of the fusion protein can be in any order.
[0144] The fusion partner may be designed and included for
essentially any desired purpose provided they do not adversely
effect the desired activity of the polypeptide. For example, in one
embodiment, a fusion partner comprises a sequence that assists in
expressing the protein (an expression enhancer) at higher yields
than the native recombinant protein. Other fusion partners may be
selected so as to increase the solubility of the protein or to
enable the protein to be targeted to desired intracellular
compartments. Still further fusion partners include affinity tags,
which facilitate purification of the protein.
[0145] Fusion proteins may generally be prepared using standard
techniques. For example, DNA sequences encoding the polypeptide
components of a desired fusion may be assembled separately, and
ligated into an appropriate expression vector. The 3' end of the
DNA sequence encoding one polypeptide component is ligated, with or
without a peptide linker, to the 5' end of a DNA sequence encoding
the second polypeptide component so that the reading frames of the
sequences are in phase. This permits translation into a single
fusion protein that retains the biological activity of both
component polypeptides.
[0146] A peptide linker sequence may be employed to separate the
first and second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures, if desired. Such a peptide linker sequence is
incorporated into the fusion protein using standard techniques well
known in the art. Certain peptide linker sequences may be chosen
based on the following factors: (1) their ability to adopt a
flexible extended conformation; (2) their inability to adopt a
secondary structure that could interact with functional epitopes on
the first and second polypeptides; and (3) the lack of hydrophobic
or charged residues that might react with the polypeptide
functional epitopes. Preferred peptide linker sequences contain
Gly, Asn and Ser residues. Other near neutral amino acids, such as
Thr and Ala may also be used in the linker sequence. Amino acid
sequences which may be usefully employed as linkers include those
disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al.,
Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No.
4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may
generally be from 1 to about 50 amino acids in length. Linker
sequences are not required when the first and second polypeptides
have non-essential N-terminal amino acid regions that can be used
to separate the functional domains and prevent steric
interference.
[0147] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
5' to the DNA sequence encoding the first polypeptides. Similarly,
stop codons required to end translation and transcription
termination signals are present 3' to the DNA sequence encoding the
second polypeptide.
[0148] In general, polypeptides and fusion polypeptides (as well as
their encoding polynucleotides) are isolated. An "isolated"
polypeptide or polynucleotide is one that is removed from its
original environment. For example, a naturally-occurring protein is
isolated if it is separated from some or all of the coexisting
materials in the natural system. Preferably, such polypeptides are
at least about 90% pure, more preferably at least about 95% pure
and most preferably at least about 99% pure. A polynucleotide is
considered to be isolated if, for example, it is cloned into a
vector that is not a part of the natural environment.
[0149] In still other embodiments, a polypeptide of the invention
may be part of a dimer. Dimers may include, for example, homodimers
between two identical AARS polypeptides, heterodimers between two
different AARS polypeptides (e.g., a full-length GlyRS polypeptide
and a truncated GlyRS polypeptide, or two different truncated AARS
polypeptides), and/or heterodimers between an AARS polypeptide and
a heterologous polypeptide. The monomers and/or dimers may be
soluble and may be isolated or purified to homogeneity. Certain
heterodimers, such as those between an AARS polypeptide and a
heterologous polypeptide, may be bi-functional.
[0150] Also included are monomeric or substantially monomeric
proteins. In certain embodiments, a monomeric protein has reduced
capacity to dimerize with itself (i.e., homodimerize) and/or
dimerize with another AARS polypeptide (i.e., heterodimerize).
[0151] In other embodiments, a polypeptide of the invention may be
part of a multi-unit complex. A multi-unit complex of the present
invention can include, for example, at least 2, 3, 4, or 5 or more
monomers. The monomers and/or multi-unit complexes may be soluble
and may be isolated or purified to homogeneity. Monomer units of a
multi-unit complex may be different, homologous, substantially
homologous, or identical to one another. However, a multi-unit
complex of the invention includes at least one monomer comprising a
polypeptide as described herein or, in other embodiments, at least
two or more polypeptides, as described herein.
[0152] Covalently linked monomers can be linked directly (by bonds)
or indirectly (e.g., via a linker). For directly linking the
polypeptide monomers herein, it may be beneficial to modify the
polypeptides herein to enhance dimerization or multimerization. For
example, one or more amino acid residues of an AARS polypeptide may
be modified by the addition or substitution by one or more
cysteines. Methods for creating amino acid substitutions, such as
cysteine substitutions, or other modifications to facilitate
linking, are well known to those skilled in the art.
[0153] Certain embodiments of the present invention also
contemplate the use of modified AARS or other polypeptides,
including modifications that improve desired characteristics of an
AARS or other polypeptide, as described herein. Illustrative
modifications of AARS polypeptides of the invention include, but
are not limited to, chemical and/or enzymatic derivatizations at
one or more constituent amino acid, including side chain
modifications, backbone modifications, and N- and C-terminal
modifications including acetylation, hydroxylation, methylation,
amidation, and the attachment of carbohydrate or lipid moieties,
cofactors, and the like. Exemplary modifications also include
pegylation of polypeptides (see, e.g., Veronese and Harris,
Advanced Drug Delivery Reviews 54: 453-456, 2002, herein
incorporated by reference).
[0154] In certain aspects, chemoselective ligation technology may
be utilized to modify truncated polypeptides of the invention, such
as by attaching polymers in a site-specific and controlled manner.
Such technology typically relies on the incorporation of
chemoselective anchors into the protein backbone by either chemical
or recombinant means, and subsequent modification with a polymer
carrying a complementary linker. As a result, the assembly process
and the covalent structure of the resulting protein-polymer
conjugate may be controlled, enabling the rational optimization of
drug properties, such as efficacy and pharmacokinetic properties
(see, e.g., Kochendoerfer, Current Opinion in Chemical Biology
9:555-560, 2005).
[0155] The AARS polypeptides described herein may be prepared by
any suitable procedure known to those of skill in the art, such as
by recombinant techniques. For example, polypeptides may be
prepared by a procedure including the steps of: (a) preparing a
construct comprising a polynucleotide sequence that encodes an AARS
polypeptide of the invention and that is operably linked to a
regulatory element; (b) introducing the construct into a host cell;
(c) culturing the host cell to express the polypeptide; and (d)
isolating the polypeptide from the host cell. Recombinant AARS
polypeptides can be conveniently prepared using standard protocols
as described for example in Sambrook, et al., (1989, supra), in
particular Sections 16 and 17; Ausubel et al., (1994, supra), in
particular Chapters 10 and 16; and Coligan et al., Current
Protocols in Protein Science (John Wiley & Sons, Inc.
1995-1997), in particular Chapters 1, 5 and 6.
[0156] In addition to recombinant production methods, polypeptides
of the invention may be produced by direct peptide synthesis using
solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154
(1963)). Protein synthesis may be performed using manual techniques
or by automation. Automated synthesis may be achieved, for example,
using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
Alternatively, various fragments may be chemically synthesized
separately and combined using chemical methods to produce the
desired molecule.
Polynucleotide Compositions
[0157] The present invention also provides isolated polynucleotides
that encode the polypeptides of the invention, as well as
compositions comprising such polynucleotides. For example,
polynucleotide sequences encoding tRNA synthetase proteins, and
other proteins described herein, are readily available via any of a
number of public sequence databases (e.g.,
http://www.ncbi.nlm.nih.gov) and can be identified, made and used
in the context of the present disclosure using techniques and
methodologies described herein and/or well established in the
art.
[0158] As used herein, the terms "DNA" and "polynucleotide" and
"nucleic acid" refer to a DNA molecule that has been isolated free
of total genomic DNA of a particular species. Therefore, a DNA
segment encoding a polypeptide refers to a DNA segment that
contains one or more coding sequences yet is substantially isolated
away from, or purified free from, total genomic DNA of the species
from which the DNA segment is obtained. Included within the terms
"DNA segment" and "polynucleotide" are DNA segments and smaller
fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phage,
viruses, and the like.
[0159] As will be understood by those skilled in the art, the
polynucleotide sequences of this invention can include genomic
sequences, extra-genomic and plasmid-encoded sequences and smaller
engineered gene segments that express, or may be adapted to
express, proteins, polypeptides, peptides and the like. Such
segments may be naturally isolated, or modified synthetically by
the hand of man.
[0160] As will be recognized by the skilled artisan,
polynucleotides may be single-stranded (coding or antisense) or
double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA
molecules. Additional coding or non-coding sequences may, but need
not, be present within a polynucleotide of the present invention,
and a polynucleotide may, but need not, be linked to other
molecules and/or support materials.
[0161] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes a polypeptide of the invention or
a portion thereof) or may comprise a variant, or a biological
functional equivalent of such a sequence. Polynucleotide variants
may contain one or more substitutions, additions, deletions and/or
insertions, as further described below, preferably such that the
desired activity of the encoded polypeptide is not substantially
diminished relative to the unmodified polypeptide. The effect on
the activity of the encoded polypeptide may generally be assessed
as described herein.
[0162] In additional embodiments, the present invention provides
isolated polynucleotides comprising various lengths of contiguous
stretches of sequence identical to or complementary a
polynucleotide encoding a polypeptide as described herein.
[0163] For example, polynucleotides are provided by this invention
that encode at least about 5, 10, 15, 20, 25, 50, 75, 100, 150,
200, 250, 300, 350, 400, 450 or 500, or more, contiguous amino acid
residues of a polypeptide of the invention, as well as all
intermediate lengths. It will be readily understood that
"intermediate lengths", in this context, means any length between
the quoted values, such as 101, 102, 103, etc.; 151, 152, 153,
etc.; 201, 202, 203, etc.
[0164] The polynucleotides of the present invention, regardless of
the length of the coding sequence itself, may be combined with
other DNA sequences, such as promoters, polyadenylation signals,
additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may
vary considerably. It is therefore contemplated that a
polynucleotide fragment of almost any length may be employed, with
the total length preferably being limited by the ease of
preparation and use in the intended recombinant DNA protocol.
[0165] Moreover, it will be appreciated by those of ordinary skill
in the art that, as a result of the degeneracy of the genetic code,
there are many nucleotide sequences that encode a polypeptide as
described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene.
Nonetheless, polynucleotides that vary due to differences in codon
usage are specifically contemplated by the present invention, for
example polynucleotides that are optimized for human and/or primate
codon selection. Further, alleles of the genes comprising the
polynucleotide sequences provided herein are within the scope of
the present invention. Alleles are endogenous genes that are
altered as a result of one or more mutations, such as deletions,
additions and/or substitutions of nucleotides. The resulting mRNA
and protein may, but need not, have an altered structure or
function. Alleles may be identified using standard techniques (such
as hybridization, amplification and/or database sequence
comparison).
[0166] Polynucleotides and fusions thereof may be prepared,
manipulated and/or expressed using any of a variety of well
established techniques known and available in the art. For example,
polynucleotide sequences which encode polypeptides of the
invention, or fusion proteins or functional equivalents thereof,
may be used in recombinant DNA molecules to direct expression of a
polypeptide in appropriate host cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences that encode
substantially the same or a functionally equivalent amino acid
sequence may be produced and these sequences may be used to clone
and express a given polypeptide.
