U.S. patent application number 11/761942 was filed with the patent office on 2008-10-09 for method of inducing apoptosis in lymphoid cells.
This patent application is currently assigned to UNIVERSITY OF MASSACHUSETTS. Invention is credited to Laxminarayana Devireddy, Michael R. Green, Fabian Richard, Jose G. Teodoro.
Application Number | 20080249010 11/761942 |
Document ID | / |
Family ID | 22880428 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080249010 |
Kind Code |
A1 |
Green; Michael R. ; et
al. |
October 9, 2008 |
Method Of Inducing Apoptosis In Lymphoid Cells
Abstract
A two-stage, transcriptionally regulated apoptotic program has
been discovered. In the first stage, IL-3 withdrawal results in
transcriptional activation of the NGAL gene followed by synthesis
and secretion of NGAL protein. In the second stage, secreted NGAL
protein induces apoptosis in lymphoid cells by an autocrine
mechanism. Based on this discovery, the invention provides a method
of inducing apoptosis in a lymphoid cell in a mammal, e.g., a human
patient. The invention includes administering a therapeutically
effective amount of an NGAL polypeptide or NGAL-like polypeptide to
a mammal.
Inventors: |
Green; Michael R.;
(Boylston, MA) ; Devireddy; Laxminarayana;
(Worcester, MA) ; Teodoro; Jose G.; (Worcester,
MA) ; Richard; Fabian; (Worcester, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
UNIVERSITY OF MASSACHUSETTS
|
Family ID: |
22880428 |
Appl. No.: |
11/761942 |
Filed: |
June 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09957801 |
Sep 21, 2001 |
7235520 |
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11761942 |
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60234216 |
Sep 21, 2000 |
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Current U.S.
Class: |
514/18.9 ;
435/375 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 38/57 20130101; A61P 37/02 20180101 |
Class at
Publication: |
514/12 ; 435/375;
514/2 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12N 5/06 20060101 C12N005/06; A61P 37/02 20060101
A61P037/02; A61K 38/02 20060101 A61K038/02 |
Claims
1. A method of inducing apoptosis in a lymphoid cell, the method
comprising administering an amount of an NGAL polypeptide effective
to induce apoptosis in the lymphoid cell.
2. The method of claim 1, wherein the lymphoid cell is a mammalian
cell.
3. The method of claim 1, wherein the polypeptide comprises an
amino acid sequence having at least 80% sequence identity with any
one of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
4. The method of claim 1, wherein the polypeptide is a composite
sequence alignable with amino acid 21 to the C-terminal amino acid
of the NGAL amino acid alignment in FIG. 9, wherein each position
in the composite sequence contains an amino acid selected from a
corresponding position in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID
NO:3.
5. The method of claim 1, wherein the polypeptide comprises SEQ ID
NO:4, wherein the blank or grey positions in SEQ ID NO:4 contain an
amino acid selected from a corresponding position in SEQ ID NO:1 or
SEQ ID NO:2.
6. The method of claim 1, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, and SEQ ID NO:7
7. The method of claim 1, wherein the lymphoid cell is a cell in
vivo.
8. The method of claim 1, wherein the lymphoid cell is a
T-lymphocyte or a B-lymphocyte.
9. The method of claim 8, wherein the T-lymphocyte or a
B-lymphocyte is leukemic.
10. The method of claim 7, wherein the polypeptide is administered
parenterally.
11. The method of claim 7, wherein the polypeptide is administered
intravenously.
12. The method of claim 7, wherein the cell is in a human.
13. A method of treating a leukemia in a mammal, the method
comprising administering to the mammal an amount of an NGAL
polypeptide effective to ameliorate a. symptom of the leukemia.
14. The method of claim 13, wherein the polypeptide comprises an
amino acid sequence having at least 80% sequence identity with any
one of SEQ ID NO:5; SEQ ID NO:6; or SEQ ID NO:7.
15. The method of claim 13, wherein the polypeptide is a composite
sequence alignable with amino acid 21 to the C-terminal amino acid
of the NGAL amino acid alignment in FIG. 9, wherein each position
in the composite sequence contains an amino acid selected from a
corresponding position in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID
NO:3.