[0167] As will be understood by those of skill in the art, it may
be advantageous in some instances to produce polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce a recombinant RNA transcript having desirable
properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
[0168] Moreover, the polynucleotide sequences of the present
invention can be engineered using methods generally known in the
art in order to alter polypeptide encoding sequences for a variety
of reasons, including but not limited to, alterations which modify
the cloning, processing, expression and/or activity of the gene
product.
[0169] In order to express a desired polypeptide, a nucleotide
sequence encoding the polypeptide, or a functional equivalent, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well
known to those skilled in the art may be used to construct
expression vectors containing sequences encoding a polypeptide of
interest and appropriate transcriptional and translational control
elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook et al.,
Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,
Current Protocols in Molecular Biology (1989).
[0170] A variety of expression vector/host systems are known and
may be utilized to contain and express polynucleotide sequences.
These include, but are not limited to, microorganisms such as
bacteria transformed with recombinant bacteriophage, plasmid, or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids); or animal cell
systems.
[0171] The "control elements" or "regulatory sequences" present in
an expression vector are those non-translated regions of the
vector--enhancers, promoters, 5' and 3' untranslated regions--which
interact with host cellular proteins to carry out transcription and
translation. Such elements may vary in their strength and
specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used. For
example, when cloning in bacterial systems, inducible promoters
such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid
(Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL,
Gaithersburg, Md.) and the like may be used. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are generally preferred. If it is necessary to generate a cell line
that contains multiple copies of the sequence encoding a
polypeptide, vectors based on SV40 or EBV may be advantageously
used with an appropriate selectable marker.
[0172] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the expressed
polypeptide. For example, when large quantities are needed, vectors
which direct high level expression of fusion proteins that are
readily purified may be used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the polypeptide of interest may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke & Schuster, J.
Biol. Chem. 264:5503 5509 (1989)); and the like. pGEX Vectors
(Promega, Madison, Wis.) may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems may be designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will.
[0173] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al., Methods Enzymol.
153:516-544 (1987).
[0174] In cases where plant expression vectors are used, the
expression of sequences encoding polypeptides may be driven by any
of a number of promoters. For example, viral promoters such as the
35S and 19S promoters of CaMV may be used alone or in combination
with the omega leader sequence from TMV (Takamatsu, EMBO J.
6:307-311 (1987)). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi et
al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science
224:838-843 (1984); and Winter et al., Results Probl. Cell Differ.
17:85-105 (1991)). These constructs can be introduced into plant
cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, e.g., Hobbs in McGraw Hill,
Yearbook of Science and Technology, pp. 191-196 (1992)).
[0175] An insect system may also be used to express a polypeptide
of interest. For example, in one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. The sequences encoding the polypeptide may be
cloned into a non-essential region of the virus, such as the
polyhedrin gene, and placed under control of the polyhedrin
promoter. Successful insertion of the polypeptide-encoding sequence
will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein. The recombinant viruses may then be
used to infect, for example, S. frugiperda cells or Trichoplusia
larvae in which the polypeptide of interest may be expressed
(Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227
(1994)).
[0176] In mammalian host cells, a number of viral-based expression
systems are generally available. For example, in cases where an
adenovirus is used as an expression vector, sequences encoding a
polypeptide of interest may be ligated into an adenovirus
transcription/translation complex consisting of the late promoter
and tripartite leader sequence. Insertion in a non-essential E1 or
E3 region of the viral genome may be used to obtain a viable virus
which is capable of expressing the polypeptide in infected host
cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A.
81:3655-3659 (1984)). In addition, transcription enhancers, such as
the Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0177] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding a polypeptide of
interest. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding the
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous translational control signals including the
ATG initiation codon should be provided. Furthermore, the
initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements
and initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system which is used, such as those described in the
literature (Scharf et al., Results Probl. Cell Differ. 20:125-162
(1994)).
[0178] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and W138, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0179] For long-term, high-yield production of recombinant
proteins, stable expression is generally preferred. For example,
cell lines which stably express a polynucleotide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0180] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., Cell
11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et
al., Cell 22:817-823 (1990)) genes which can be employed in tk- or
aprt-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler et
al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which
confers resistance to the aminoglycosides, neomycin and G-418
(Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or
pat, which confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine
(Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51
(1988)). The use of visible markers has gained popularity with such
markers as anthocyanins, .beta.-glucuronidase and its substrate
GUS, and luciferase and its substrate luciferin, being widely used
not only to identify transformants, but also to quantify the amount
of transient or stable protein expression attributable to a
specific vector system (Rhodes et al., Methods Mol. Biol.
55:121-131 (1995)).
[0181] A variety of protocols for detecting and measuring the
expression of polynucleotide-encoded products, using either
polyclonal or monoclonal antibodies specific for the product are
known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). These and other assays are described, among
other places, in Hampton et al., Serological Methods, a Laboratory
Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216
(1983).
[0182] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the sequences, or any portions thereof may be cloned
into a vector for the production of an mRNA probe. Such vectors are
known in the art, are commercially available, and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These
procedures may be conducted using a variety of commercially
available kits. Suitable reporter molecules or labels, which may be
used include radionuclides, enzymes, fluorescent, chemiluminescent,
or chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0183] Host cells transformed with a polynucleotide sequence of
interest may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a recombinant cell may be secreted or contained
intracellularly depending on the sequence and/or the vector used.
As will be understood by those of skill in the art, expression
vectors containing polynucleotides of the invention may be designed
to contain signal sequences which direct secretion of the encoded
polypeptide through a prokaryotic or eukaryotic cell membrane.
Other recombinant constructions may be used to join sequences
encoding a polypeptide of interest to nucleotide sequence encoding
a polypeptide domain which will facilitate purification of soluble
proteins.
[0184] In addition to recombinant production methods, polypeptides
of the invention, and fragments thereof, may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield, J. Am.
Chem. Soc. 85:2149-2154 (1963)). Protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer). Alternatively, various fragments may be
chemically synthesized separately and combined using chemical
methods to produce the full length molecule.
[0185] According to another aspect of the invention,
polynucleotides encoding polypeptides of the invention may be
delivered to a subject in vivo, e.g., using gene therapy
techniques. Gene therapy refers generally to the transfer of
heterologous nucleic acids to the certain cells, target cells, of a
mammal, particularly a human, with a disorder or conditions for
which such therapy is sought. The nucleic acid is introduced into
the selected target cells in a manner such that the heterologous
DNA is expressed and a therapeutic product encoded thereby is
produced.
[0186] Various viral vectors that can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia,
adeno-associated virus (AAV), or, preferably, an RNA virus such as
a retrovirus. Preferably, the retroviral vector is a derivative of
a murine or avian retrovirus, or is a lentiviral vector. The
preferred retroviral vector is a lentiviral vector. Examples of
retroviral vectors in which a single foreign gene can be inserted
include, but are not limited to: Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. By inserting a zinc finger derived-DNA binding
polypeptide sequence of interest into the viral vector, along with
another gene that encodes the ligand for a receptor on a specific
target cell, for example, the vector may be made target specific.
Retroviral vectors can be made target specific by inserting, for
example, a polynucleotide encoding a protein (dimer). Illustrative
targeting may be accomplished by using an antibody to target the
retroviral vector. Those of skill in the art will know of, or can
readily ascertain without undue experimentation, specific
polynucleotide sequences which can be inserted into the retroviral
genome to allow target specific delivery of the retroviral vector
containing the zinc finger-nucleotide binding protein
polynucleotide.
[0187] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsulation. Helper cell lines which have deletions of the
packaging signal include but are not limited to PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced. The vector virions produced by
this method can then be used to infect a tissue cell line, such as
NIH 3T3 cells, to produce large quantities of chimeric retroviral
virions.
[0188] "Non-viral" delivery techniques for gene therapy can also be
used including, for example, DNA-ligand complexes,
adenovirus-ligand-DNA complexes, direct injection of DNA,
CaPO.sub.4 precipitation, gene gun techniques, electroporation,
liposomes, lipofection, and the like. Any of these methods are
widely available to one skilled in the art and would be suitable
for use in the present invention. Other suitable methods are
available to one skilled in the art, and it is to be understood
that the present invention can be accomplished using any of the
available methods of transfection. Lipofection can be accomplished
by encapsulating an isolated DNA molecule within a liposomal
particle and contacting the liposomal particle with the cell
membrane of the target cell. Liposomes are self-assembling,
colloidal particles in which a lipid bilayer, composed of
amphiphilic molecules such as phosphatidyl serine or phosphatidyl
choline, encapsulates a portion of the surrounding media such that
the lipid bilayer surrounds a hydrophilic interior. Unilammellar or
multilammellar liposomes can be constructed such that the interior
contains a desired chemical, drug, or, as in the instant invention,
an isolated DNA molecule.
[0189] Embodiments of the present invention also include
oligonucleotides (e.g., antisense oligomers, probes, primers),
whether for detection, amplification, antisense therapies, or other
purpose. Oligonucleotides typically comprise or are complementary
to at least a portion of an AARS polynucleotide sequence. For these
and related purposes, the term "oligonucleotide" or "oligo" or
"oligomer" is intended to encompass a singular "oligonucleotide" as
well as plural "oligonucleotides," and refers to any polymer of two
or more of nucleotides, nucleosides, nucleobases or related
compounds used as a reagent in the amplification methods of the
present invention, as well as subsequent detection methods. The
oligonucleotide may be DNA and/or RNA and/or analogs thereof.
[0190] In certain embodiments, oligomers such as antisense
oligomers as long as 40 bases may be suitable, where at least a
minimum number of bases, e.g., 10-12 bases, are complementary to
the target sequence (e.g., an AARS polynucleotide sequence). In
general, however, facilitated or active uptake in cells is
optimized at oligomer lengths less than about 30. For certain
oligomers, described further below, an optimum balance of binding
stability and uptake generally occurs at lengths of 18-25 bases.
Included are antisense oligomers (e.g., PNAs, LNAs, 2'-OMe, MOE,
morpholinos) that consist of about 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, or 40 bases, in which at least about 6, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40
contiguous or non-contiguous bases are complementary to their AARS
target sequence, or variants thereof.
[0191] Certain embodiments relate to RNA interference (RNAi)
agents, such as short-interfering RNA (siRNA) or other siRNA
agents, that target one or more mRNA transcripts of an AARS
polynucleotide. For certain siRNA-related embodiments, each strand
of an siRNA agent can be equal to or less than 35, 30, 25, 24, 23,
22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. The strand
is preferably at least 19 nucleotides in length. For example, each
strand can be between 21 and 25 nucleotides in length. Preferred
siRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23,
24, or 25 nucleotide pairs, and one or more overhangs, preferably
one or two 3' overhangs, of 2-3 nucleotides. Also included are
methods of use thereof to modulate the levels of a selected AARS
transcript, such as an AARS polynucleotide that encodes a motif as
described herein.
[0192] As noted above, the AARS polynucleotides of the present
invention can be used in any of the diagnostic, drug discovery, or
therapeutic methods described herein.
Antibody Compositions, Fragments Thereof and Other Binding
Agents
[0193] According to another aspect, the present invention further
provides binding agents, such as antibodies and antigen-binding
fragments thereof, soluble receptors, peptides, peptide mimetics,
aptamers, etc., that exhibit binding specificity for a polypeptide
disclosed herein, or to a portion, variant or derivative thereof,
and methods of using same. Also included are binding agents that
exhibit binding specificity for a cellular binding partner of a
polypeptide disclosed herein. Preferably, such binding agents are
effective for modulating one or more of the non-canonical
activities mediated by a polypeptide of the invention.
[0194] In certain embodiments, for example, the binding agent is
one that binds to a polypeptide of the invention and inhibits its
ability to bind to one or more of its cellular binding partners.
Accordingly, such binding agents may be used to treat or prevent
diseases, disorders or other conditions that are mediated by a
polypeptide of the invention by antagonizing its activity.
[0195] An antibody, or antigen-binding fragment thereof, is said to
"specifically bind," "immunogically bind," and/or is
"immunologically reactive" to a polypeptide of the invention if it
reacts at a detectable level (within, for example, an ELISA assay)
with the polypeptide, and does not react detectably with unrelated
polypeptides under similar conditions.
[0196] Immunological binding, as used in this context, generally
refers to the non-covalent interactions of the type which occur
between an immunoglobulin molecule and an antigen for which the
immunoglobulin is specific. The strength, or affinity of
immunological binding interactions can be expressed in terms of the
dissociation constant (K.sub.d) of the interaction, wherein a
smaller K.sub.d represents a greater affinity. Immunological
binding properties of selected polypeptides can be quantified using
methods well known in the art. One such method entails measuring
the rates of antigen-binding site/antigen complex formation and
dissociation, wherein those rates depend on the concentrations of
the complex partners, the affinity of the interaction, and on
geometric parameters that equally influence the rate in both
directions. Thus, both the "on rate constant" (k.sub.on) and the
"off rate constant" (k.sub.off) can be determined by calculation of
the concentrations and the actual rates of association and
dissociation. The ratio of k.sub.off/k.sub.on enables cancellation
of all parameters not related to affinity, and is thus equal to the
dissociation constant K.sub.d. See, generally, Davies et al. (1990)
Annual Rev. Biochem. 59:439-473.
[0197] An "antigen-binding site," or "binding portion" of an
antibody refers to the part of the immunoglobulin molecule that
participates in antigen binding. The antigen binding site is formed
by amino acid residues of the N-terminal variable ("V") regions of
the heavy ("H") and light ("L") chains. Three highly divergent
stretches within the V regions of the heavy and light chains are
referred to as "hypervariable regions" which are interposed between
more conserved flanking stretches known as "framework regions," or
"FRs". Thus the term "FR" refers to amino acid sequences which are
naturally found between and adjacent to hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs."
[0198] A binding agent may be, for example, a ribosome, with or
without a peptide component, an RNA molecule or a polypeptide. In a
preferred embodiment, a binding agent is an antibody or an
antigen-binding fragment thereof. Antibodies may be prepared by any
of a variety of techniques known to those of ordinary skill in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. In general, antibodies can be
produced by cell culture techniques, including the generation of
monoclonal antibodies as described herein, or via transfection of
antibody genes into suitable bacterial or mammalian cell hosts, in
order to allow for the production of recombinant antibodies. In one
technique, an immunogen comprising the polypeptide is initially
injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits, sheep or goats). In this step, the polypeptides of this
invention may serve as the immunogen without modification.
Alternatively, particularly for relatively short polypeptides, a
superior immune response may be elicited if the polypeptide is
joined to a carrier protein, such as bovine serum albumin or
keyhole limpet hemocyanin. The immunogen is injected into the
animal host, preferably according to a predetermined schedule
incorporating one or more booster immunizations, and the animals
are bled periodically. Polyclonal antibodies specific for the
polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to a
suitable solid support.
[0199] Monoclonal antibodies specific for a polypeptide of interest
may be prepared, for example, using the technique of Kohler and
Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements
thereto. Briefly, these methods involve the preparation of immortal
cell lines capable of producing antibodies having the desired
specificity (i.e., reactivity with the polypeptide of interest).
Such cell lines may be produced, for example, from spleen cells
obtained from an animal immunized as described above. The spleen
cells are then immortalized by, for example, fusion with a myeloma
cell fusion partner, preferably one that is syngeneic with the
immunized animal. A variety of fusion techniques may be employed.
For example, the spleen cells and myeloma cells may be combined
with a nonionic detergent for a few minutes and then plated at low
density on a selective medium that supports the growth of hybrid
cells, but not myeloma cells. A preferred selection technique uses
HAT (hypoxanthine, aminopterin, thymidine) selection. After a
sufficient time, usually about 1 to 2 weeks, colonies of hybrids
are observed. Single colonies are selected and their culture
supernatants tested for binding activity against the polypeptide.
Hybridomas having high reactivity and specificity are
preferred.
[0200] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0201] A number of therapeutically useful molecules are known in
the art which comprise antigen-binding sites that are capable of
exhibiting immunological binding properties of an antibody
molecule. The proteolytic enzyme papain preferentially cleaves IgG
molecules to yield several fragments, two of which (the "F(ab)"
fragments) each comprise a covalent heterodimer that includes an
intact antigen-binding site. The enzyme pepsin is able to cleave
IgG molecules to provide several fragments, including the
"F(ab').sub.2" fragment which comprises both antigen-binding sites.
An "Fv" fragment can be produced by preferential proteolytic
cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin
molecule. Fv fragments are, however, more commonly derived using
recombinant techniques known in the art. The Fv fragment includes a
non-covalent V.sub.H::V.sub.L heterodimer including an
antigen-binding site which retains much of the antigen recognition
and binding capabilities of the native antibody molecule. Inbar et
al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem
19:4091-4096.
[0202] A single chain Fv ("sFv") polypeptide is a covalently linked
V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion
including V.sub.H- and V.sub.L-encoding genes linked by a
peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to
discern chemical structures for converting the naturally
aggregated--but chemically separated--light and heavy polypeptide
chains from an antibody V region into an sFv molecule which will
fold into a three dimensional structure substantially similar to
the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.
5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No.
4,946,778, to Ladner et al.
[0203] Each of the above-described molecules includes a heavy chain
and a light chain CDR set, respectively interposed between a heavy
chain and a light chain FR set which provide support to the CDRS
and define the spatial relationship of the CDRs relative to each
other. As used herein, the term "CDR set" refers to the three
hypervariable regions of a heavy or light chain V region.
Proceeding from the N-terminus of a heavy or light chain, these
regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An
antigen-binding site, therefore, includes six CDRs, comprising the
CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3)
is referred to herein as a "molecular recognition unit."
Crystallographic analysis of a number of antigen-antibody complexes
has demonstrated that the amino acid residues of CDRs form
extensive contact with bound antigen, wherein the most extensive
antigen contact is with the heavy chain CDR3. Thus, the molecular
recognition units are primarily responsible for the specificity of
an antigen-binding site.
[0204] As used herein, the term "FR set" refers to the four
flanking amino acid sequences which frame the CDRs of a CDR set of
a heavy or light chain V region. Some FR residues may contact bound
antigen; however, FRs are primarily responsible for folding the V
region into the antigen-binding site, particularly the FR residues
directly adjacent to the CDRS. Within FRs, certain amino residues
and certain structural features are very highly conserved. In this
regard, all V region sequences contain an internal disulfide loop
of around 90 amino acid residues. When the V regions fold into a
binding-site, the CDRs are displayed as projecting loop motifs
which form an antigen-binding surface. It is generally recognized
that there are conserved structural regions of FRs which influence
the folded shape of the CDR loops into certain "canonical"
structures--regardless of the precise CDR amino acid sequence.
Further, certain FR residues are known to participate in
non-covalent interdomain contacts which stabilize the interaction
of the antibody heavy and light chains.
[0205] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent V
regions and their associated CDRs fused to human constant domains
(Winter et al. (1991) Nature 349:293-299; Lobuglio et al. (1989)
Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J.
Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res.
47:3577-3583), rodent CDRs grafted into a human supporting FR prior
to fusion with an appropriate human antibody constant domain
(Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.
(1988) Science 239:1534-1536; and Jones et al. (1986) Nature
321:522-525), and rodent CDRs supported by recombinantly veneered
rodent FRs (European Patent Publication No. 519,596, published Dec.
23, 1992). These "humanized" molecules are designed to minimize
unwanted immunological response toward rodent antibody molecules
which limits the duration and effectiveness of therapeutic
applications of those moieties in human recipients.
[0206] As used herein, the terms "veneered FRs" and "recombinantly
veneered FRs" refer to the selective replacement of FR residues
from, e.g., a rodent heavy or light chain V region, with human FR
residues in order to provide a xenogeneic molecule comprising an
antigen-binding site which retains substantially all of the native
FR polypeptide folding structure. Veneering techniques are based on
the understanding that the ligand binding characteristics of an
antigen-binding site are determined primarily by the structure and
relative disposition of the heavy and light chain CDR sets within
the antigen-binding surface. Davies et al. (1990) Ann. Rev.
Biochem. 59:439-473. Thus, antigen binding specificity can be
preserved in a humanized antibody only wherein the CDR structures,
their interaction with each other, and their interaction with the
rest of the V region domains are carefully maintained. By using
veneering techniques, exterior (e.g., solvent-accessible) FR
residues which are readily encountered by the immune system are
selectively replaced with human residues to provide a hybrid
molecule that comprises either a weakly immunogenic, or
substantially non-immunogenic veneered surface.
[0207] In another embodiment of the invention, monoclonal
antibodies or other binding agents of the present invention may be
coupled to one or more agents of interest. For example, a
therapeutic agent may be coupled (e.g., covalently bonded) to a
suitable monoclonal antibody either directly or indirectly (e.g.,
via a linker group). A direct reaction between an agent and an
antibody is possible when each possesses a substituent capable of
reacting with the other. For example, a nucleophilic group, such as
an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other.
[0208] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0209] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
[0210] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0211] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
that provide multiple sites for attachment can be used.
[0212] As noted above, "peptides" are included as binding agents.
The term peptide typically refers to a polymer of amino acid
residues and to variants and synthetic analogues of the same. In
certain embodiments, the term "peptide" refers to relatively short
polypeptides, including peptides that consist of about 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, or 50 amino acids, including all integers and ranges (e.g.,
5-10, 8-12, 10-15) in between, and interact with an AARS
polypeptide, its cellular binding partner, or both. Peptides can be
composed of naturally-occurring amino acids and/or non-naturally
occurring amino acids, as described herein.
[0213] A binding agent may include a peptide mimetic or other small
molecule. A "small molecule" refers to an organic compound that is
of synthetic or biological origin (biomolecule), but is typically
not a polymer. Organic compounds refer to a large class of chemical
compounds whose molecules contain carbon, typically excluding those
that contain only carbonates, simple oxides of carbon, or cyanides.
A "biomolecule" refers generally to an organic molecule that is
produced by a living organism, including large polymeric molecules
(biopolymers) such as peptides, polysaccharides, and nucleic acids
as well, and small molecules such as primary secondary metabolites,
lipids, phospholipids, glycolipids, sterols, glycerolipids,
vitamins, and hormones. A "polymer" refers generally to a large
molecule or macromolecule composed of repeating structural units,
which are typically connected by covalent chemical bond.
[0214] In certain embodiments, a small molecule has a molecular
weight of less than 1000 Daltons, typically between about 300 and
700 Daltons, and including about 50, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, or 1000
Daltons.
[0215] Aptamers are also included as binding agents. Examples of
aptamers included nucleic acid aptamers (e.g., DNA aptamers, RNA
aptamers) and peptide aptamers. Nucleic acid aptamers refer
generally to nucleic acid species that have been engineered through
repeated rounds of in vitro selection or equivalent method, such as
SELEX (systematic evolution of ligands by exponential enrichment),
to bind to various molecular targets such as small molecules,
proteins, nucleic acids, and even cells, tissues and organisms.
Hence, included are nucleic acid aptamers that bind to the AARS
polypeptides described herein and/or their cellular binding
partners.
[0216] Peptide aptamers typically include a variable peptide loop
attached at both ends to a protein scaffold, a double structural
constraint that typically increases the binding affinity of the
peptide aptamer to levels comparable to that of an antibody's
(e.g., in the nanomolar range). In certain embodiments, the
variable loop length may be composed of about 10-20 amino acids
(including all integers in between), and the scaffold may include
any protein that has good solubility and compacity properties.
Certain exemplary embodiments may utilize the bacterial protein
Thioredoxin-A as a scaffold protein, the variable loop being
inserted within the reducing active site (-Cys-Gly-Pro-Cys-loop in
the wild protein), with the two cysteines lateral chains being able
to form a disulfide bridge. Hence, included are peptide aptamers
that bind to the AARS polypeptides described herein and/or their
cellular binding partners. Peptide aptamer selection can be
performed using different systems known in the art, including the
yeast two-hybrid system.
[0217] As noted above, the AARS polypeptides and binding agents of
the present invention can be used in any of the diagnostic, drug
discovery, or therapeutic methods described herein.
Formulation and Administration
[0218] The compositions of the invention (e.g., polypeptides,
polynucleotides, antibodies, etc.) are generally formulated in
pharmaceutically-acceptable or physiologically-acceptable solutions
for administration to a cell, tissue or animal, either alone, or in
combination with one or more other modalities of therapy. It will
also be understood that, if desired, the compositions of the
invention may be administered in combination with other agents as
well, such as, e.g., other proteins or polypeptides or various
pharmaceutically-active agents. There is virtually no limit to
other components that may also be included in the compositions,
provided that the additional agents do not adversely affect
properties of a polypeptide of the invention.
[0219] In the pharmaceutical compositions of the invention,
formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the
development of suitable dosing and treatment regimens for using the
particular compositions described herein in a variety of treatment
regimens, including e.g., oral, parenteral, intravenous,
intranasal, intracranial and intramuscular administration and
formulation.
[0220] In certain applications, the pharmaceutical compositions
disclosed herein may be delivered via oral administration to a
subject. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0221] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally as
described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No.
5,641,515 and U.S. Pat. No. 5,399,363 (each specifically
incorporated herein by reference in its entirety). Solutions of the
active compounds as free base or pharmacologically acceptable salts
may be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0222] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form should
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and should be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be facilitated by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0223] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage may be dissolved in 1 ml of isotonic NaCl solution and
either added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion (see, e.g., Remington's Pharmaceutical
Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and the general
safety and purity standards as required by FDA Office of Biologics
standards.
[0224] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with the various other ingredients enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0225] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug-release capsules,
and the like.
[0226] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0227] The phrase "pharmaceutically-acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0228] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering genes, polynucleotides,
and peptide compositions directly to the lungs via nasal aerosol
sprays has been described e.g., in U.S. Pat. No. 5,756,353 and U.S.
Pat. No. 5,804,212 (each specifically incorporated herein by
reference in its entirety). Likewise, the delivery of drugs using
intranasal microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
[0229] In certain embodiments, the delivery may occur by use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the compositions of the present invention may be
formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The
formulation and use of such delivery vehicles can be carried out
using known and conventional techniques.
Kits Comprising Compositions of the Invention
[0230] The invention, in other aspects, provides kits comprising
one or more containers filled with one or more of the polypeptides,
polynucleotides, antibodies, multiunit complexes, compositions
thereof, etc., of the invention, as described herein. The kits can
include written instructions on how to use such compositions (e.g.,
to modulate cellular signaling, angiogenesis, cancer, inflammatory
conditions, etc.).
[0231] The kits herein may also include a one or more additional
therapeutic agents or other components suitable or desired for the
indication being treated. An additional therapeutic agent may be
contained in a second container, if desired. Examples of additional
therapeutic agents include, but are not limited to antineoplastic
agents, anti-inflammatory agents, antibacterial agents, antiviral
agents, angiogenic agents, etc.
[0232] The kits herein can also include one or more syringes or
other components necessary or desired to facilitate an intended
mode of delivery (e.g., stents, implantable depots, etc.).
Methods of Use
[0233] Embodiments of the present invention also include methods of
using the aminoacyl-tRNA (AARS) "agents" described herein for
diagnostic, drug discovery, and/or therapeutic purposes. The term
AARS "agents" refers generally to the AARS polynucleotides, AARS
polypeptides, binding agents such as peptide mimetics, and other
compounds described herein. For diagnostic purposes, the AARS
agents can be used in a variety of non-limiting ways, such as to
distinguish between different cell types or different cellular
states, or to identify subjects having a relevant disease or
condition. For drug discovery purposes, the AARS agents can be used
to identify one or more cellular "binding partners" of an AARS
polypeptide, characterize one or more "non-canonical" activities of
an AARS polypeptide, identify agents that selectively or
non-selectively agonize or antagonize the interaction of an AARS
polypeptide with its binding partner(s), and/or identify agents
that selectively or non-selectively agonize or antagonize one or
more "non-canonical" activities of an AARS polypeptide. For
therapeutic purposes, the AARS agents or compositions provided
herein can be used to treat a variety of diseases or conditions,
detailed below.
[0234] Drug Discovery
[0235] Certain embodiments relate to the use of the aminoacyl-tRNA
synthetase (AARS) polypeptide sequences described herein in drug
discovery, typically to identify agents that modulate one or more
of the non-canonical activities of the polypeptide. For example,
certain embodiments include methods of identifying one or more
"binding partners" of an AARS polypeptide of the present invention,
such as a cellular protein or other host molecule that associates
with the polypeptide and participates in its non-canonical activity
or activities. Also included are methods of identifying a compound
(e.g., polypeptide) or other agent that agonizes or antagonizes the
non-canonical activity of a reference polypeptide or active variant
thereof, such as by interacting with the polypeptide and/or one or
more of its cellular binding partners.
[0236] Certain embodiments therefore include methods of identifying
a binding partner of an AARS polypeptide, comprising a) combining
the AARS polypeptide with a biological sample under suitable
conditions, and b) detecting specific binding of the AARS
polypeptide to a binding partner, thereby identifying a binding
partner that specifically binds to the AARS polypeptide. Also
included are methods of screening for a compound that specifically
binds to an AARS polypeptide or a binding partner of the
polypeptide, comprising a) combining the polypeptide or the binding
partner with at least one test compound under suitable conditions,
and b) detecting binding of the polypeptide or the binding partner
to the test compound, thereby identifying a compound that
specifically binds to the polypeptide or its binding partner. In
certain embodiments, the compound is a polypeptide or peptide. In
certain embodiments, the compound is a small molecule or other
(e.g., non-biological) chemical compound. In certain embodiments,
the compound is a peptide mimetic.
[0237] Any method suitable for detecting protein-protein
interactions may be employed for identifying cellular proteins that
interact with an AARS polypeptide, interact with one or more of its
cellular binding partners, or both. Examples of traditional methods
that may be employed include co-immunoprecipitation, cross-linking,
and co-purification through gradients or chromatographic columns of
cell lysates or proteins obtained from cell lysates, mainly to
identify proteins in the lysate that interact with the AARS
polypeptide.
[0238] In these and related embodiments, at least a portion of the
amino acid sequence of a protein that interacts with an AARS
polypeptide or its binding partner can be ascertained using
techniques well known to those of skill in the art, such as via the
Edman degradation technique. See, e.g., Creighton Proteins:
Structures and Molecular Principles, W. H. Freeman & Co., N.Y.,
pp. 34 49, 1983. The amino acid sequence obtained may be used as a
guide for the generation of oligonucleotide mixtures that can be
used to screen for gene sequences encoding such proteins. Screening
may be accomplished, for example, by standard hybridization or PCR
techniques, as described herein and known in the art. Techniques
for the generation of oligonucleotide mixtures and the screening
are well known. See, e.g., Ausubel et al. Current Protocols in
Molecular Biology Green Publishing Associates and Wiley
Interscience, N.Y., 1989; and Innis et al., eds. PCR Protocols: A
Guide to Methods and Applications Academic Press, Inc., New York,
1990.
[0239] Additionally, methods may be employed in the simultaneous
identification of genes that encode the binding partner or other
polypeptide. These methods include, for example, probing expression
libraries, in a manner similar to the well known technique of
antibody probing of lambda-gt11 libraries, using labeled AARS
protein, or another polypeptide, peptide or fusion protein, e.g., a
variant AARS polypeptide or AARS domain fused to a marker (e.g., an
enzyme, fluor, luminescent protein, or dye), or an Ig-Fc
domain.
[0240] One method that detects protein interactions in vivo, the
two-hybrid system, is described in detail for illustration only and
not by way of limitation. One example of this system has been
described (Chien et al., PNAS USA 88:9578 9582, 1991) and is
commercially available from Clontech (Palo Alto, Calif.).
[0241] Briefly, utilizing such a system, plasmids may be
constructed that encode two hybrid proteins: one plasmid consists
of nucleotides encoding the DNA-binding domain of a transcription
activator protein fused to an AARS reference nucleotide sequence
(or, in certain embodiments, its binding partner), or a variant
thereof, and the other plasmid consists of nucleotides encoding the
transcription activator protein's activation domain fused to a cDNA
(or collection of cDNAs) encoding an unknown protein(s) that has
been recombined into the plasmid as part of a cDNA library. The
DNA-binding domain fusion plasmid and the activator cDNA library
may be transformed into a strain of the yeast Saccharomyces
cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose
regulatory region contains the transcription activator's binding
site. Either hybrid protein alone cannot activate transcription of
the reporter gene: the DNA-binding domain hybrid cannot because it
does not provide activation function and the activation domain
hybrid cannot because it cannot localize to the activator's binding
sites. Interaction of the two hybrid proteins reconstitutes the
functional activator protein and results in expression of the
reporter gene, which is detected by an assay for the reporter gene
product.
[0242] The two-hybrid system or other such methodology may be used
to screen activation domain libraries for proteins that interact
with the "bait" gene product. By way of example, and not by way of
limitation, an AARS reference polypeptide or variant may be used as
the bait gene product. An AARS binding partner may also be used as
a "bait" gene product. Total genomic or cDNA sequences are fused to
the DNA encoding an activation domain. This library and a plasmid
encoding a hybrid of a bait AARS gene product fused to the
DNA-binding domain are co-transformed into a yeast reporter strain,
and the resulting transformants are screened for those that express
the reporter gene.
[0243] A cDNA library of the cell line from which proteins that
interact with bait AARS gene products are to be detected can be
made using methods routinely practiced in the art. For example, the
cDNA fragments can be inserted into a vector such that they are
translationally fused to the transcriptional activation domain of
GAL4. This library can be co-transformed along with the bait
gene-GAL4 fusion plasmid into a yeast strain, which contains a lacZ
gene driven by a promoter that contains GAL4 activation sequence. A
cDNA encoded protein, fused to GAL4 transcriptional activation
domain, that interacts with bait gene product will reconstitute an
active GAL4 protein and thereby drive expression of the HIS3 gene.
Colonies, which express HIS3, can be detected by their growth on
Petri dishes containing semi-solid agar based media lacking
histidine. The cDNA can then be purified from these strains, and
used to produce and isolate the bait AARS gene-interacting protein
using techniques routinely practiced in the art.
[0244] Also included are three-hybrid systems, which allow the
detection of RNA-protein interactions in yeast. See, e.g., Hook et
al., RNA. 11:227-233, 2005. Accordingly, these and related methods
can be used to identify a cellular binding partner of an AARS
polypeptide, and to identify other proteins or nucleic acids that
interact with the AARS polypeptide, the cellular binding partner,
or both.
[0245] As noted above, once isolated, binding partners can be
identified and can, in turn, be used in conjunction with standard
techniques to identify proteins or other compounds with which it
interacts. Certain embodiments thus relate to methods of screening
for a compound that specifically binds to the binding partner of an
AARS reference polypeptide, comprising a) combining the binding
partner with at least one test compound under suitable conditions,
and b) detecting binding of the binding partner to the test
compound, thereby identifying a compound that specifically binds to
the binding partner. In certain embodiments, the test compound is a
polypeptide. In certain embodiments, the test compound is a
chemical compound, such as a small molecule compound or peptide
mimetic.
[0246] Certain embodiments include methods of screening for a
compound that modulates the activity of an AARS polypeptide,
comprising a) combining the polypeptide with at least one test
compound under conditions permissive for the activity of the
polypeptide, b) assessing the activity of the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the test compound with the activity
of the polypeptide in the absence of the test compound, wherein a
change in the activity of the polypeptide in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide. Certain embodiments include methods of
screening for a compound that modulates the activity of a binding
partner of an AARS polypeptide, comprising a) combining the
polypeptide with at least one test compound under conditions
permissive for the activity of the binding partner, b) assessing
the activity of the binding partner in the presence of the test
compound, and c) comparing the activity of the binding partner in
the presence of the test compound with the activity of the binding
partner in the absence of the test compound, wherein a change in
the activity of the binding partner in the presence of the test
compound is indicative of a compound that modulates the activity of
the binding partner. Typically, these and related embodiments
include assessing a selected non-canonical activity that is
associated with the AARS polypeptide or its binding partner.
Included are in vitro and in vivo conditions, such as cell culture
conditions.
[0247] Certain embodiments include methods of screening a compound
for effectiveness as a full or partial agonist of an AARS
polypeptide or an active fragment or variant thereof, comprising a)
exposing a sample comprising the polypeptide to a compound, and b)
detecting agonist activity in the sample, typically by measuring an
increase in the non-canonical activity of the AARS polypeptide.
Certain methods include a) exposing a sample comprising a binding
partner of the AARS polypeptide to a compound, and b) detecting
agonist activity in the sample, typically by measuring an increase
in the selected non-canonical activity of the AARS polypeptide.
Certain embodiments include compositions that comprise an agonist
compound identified by the method and a pharmaceutically acceptable
carrier or excipient.
[0248] Also included are methods of screening a compound for
effectiveness as a full or partial antagonist of an AARS
polypeptide, comprising a) exposing a sample comprising the
polypeptide to a compound, and b) detecting antagonist activity in
the sample, typically by measuring a decrease in the non-canonical
activity of the AARS polypeptide. Certain methods include a)
exposing a sample comprising a binding partner of the AARS
polypeptide to a compound, and b) detecting antagonist activity in
the sample, typically by measuring a decrease in the selected
non-canonical activity of the AARS polypeptide. Certain embodiments
include compositions that comprise an antagonist compound
identified by the method and a pharmaceutically acceptable carrier
or excipient.
[0249] In certain embodiments, in vitro systems may be designed to
identify compounds capable of interacting with or modulating an
AARS reference sequence or its binding partner. Certain of the
compounds identified by such systems may be useful, for example, in
modulating the activity of the pathway, and in elaborating
components of the pathway itself. They may also be used in screens
for identifying compounds that disrupt interactions between
components of the pathway; or may disrupt such interactions
directly. One exemplary approach involves preparing a reaction
mixture of the AARS polypeptide and a test compound under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex that can be removed from and/or
detected in the reaction mixture
[0250] In vitro screening assays can be conducted in a variety of
ways. For example, an AARS polypeptide, a cellular binding partner,
or test compound(s) can be anchored onto a solid phase. In these
and related embodiments, the resulting complexes may be captured
and detected on the solid phase at the end of the reaction. In one
example of such a method, the AARS polypeptide and/or its binding
partner are anchored onto a solid surface, and the test
compound(s), which are not anchored, may be labeled, either
directly or indirectly, so that their capture by the component on
the solid surface can be detected. In other examples, the test
compound(s) are anchored to the solid surface, and the AARS
polypeptide and/or its binding partner, which are not anchored, are
labeled or in some way detectable. In certain embodiments,
microtiter plates may conveniently be utilized as the solid phase.
The anchored component (or test compound) may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0251] To conduct an exemplary assay, the non-immobilized component
is typically added to the coated surface containing the anchored
component. After the reaction is complete, un-reacted components
are removed (e.g., by washing) under conditions such that any
specific complexes formed will remain immobilized on the solid
surface. The detection of complexes anchored on the solid surface
can be accomplished in a number of ways. For instance, where the
previously non-immobilized component is pre-labeled, the detection
of label immobilized on the surface indicates that complexes were
formed. Where the previously non-immobilized component is not
pre-labeled, an indirect label can be used to detect complexes
anchored on the surface; e.g., using a labeled antibody specific
for the previously non-immobilized component (the antibody, in
turn, may be directly labeled or indirectly labeled with a labeled
anti-Ig antibody).
[0252] Alternatively, the presence or absence of binding of a test
compound can be determined, for example, using surface plasmon
resonance (SPR) and the change in the resonance angle as an index,
wherein an AARS polypeptide or a cellular binding partner is
immobilized onto the surface of a commercially available sensorchip
(e.g., manufactured by Biacore.TM.) according to a conventional
method, the test compound is contacted therewith, and the
sensorchip is illuminated with a light of a particular wavelength
from a particular angle. The binding of a test compound can also be
measured by detecting the appearance of a peak corresponding to the
test compound by a method wherein an AARS polypeptide or a cellular
binding partner is immobilized onto the surface of a protein chip
adaptable to a mass spectrometer, a test compound is contacted
therewith, and an ionization method such as MALDI-MS, ESI-MS,
FAB-MS and the like is combined with a mass spectrometer (e.g.,
double-focusing mass spectrometer, quadrupole mass spectrometer,
time-of-flight mass spectrometer, Fourier transformation mass
spectrometer, ion cyclotron mass spectrometer and the like).
[0253] In certain embodiments, cell-based assays, membrane
vesicle-based assays, or membrane fraction-based assays can be used
to identify compounds that modulate interactions in the
non-canonical pathway of the selected AARS polypeptide. To this
end, cell lines that express an AARS polypeptide and/or a binding
partner, or a fusion protein containing a domain or fragment of
such proteins (or a combination thereof), or cell lines (e.g., COS
cells, CHO cells, HEK293 cells, Hela cells etc.) that have been
genetically engineered to express such protein(s) or fusion
protein(s) can be used. Test compound(s) that influence the
non-canonical activity can be identified by monitoring a change
(e.g., a statistically significant change) in that activity as
compared to a control or a predetermined amount.
[0254] For embodiments that relate to antisense and RNAi agents,
for example, also included are methods of screening a compound for
effectiveness in altering expression of an AARS polynucleotide
sequence, comprising a) exposing a sample comprising the AARS
polynucleotide to a compound such as a potential antisense
oligonucleotide, and b) detecting altered expression of the AARS
polynucleotide. In certain non-limiting examples, these and related
embodiments can be employed in cell-based assays or in cell-free
translation assays, according to routine techniques in the art.
Also included are the antisense oligonucleotides and RNAi agents
identified by such methods.
[0255] Also included are any of the above methods, or other
screening methods known in the art, which are adapted for
high-throughput screening (HTS). HTS typically uses automation to
run a screen of an assay against a library of candidate compounds,
for instance, an assay that measures an increase or a decrease in a
non-canonical activity, as described herein.
[0256] Methods of Treatment
[0257] In another aspect, the present invention relates to methods
of using the compositions of the present invention for treating a
cell, tissue or subject with a composition as described herein. The
cells or tissues that may be modulated by the present invention are
preferably mammalian cells or tissues, or more preferably human
cells or tissues. Such cells or tissues can be of a healthy state
or of a diseased state.
[0258] In certain embodiments, for example, methods are provided
for modulating therapeutically relevant cellular activities
including, but not limited to, cellular metabolism, cell
differentiation, cell proliferation, cell death, cell mobilization,
cell migration, gene transcription, mRNA translation, cell
impedance, and the like, comprising contacting a cell with a
composition as described herein. Examples of cells or tissues that
may be modulated include those of the skin (e.g., dermis,
epidermis, subcutaneous layer), hair follicles, nervous system
(e.g., brain, spinal cord, peripheral nerves), auditory system or
balance organs (e.g., inner ear, middle ear, outer ear),
respiratory system (e.g., nose, trachea, lungs), gastroesophogeal
tissues, the gastrointestinal system (e.g., mouth, esophagus,
stomach, small intestines, large intestines, rectum), vascular
system (e.g., heart, blood vessels and arteries), liver,
gallbladder, lymphatic/immune system (e.g., lymph nodes, lymphoid
follicles, spleen, thymus, bone marrow), uro-genital system (e.g.,
kidneys, ureter, bladder, urethra, cervix, Fallopian tubes,
ovaries, uterus, vulva, prostate, bulbourethral glands, epidiymis,
prostate, seminal vesicles, testicles), musculoskeletal system
(e.g., skeletal muscles, smooth muscles, bone, cartilage, tendons,
ligaments), adipose tissue, mammaries, and the endocrine system
(e.g., hypothalamus, pituitary, thyroid, pancreas, adrenal glands).
Accordingly, the compositions may be employed in treating
essentially any cell or tissue or subject that would benefit from
modulation of one or more such exemplary activities, or one or more
of such exemplary cells or tissues.
[0259] The compositions may also be used in any of a number of
therapeutic contexts including, for example, those relating to the
treatment or prevention of neoplastic diseases, immune system
diseases (e.g., autoimmune diseases and inflammation), infectious
diseases, metabolic diseases, neuronal/neurological diseases,
muscular/cardiovascular diseases, diseases associated with aberrant
hematopoiesis, diseases associated with aberrant angiogenesis,
diseases associated with aberrant cell survival, and others.
[0260] For example, in certain illustrative embodiments, the
compositions of the invention may be used to modulate angiogenesis,
e.g., via modulation of endothelial cell proliferation and/or
signaling. Endothelial cell proliferation and/or signaling may be
monitored using an appropriate cell line (e.g., human microvascular
endothelial lung cells (HMVEC-L) and human umbilical vein
endothelial cells (HUVEC)), and using an appropriate assay (e.g.,
endothelial cell migration assays, endothelial cell proliferation
assays, tube-forming assays, matrigel plug assays, etc.), many of
which are known and available in the art.
[0261] Therefore, in related embodiments, the compositions of the
invention may be employed in the treatment of essentially any cell
or tissue or subject that would benefit from modulation of
angiogenesis. For example, in some embodiments, a cell or tissue or
subject experiencing or susceptible to angiogenesis (e.g., an
angiogenic condition) may be contacted with a suitable composition
of the invention to inhibit an angiogenic condition. In other
embodiments, a cell or tissue experiencing or susceptible to
insufficient angiogenesis (e.g., an angiostatic condition) may be
contacted with an appropriate composition of the invention in order
to interfere with angiostatic activity and/or promote
angiogenesis.
[0262] Illustrative examples of angiogenic conditions include, but
are not limited to, age-related macular degeneration (AMD), cancer
(both solid and hematologic), developmental abnormalities
(organogenesis), diabetic blindness, endometriosis, ocular
neovascularization, psoriasis, rheumatoid arthritis (RA), and skin
discolorations (e.g., hemangioma, nevus flammeus or nevus simplex).
Examples of anti-angiogenic conditions include, but are not limited
to, cardiovascular disease, restenosis, tissue damage after
reperfusion of ischemic tissue or cardiac failure, chronic
inflammation and wound healing.
[0263] The compositions of the invention may also be useful as
immunomodulators for treating anti- or pro-inflammatory indications
by modulating the cells that mediate, either directly or
indirectly, autoimmune and/or inflammatory diseases, conditions and
disorders. The utility of the compositions of the invention as
immunomodulators can be monitored using any of a number of known
and available techniques in the art including, for example,
migration assays (e.g., using leukocytes or lymphocytes) or cell
viability assays (e.g., using B-cells, T-cells, monocytes or NK
cells). In certain embodiments, the compositions of the invention
may modulate inflammatory cytokines, such as TNF-.alpha.. In
certain embodiments, the compositions of the invention may modulate
chemotaxis of immune cells, such as monocytes.
[0264] "Inflammation" refers generally to the biological response
of tissues to harmful stimuli, such as pathogens, damaged cells
(e.g., wounds), and irritants. The term "inflammatory response"
refers to the specific mechanisms by which inflammation is achieved
and regulated, including, merely by way of illustration, immune
cell activation or migration, cytokine production, vasodilation,
including kinin release, fibrinolysis, and coagulation, among
others described herein and known in the art. Ideally, inflammation
is a protective attempt by the body to both remove the injurious
stimuli and initiate the healing process for the affected tissue or
tissues. In the absence of inflammation, wounds and infections
would never heal, creating a situation in which progressive
destruction of the tissue would threaten survival. On the other
hand, excessive or chronic inflammation may associate with a
variety of diseases, such as hay fever, atherosclerosis, and
rheumatoid arthritis, among others described herein and known in
the art.
[0265] The compositions of the invention may modulate acute
inflammation, chronic inflammation, or both. Certain embodiments
relate to increasing acute inflammation or acute inflammatory
responses, and certain embodiments relate to increasing chronic
inflammation or chronic inflammatory responses. Depending on the
needs of the subject, certain embodiments relate to reducing acute
inflammation or inflammatory responses, and certain embodiments
relate to reducing chronic inflammation or chronic inflammatory
responses.
[0266] Acute inflammation relates to the initial response of the
body to presumably harmful stimuli and involves increased movement
of plasma and leukocytes from the blood into the injured tissues.
It is a short-term process, typically beginning within minutes or
hours and ending upon the removal of the injurious stimulus. Acute
inflammation may be characterized by any one or more of redness,
increased heat, swelling, pain, and loss of function. Redness and
heat are due mainly to increased blood flow at body core
temperature to the inflamed site, swelling is caused by
accumulation of fluid, pain is typically due to release of
chemicals that stimulate nerve endings, and loss of function has
multiple causes.
[0267] Acute inflammatory responses are initiated mainly by local
immune cells, such as resident macrophages, dendritic cells,
histiocytes, Kuppfer cells and mastocytes. At the onset of an
infection, burn, or other injuries, these cells undergo activation
and release inflammatory mediators responsible for the clinical
signs of inflammation, such as vasoactive amines and eicosanoids.
Vasodilation and its resulting increased blood flow cause the
redness and increased heat. Increased permeability of the blood
vessels results in an exudation or leakage of plasma proteins and
fluid into the tissue, which creates swelling. Certain released
mediators such as bradykinin increase sensitivity to pain, and
alter the blood vessels to permit the migration or extravasation of
leukocytes, such as neutrophils, which typically migrate along a
chemotactic gradient created by the local immune cells.
[0268] Acute inflammatory responses also includes one or more
acellular biochemical cascade systems, consisting of preformed
plasma proteins modulate, which act in parallel to initiate and
propagate the inflammatory response. These systems include the
complement system, which is mainly activated by bacteria, and the
coagulation and fibrinolysis systems, which are mainly activated by
necrosis, such as the type of tissue damage that is caused by
certain infections, burns, or other trauma. Hence, the compositions
of the invention may be used to modulate acute inflammation, or any
of one or more of the individual acute inflammatory responses.
[0269] Chronic inflammation, a prolonged and delayed inflammatory
response, is characterized by a progressive shift in the type of
cells that are present at the site of inflammation, and often leads
to simultaneous or near simultaneous destruction and healing of the
tissue from the inflammatory process. At the cellular level,
chronic inflammatory responses involve a variety of immune cells
such as monocytes, macrophages, lymphocytes, plasma cells, and
fibroblasts, though in contrast to acute inflammation, which is
mediated mainly by granulocytes, chronic inflammation is mainly
mediated by mononuclear cells such as monocytes and lymphocytes.
Chronic inflammation also involves a variety of inflammatory
mediators, such as IFN-.gamma. and other cytokines, growth factors,
reactive oxygen species, and hydrolytic enzymes. Chronic
inflammation may last for many months or years, and may result in
undesired tissue destruction and fibrosis.
[0270] Clinical signs of chronic inflammation are dependent upon
duration of the illness, inflammatory lesions, cause and anatomical
area affected. (see, e.g., Kumar et al., Robbins Basic
Pathology-8.sup.th Ed., 2009 Elsevier, London; Miller, L M,
Pathology Lecture Notes, Atlantic Veterinary College,
Charlottetown, PEI, Canada). Chronic inflammation is associated
with a variety of pathological conditions or diseases, including,
for example, allergies, Alzheimer's disease, anemia, aortic valve
stenosis, arthritis such as rheumatoid arthritis and
osteoarthritis, cancer, congestive heart failure, fibromyalgia,
fibrosis, heart attack, kidney failure, lupus, pancreatitis,
stroke, surgical complications, inflammatory lung disease,
inflammatory bowel disease, atherosclerosis, and psoriasis, among
others described herein and known in the art. Hence, the
compositions provided herein may be used to treat or manage chronic
inflammation, modulate any of one or more of the individual chronic
inflammatory responses, or treat any one or more diseases or
conditions associated with chronic inflammation.
[0271] The compositions of the invention may also modulate
proliferative inflammation, an inflammatory process characterized
by an increase in the number of tissue cells. These can encompass
skin conditions such as psoriasis, seborrhea or eczema, or can also
be thought of in terms of cancers and abnormal growths especially
in light of accumulating evidence based on more efficient molecular
methods to document even low grade chronic inflammation.
[0272] Criteria for assessing the signs and symptoms of
inflammatory and other conditions, including for purposes of making
differential diagnosis and also for monitoring treatments such as
determining whether a therapeutically effective dose has been
administered in the course of treatment, e.g., by determining
improvement according to accepted clinical criteria, will be
apparent to those skilled in the art and are exemplified by the
teachings of e.g., Berkow et al., eds., The Merck Manual, 16.sup.th
edition, Merck and Co., Rahway, N.J., 1992; Goodman et al., eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
10.sup.th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001);
Avery's Drug Treatment: Principles and Practice of Clinical
Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd.,
Williams and Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology,
Little, Brown and Co., Boston, (1985); Osolci al., eds.,
Remington's Pharmaceutical Sciences, 18.sup.th edition, Mack
Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical
Pharmacology, Appleton and Lange, Norwalk, Conn. (1992).
[0273] Illustrative immune system diseases, disorders or conditions
that may be treated according to the present invention include, but
are not limited to, primary immunodeficiencies, immune-mediated
thrombocytopenia, Kawasaki syndrome, bone marrow transplant (for
example, recent bone marrow transplant in adults or children),
chronic B cell lymphocytic leukemia, HIV infection (for example,
adult or pediatric HIV infection), chronic inflammatory
demyelinating polyneuropathy, post-transfusion purpura, and the
like.
[0274] As noted above, certain embodiments may employ the
compositions of the invention to increase inflammation or increase
an immune response. For instance, depending on the needs of the
subject, certain embodiments may increase acute inflammation or
increase acute inflammatory responses or both. Certain embodiments
may increase chronic inflammation or chronic inflammatory responses
or both. Certain embodiments may increase both acute and chronic
inflammation. Certain embodiments may increase local or systemic
inflammation or both.
[0275] In certain embodiments, compositions may be used to treat or
manage immunodeficiencies, including primary immunodeficiencies and
secondary immunodeficiencies, in which the body may not mount an
adequate inflammatory response. Examples of primary
immunodeficiencies include various autosomal recessive and X-linked
genetic conditions such as T-cell and B-cell immunodeficiencies,
including combined T-cell and B-cell immunodeficiencies, antibody
deficiencies, well-defined syndromes, immune dysregulation
diseases, phagocyte disorders, innate immunity disorders, and
complement deficiencies.
[0276] Examples of T-cell and B-cell immunodeficiencies include
T-/B+ deficiencies such as .gamma.c deficiency, JAK3 deficiency,
interleukin 7 receptor chain a deficiency, CD45 deficiency,
CD3.delta./CD3.epsilon. deficiency; and T-/B- deficiencies such as
RAG 1/2 deficiency, DCLRE1C deficiency, adenosine deaminase (ADA)
deficiency, reticular dysgenesis. Additional examples include Omenn
syndrome, DNA ligase type IV deficiency, CD40 ligand deficiency,
CD40 deficiency, purine nucleoside phosphorylase (PNP) deficiency,
MHC class II deficiency, CD3.gamma. deficiency, CD8 deficiency,
ZAP-70 deficiency, TAP-1/2 deficiency, and winged helix
deficiency.
[0277] Examples of antibody deficiencies include X-linked
agammaglobulinemia (btk deficiency, or Bruton's
agammaglobulinemia), .mu.-Heavy chain deficiency, I-5 deficiency,
Ig.alpha. deficiency, BLNK deficiency, thymoma with
immunodeficiency, common variable immunodeficiency (LVID), ICOS
deficiency, CD19 deficiency, TACI (TNFRSF13B) deficiency, and BAFF
receptor deficiency. Additional examples include AID deficiency,
UNG deficiency, heavy chain deletions, kappa chain deficiency,
isolated IgG subclass deficiency, IgA with IgG subclass deficiency,
selective immunoglobulin A deficiency, and transient
hypogammaglobulinemia of infancy (THI).
[0278] Examples of "well-defined syndromes" include Wiskott-Aldrich
syndrome, ataxia telangiectasia, ataxia-like syndrome, Nijmegen
breakage syndrome, Bloom syndrome, DiGeorge syndrome,
immuno-osseous dysplasias such as cartilage-hair hypoplasia,
Schimke syndrome, Hermansky-Pudlak syndrome type 2, Hyper-IgE
syndrome, chronic mucocutaneous candidiasis.
[0279] Examples of immune dysregulation diseases include
immunodeficiency with hypopigmentation or albinism such as
Chediak-Higashi syndrome and Griscelli syndrome type 2, familial
hemophagocytic lymphohistiocytosis such as perforin deficiency,
MUNC13D deficiency, and syntaxin 11 deficiency, X-linked
lymphoproliferative syndrome, autoimmune lymphoproliferative
syndrome such as type 1a (CD95 defects), type 1b (Fas ligand
defects), type 2a (CASP10 defects), and type 2b (CASP8 defects),
autoimmune polyendocrinopathy with candidiasis and ectodermal
dystrophy, and immunodysregulation polyendocrinopathy enteropathy
X-linked syndrome. Additionally, diseases affecting the bone marrow
may result in abnormal or few leukocytes, such as leukopenia.
Leukopenia can be induced by certain infections and diseases,
including viral infection, Rickettsia infection, some protozoa,
tuberculosis, and certain cancers
[0280] Examples of phagocyte disorders include severe congenital
neutropenia such as ELA2 deficiency (e.g., with myelodysplasia),
GFI1 deficiency (with T/B lymphopenia) or G-CSFR deficiency
(G-CSF-unresponsive), Kostmann syndrome, cyclic neutropenia,
X-linked neutropenia/myelodysplasia, leukocyte adhesion deficiency
types 1, 2 and 3, RAC2 deficiency, .beta.-actin deficiency,
localized juvenile periodontitis, Papillon-Lefevre syndrome,
specific granule deficiency, Shwachman-Diamond syndrome, chronic
granulomatous disease, including X-linked and autosomal forms,
neutrophil glucose-6-phosphate dehydrogenase deficiency, IL-12 and
IL-23 .beta.1 chain deficiency, IL-12p40 deficiency, interferon
.gamma. receptor 1 deficiency, interferon .gamma. receptor 2
deficiency, and STAT1 deficiency.
[0281] Examples of innate immunity deficiencies include
hypohidrotic ectodermal dysplasia such as NEMO deficiency and IKBA
deficiency, IRAK-4 deficiency, WHIM syndrome (warts,
hypogammaglobulinaemia, infections, myleokathexis), and
epidermodysplasia verruciformis. Examples of complement
deficiencies and exemplary associated conditions include C1q
deficiency (e.g., lupus-like syndrome, rheumatoid disease,
infections), C1r deficiency, C4 deficiency, C2 deficiency (e.g.,
lupus-like syndrome, vasculitis, polymyositis, pyogenic
infections), C3 deficiency (e.g., recurrent pyogenic infections),
C5 deficiency (e.g., neisserial infections), C6 deficiency, C7
deficiency (e.g., vasculitis), C8a and C8b deficiency, C9
deficiency (e.g., neisserial infections), C1-inhibitor deficiency
(e.g., hereditary angioedema), Factor I deficiency (pyogenic
infections), Factor H deficiency (e.g., haemolytic-uraemic
syndrome, membranoproliferative glomerulonephritis), Factor D
deficiency (e.g., neisserial infections), Properdin deficiency
(e.g., neisserial infections), MBP deficiency (e.g., pyogenic
infections), and MASP2 deficiency.
[0282] Primary immune deficiencies can be diagnosed according to
routine techniques in the art. Exemplary diagnostic tests include,
without limitation, performing counts of the different types of
mononuclear cells in the blood (e.g., lymphocytes and monocytes,
including lymphocytes, different groups of B lymphocytes such as
CD19+, CD20+, and CD21+ lymphocytes, natural killer cells, and
monocytes positive for CD15+), measuring the presence of activation
markers (e.g., HLA-DR, CD25, CD80), performing tests for T cell
function such as skin tests for delayed-type hypersensitivity, cell
responses to mitogens and allogeneic cells, cytokine production by
cells, performing tests for B cell function such as by identifying
antibodies to routine immunizations and commonly acquired
infections and by quantifying IgG subclasses, and performing tests
or phagocyte function, such as by measuring the reduction of nitro
blue tetrazolium chloride, and performing assays of chemotaxis and
bactericidal activity. AARS polypeptides may therefore be used to
stimulate or maintain acute inflammation or acute inflammatory
responses in subjects with a primary immunodeficiency, as described
herein and known in the art.
[0283] Examples of causes of secondary immunodeficiencies include
malnutrition, aging, and medications (e.g., chemotherapy,
disease-modifying anti-rheumatic drugs, immunosuppressive drugs
after organ transplants, glucocorticoids). Additional causes
include various cancers, including cancers of the bone marrow and
blood cells (e.g., leukemia, lymphoma, multiple myeloma), and
certain chronic infections, such as acquired immunodeficiency
syndrome (AIDS), caused by the human immunodeficiency virus (HIV).
AARS polypeptides may be used to stimulate or maintain acute
inflammation or acute inflammatory responses in subjects with an
immunodeficiency, as described herein and known in the art. AARS
polypeptides may also be used to stimulate or maintain chronic
inflammation or chronic inflammatory responses in subjects with a
secondary immunodeficiency, as described herein and known in the
art.
[0284] Additionally, further diseases, disorders and conditions
include Guillain-Barre syndrome, anemia (for example, anemia
associated with parvovirus B19, patients with stable multiple
myeloma who are at high risk for infection (for example, recurrent
infection), autoimmune hemolytic anemia (for example, warm-type
autoimmune hemolytic anemia), thrombocytopenia (for example,
neonatal thrombocytopenia), and immune-mediated neutropenia,
transplantation (for example, cytomegalovirus (CMV)-negative
recipients of CMV-positive organs), hypogammaglobulinemia (for
example, hypogammaglobulinemic neonates with risk factor for
infection or morbidity), epilepsy (for example, intractable
epilepsy), systemic vasculitic syndromes, myasthenia gravis (for
example, decompensation in myasthenia gravis), dermatomyositis, and
polymyositis.
[0285] Further autoimmune diseases, disorders and conditions
include, but are not limited to, autoimmune hemolytic anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid
syndrome, dermatitis, allergic encephalomyelitis, myocarditis,
relapsing polychondritis, rheumatic heart disease,
glomerulonephritis (for example, IgA nephropathy), multiple
sclerosis, neuritis, uveitis ophthalmia, polyendocrinopathies,
purpura (for example, Henloch-Scoenlein purpura), Reiter's disease,
stiff-man syndrome, autoimmune pulmonary inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye disease.
[0286] Additional autoimmune diseases, disorders or conditions
include, but are not limited to, autoimmune thyroiditis;
hypothyroidism, including Hashimoto's thyroiditis and thyroiditis
characterized, for example, by cell-mediated and humoral thyroid
cytotoxicity; SLE (which is often characterized, for example, by
circulating and locally generated immune complexes); Goodpasture's
syndrome (which is often characterized, for example, by
anti-basement membrane antibodies); pemphigus (which is often
characterized, for example, by epidermal acantholytic antibodies);
receptor autoimmunities such as, for example, Graves' disease
(which is often characterized, for example, by antibodies to a
thyroid stimulating hormone receptor) myasthenia gravis, (which is
often characterized, for example, by acetylcholine receptor
antibodies); insulin resistance (which is often characterized, for
example, by insulin receptor antibodies); autoimmune hemolytic
anemia (which is often characterized, for example, by phagocytosis
of antibody-sensitized red blood cells); and autoimmune
thrombocytopenic purpura (which is often characterized, for
example, by phagocytosis of antibody-sensitized platelets).
[0287] Further autoimmune diseases, disorders or conditions
include, but are not limited to, rheumatoid arthritis (which is
often characterized, for example, by immune complexes in joints);
scleroderma with anti-collagen antibodies (which is often
characterized, for example, by nucleolar and other nuclear
antibodies); mixed connective tissue disease, (which is often
characterized, for example, by antibodies to extractable nuclear
antigens, for example, ribonucleoprotein);
polymyositis/dermatomyositis (which is often characterized, for
example, by nonhistone anti-nuclear antibodies); pernicious anemia
(which is often characterized, for example, by antiparietal cell,
antimicrosome, and anti-intrinsic factor antibodies); idiopathic
Addison's disease (which is often characterized, for example, by
humoral and cell-mediated adrenal cytotoxicity); infertility (which
is often characterized, for example, by antispennatozoal
antibodies); glomerulonephritis (which is often characterized, for
example, by glomerular basement membrane antibodies or immune
complexes); primary glomerulonephritis, IgA nephropathy; bullous
pemphigoid (which is often characterized, for example, by IgG and
complement in the basement membrane); Sjogren's syndrome (which is
often characterized, for example, by multiple tissue antibodies
and/or the specific nonhistone antinuclear antibody (SS-B));
diabetes mellitus (which is often characterized, for example, by
cell-mediated and humoral islet cell antibodies); and adrenergic
drug resistance, including adrenergic drug resistance with asthma
or cystic fibrosis (which is often characterized, for example, by
beta-adrenergic receptor antibodies).
[0288] Still further autoimmune diseases, disorders or conditions
include, but are not limited to chronic active hepatitis (which is
often characterized, for example by smooth muscle antibodies);
primary biliary cirrhosis (which is often characterized, for
example, by anti-mitchondrial antibodies); other endocrine gland
failure (which is characterized, for example, by specific tissue
antibodies in some cases); vitiligo (which is often characterized,
for example, by anti-melanocyte antibodies); vasculitis (which is
often characterized, for example, by immunoglobulin and complement
in vessel walls and/or low serum complement); post-myocardial
infarction conditions (which are often characterized, for example,
by anti-myocardial antibodies); cardiotomy syndrome (which is often
characterized, for example, by anti-myocardial antibodies);
urticaria (which is often characterized, for example, by IgG and
IgM antibodies to IgE); atopic dermatitis (which is often
characterized, for example, by IgG and IgM antibodies to IgE);
asthma (which is often characterized, for example, by IgG and IgM
antibodies to IgE); inflammatory myopathies; and other
inflammatory, granulomatous, degenerative, and atrophic
disorders.
[0289] In other embodiments, the compositions of the invention may
be used to modulate cellular proliferation and/or survival and,
accordingly, for treating or preventing diseases, disorders or
conditions characterized by abnormalities in cellular proliferation
and/or survival. For example, in certain embodiments, the
compositions may be used to modulate apoptosis and/or to treat
diseases or conditions associated with abnormal apoptosis.
Apoptosis is the term used to describe the cell signaling cascade
known as programmed cell death. Various therapeutic indications
exist for molecules that induce apoptosis (e.g., cancer), as well
as those that inhibit apoptosis (e.g., stroke, myocardial
infarction, sepsis, etc.). Apoptosis can be monitored by any of a
number of available techniques known and available in the art
including, for example, assays that measure fragmentation of DNA,
alterations in membrane asymmetry, activation of apoptotic caspases
and/or release of cytochrome C and AIF.
[0290] Illustrative diseases associated with increased cell
survival, or the inhibition of apoptosis include, but are not
limited to, cancers (such as follicular lymphomas, carcinomas, and
hormone-dependent tumors, including, but not limited to colon
cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Graves' disease,
Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis, autoimmune
gastritis, autoimmune thrombocytopenic purpura, and rheumatoid
arthritis), viral infections (such as herpes viruses, pox viruses
and adenoviruses), inflammation, graft vs. host disease (acute
and/or chronic), acute graft rejection, and chronic graft
rejection.
[0291] Further illustrative diseases or conditions associated with
increased cell survival include, but are not limited to,
progression and/or metastases of malignancies and related disorders
such as leukemia (including acute leukemias (for example, acute
lymphocytic leukemia, acute myelocytic leukemia, including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (for example, chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia), myelodysplastic syndrome, polycythemia vera, lymphomas
(for example, Hodgkin's disease and non-Hodgkin's disease),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
diseases, and solid tumors including, but not limited to, sarcomas
and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0292] Illustrative diseases associated with increased apoptosis
include, but are not limited to, AIDS (such as HIV-induced
nephropathy and HIV encephalitis), neurodegenerative disorders
(such as Alzheimer's disease, Parkinson's disease, amyotrophic
lateral sclerosis, retinitis pigmentosa, cerebellar degeneration
and brain tumor or prior associated disease), autoimmune disorders
such as multiple sclerosis, Sjogren's syndrome, Graves' disease,
Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus, immune-related glomerulonephritis, autoimmune
gastritis, thrombocytopenic purpura, rheumatoid arthritis,
myelodysplastic syndromes (such as aplastic anemia), graft vs. host
disease (acute and/or chronic), ischemic injury (such as that
caused by myocardial infarction, stroke and reperfusion injury),
liver injury or disease (for example, hepatitis related liver
injury, cirrhosis, ischemia/reperfusion injury, cholestosis (bile
duct injury) and liver cancer), toxin-induced liver disease (such
as that caused by alcohol), septic shock, ulcerative colitis,
cachexia, and anorexia.
[0293] In still further embodiments, the compositions of the
invention may be used in the treatment of neuronal/neurological
diseases or disorders, illustrative examples of which include
Parkinson's disease, Alzheimer's disease, Pick's Disease,
Creutzfeldt-Jacob disease, Huntington's chorea, alternating
hemiplegia, amyotrophic lateral sclerosis, ataxia, cerebral palsy,
chronic fatigue syndrome, chronic pain syndromes, congenital
neurological anomalies, cranial nerve diseases, delirium, dementia,
demyelinating diseases, dysautonomia, epilepsy, headaches,
Huntington's disease, hydrocephalus, meningitis, movement
disorders, muscle diseases, nervous system neoplasms,
neurocutaneous syndromes, neurodegenerative diseases, neurotoxicity
syndromes, ocular motility disorders, peripheral nervous system
disorders, pituitary disorders, porencephaly, Rett syndrome, sleep
disorders, spinal cord disorders, stroke, sydenham's chorea,
tourette syndrome, nervous system trauma and injuries, etc.
[0294] Furthermore, additional embodiments relate to the use of the
compositions of the invention in the treatment of metabolic
disorders such as adrenoleukodystrophy, Krabbe's disease (globoid
cell leukodystrophy), metachromatic leukodystrophy, Alexander's
disease, Canavan's disease (spongiform leukodystrophy),
Pelizaeus-Merzbacher disease, Cockayne's syndrome, Hurler's
disease, Lowe's syndrome, Leigh's disease, Wilson's disease,
Hallervorden-Spatz disease, Tay-Sachs disease, etc. The utility of
the compositions of the invention in modulating metabolic processes
may be monitored using any of a variety of techniques known and
available in the art including, for example, assays which measure
adipocyte lipogenesis or adipocyte lipolysis.
[0295] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0296] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. The
following examples are provided by way of illustration only and not
by way of limitation. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed
or modified to yield essentially similar results.
EXAMPLES
Example 1
Identification of Structural Motif within a GlyRS Polypeptide
Fragment Having Cell Signaling Activity
[0297] As shown in FIGS. 2A-2C, a fragment of human glycyl-tRNA
synthetase (GlyRS) referred to as G6, and corresponding to residues
214-439 of human GlyRS (SEQ ID NO: 1), was unexpectedly discovered
to be effective in inducing GPCR-dependent chemotaxis of THP-1
monocytes. Further analysis of this active fragment revealed the
presence of a structural motif, comprising 3 antiparallel
.beta.-sheets flanked by an .alpha.-helix at each end, believed to
be responsible for the observed chemokine activity. The structural
motif was identified as being contained within amino acid residues
345-438 of the human GlyRS protein sequence set forth in SEQ ID NO:
1.
Example 2
Identification of Structural Motif in Other Class II AARS
Proteins
[0298] The structural motif identified above in Example 1 was also
found in similar regions of other human Class II AARS. These
included AspRS, HisRS, ThrRS, GluProRS and SerRS. This region of
Class II AARS proteins is highly conserved structurally among
members of this AARS class indicating that the region contains the
same basic structural architecture, but is not well conserved in
residue sequence identity, creating the potential to display
different sequences which may have different signaling activities
on this architecture. (e.g., FIG. 4, adapted from O'Donoghue and
Luthey-Schulten, Microbiology and Molecular Biology Reviews,
December 2003)
[0299] The structural motif was identified as being contained
within amino acid residues 367-448 of the human AspRS protein
sequence set forth in SEQ ID NO: 2; within amino acid residues
294-372 of the human HisRS protein set forth in SEQ ID NO: 3;
within amino acid residues 469-586 of the human ThrRS protein set
forth in SEQ ID NO: 4; within amino acid residues 1171-1253 of the
human GluProRS protein set forth in SEQ ID NO: 5; within amino acid
residues 325-410 of the human SerRS protein set forth in SEQ ID NO:
6; within amino acid residues 380-449 of the human PheRS protein
set forth in SEQ ID NO: 7; within amino acid residues 425-523 of
the human LysRS protein set forth in SEQ ID NO: 8; within amino
acid residues 416-494 of the human AsnRS protein set forth in SEQ
ID NO: 9; and within amino acid residues 148-258 of the human AlaRS
protein set forth in SEQ ID NO: 10.
Example 3
Illustrative Non-AARS Proteins Containing the Structural Motif
[0300] Further analysis identified the presence of this same
structural motif comprising 3 antiparallel .beta.-sheets and two
.alpha.-helices in several non-AARS proteins (FIG. 5). For example,
the motif was identified in human thioredoxin, contained within
residues 20-105 of the human thioredoxin sequence of SEQ ID NO: 11,
as well as within residues 20-84 of Trx80, which is a truncated,
secreted form of thioredoxin containing the N-terminal 84 amino
acid residues.
[0301] The motif was also identified in the human macrophage
inhibitory factor protein, contained within residues 1-90 of the
human thioredoxin sequence of SEQ ID NO: 12.
[0302] The motif was also identified in the human peroxiredoxin 5
isoform B protein, contained within non-contiguous fragments
corresponding to residues 32-68 and 125-161 of the human
peroxiredoxin 5 isoform B sequence of SEQ ID NO: 13.
[0303] Thus, by screening non-AARS proteins for the presence of the
identified structural motif, the present invention provides a means
for identifying protein fragments having potential non-canonical
biological activities of therapeutic relevance.
Example 4
Identification of Structural Motif within a SerRS Polypeptide
Fragment Having Cell Signaling Activity
[0304] As shown in FIG. 7, a fragment of human seryl-tRNA
synthetase (SerRS) referred to as S3, and corresponding to residues
253-484 of full-length human SerRS (SEQ ID NO: 6), was unexpectedly
discovered to be effective in binding to human monocytes and
B-cells, stimulating secretion of TNF-.alpha. from a human
monocytic cell line, and signaling through Toll-like receptor 2.
Further analysis of this active fragment revealed the presence of a
structural motif, comprising 3 antiparallel .beta.-sheets flanked
by an .alpha.-helix at each end, believed to be responsible for the
observed activity. The structural motif was identified as being
contained within amino acid residues 325-410 of the human SerRS
protein sequence set forth in SEQ ID NO: 6.
[0305] For S3 binding, peripheral blood mononuclear cells (PBMCs)
were purified from a healthy blood donor and resuspended in RPMI
with 10% FBS at 1.times.10.sup.7 per ml. 1.times.10.sup.6 cells in
100 .mu.l of media were then treated with PBS or 500 nM S3. After
45 minutes cells were washed twice with 1 mL of staining buffer
(PBS+2% FBS) and stained with .alpha.V5-FITC antibody from
Invitrogen (Catalog #R96325), CD19 for B-cells (BD catalog
#555413), in staining buffer with 200 .mu.g/ml human IgG for 30
minutes. Cells were then washed twice with 1 mL staining buffer,
resuspended and analyzed by FACS. The results are shown in FIG. 7A,
which shows S3 binding to human monocytes, and FIG. 7B, which shows
S3 binding to human B-cells, as compared to a PBS negative
control.
[0306] For TNF.alpha. secretion, THP cells were cultured in RPMI
media with 10% FBS and 0.5 mM BME. One million cells were treated
with PBS, S3 (100 nM), or Lipopolysaccharide (LPS, 1 ug/mL). After
4 hours, cell supernatants were collected by centrifugation at
2000.times.g for 10 minutes and evaluated in a TNF.alpha. ELISA
(R&D Systems; Cat.#DTA00C) per kit directions. The results are
shown in FIG. 7C, which shows induction of TNF-.alpha. secretion
from human monocytic cell line (THP-1), as compared to an LPS
positive control and a PBS negative control.
[0307] For signaling through Toll-like receptor 2, HEK-Blue 2 cells
(Invivogen #hb2-cells) stably transfected with mouse toll-like
receptor 2 (mTLR2) linked to an NF.kappa.B alkaline phosphatase
reporter gene were treated overnight with varying doses of S3
protein. The following day, media was removed and assayed four
hours for secreted alkaline phosphatase using QUANTI-Blue
(Invivogen #rep-qb). As shown in FIG. 8, treatment elicited a
strong secretion of alkaline phoshpatase in a dose-dependent
manner, indicating activation of the receptor and its associated
NF.kappa.B signaling pathway.
[0308] In another experiment, the same cells were pre-treated with
5 ug/ml MAb mTLR2 (Invivogen #mab-mtlr2) (labeled (+) TLR-2) or
untreated (S3) for 30 minutes, followed by overnight treatment with
250 nM S3. The following day, media was removed and assayed four
hours for secreted alkaline phosphatase using QUANTI-Blue
(Invivogen #rep-qb). As shown in FIG. 9, the activation of
Toll-like receptor 2 was inhibited by pre-treatment with a
monoclonal antibody that blocks binding to mTLR2.
* * * * *
References