16. The method of claim 13, wherein the polypeptide comprises SEQ
ID NO:4, wherein the blank or grey positions in SEQ ID NO:4 contain
an amino acid selected from a corresponding position in SEQ ID NO:1
or SEQ ID NO:2.
17. The method of claim 13, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:6, and SEQ ID NO:7.
18. The method of claim 13, wherein the polypeptide is administered
parenterally.
19. The method of claim 13, wherein the polypeptide is administered
intravenously.
20. The method of claim 13, wherein the mammal is a human.
21. A method of treating an immune disorder in a mammal, the method
comprising administering to the mammal an amount of an NGAL
polypeptide effective to reduce a symptom of the immune disorder in
the mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. application Ser. No. 09/957,801, filed on Sep. 21, 2001, which
in turn claims priority to provisional U.S. Application Ser. No.
60/234,216, filed on Sep. 21, 2000. The contents of both
applications are incorporated by reference in their entirety.
TECHNICAL FIELD
[0002] This invention relates to molecular biology, cell biology
immunology and oncology.
BACKGROUND
[0003] Apoptosis is a physiological form of cell death that plays a
role in various biological processes, including normal development,
tissue homeostasis, and defense against pathogens (Thompson et al.,
1995, Science 267:1456-1462). Different forms of apoptosis can be
distinguished according to whether transcription and translation,
i.e., gene expression, are involved. For example, Fas ligand (FasL)
and tumor necrosis factor (TNF) promote cell death by recruiting
and activating caspases at the plasma membrane in the absence of
transcription and translation (Rathmell et al., 1999, Annu. Rev
Immunol. 17:781-828). In contrast, other apoptotic programs require
gene expression. The p53 tumor suppressor induces apoptosis in
response to genotoxic agents, resulting, at least in part, from
transcriptional activation of p53-dependent genes (Polyak et al.,
1997, Nature 389:300-305). Other transcription-dependent apoptosis
programs include glucocorticoid-induced killing of thymocytes
(Cohen et al., 1984, J. Immunol. 132:3842) and cell death induced
by signaling through the T-cell receptor (TCR) (Lenardo et al.,
1999, Annu. Rev. Immunol 17:221-253).
[0004] In some cells, apoptosis can be induced by deprivation of
trophic factors. For example, transcription-dependent cell death
occurs following withdrawal of nerve growth factor (NGF) (Martin et
al., 1988, J. Cell Biol. 106:829-344). IL-3-dependent cell lines
undergo apoptosis upon cytokine withdrawal, and IL-3 promotes
survival of several lymphoid progenitors (Palacios et al., 1987, J.
Exp. Med. 166:12-32; Palacios et al., 1985, Cell 41:727-734).
[0005] Neutrophil gelatinase associated lipocalin (NGAL), a member
of the lipocalin family of proteins, is a secreted 25 kDa
glycoprotein found in granules of human neutrophils (Kjeldsen et
al., 1993, J. Biol. Chem. 268:10425-10432). Lipocalins have been
characterized by their ability to bind small lipophilic substances.
Lipocalins share a common three-dimensional .beta.-barrel structure
which functions, in at least some lipocalins, in binding a
lipophilic ligand, e.g., a steroid, bilin, retinoid, or other
lipid. For a review of structure and function in the lipocalin
family, see Flower, 1996, Biochem. J. 318:1-14. Murine forms of
NGAL (homologs) from mice and rats are known. NGAL in mice is known
by various designations, including NGAL, 24p3 protein, SIP24, P25,
lipocalin 2, and uterocalin. NGAL in rats is known as NGAL or alpha
2-microglobulin. NGAL increases 7- to10-fold in cultured mouse
kidney cells in response to viral infection (Hraba-Renevey et al.,
1989, Oncogene 4:601-608). NGAL is a major secretory product of
lipopolysaccharide-stimulated, cultured mouse macrophages (Meheus
et al., 1993, J. Immunol. 151:1535-1547).
[0006] NGAL is a positive acute phase protein. It has been
suggested that NGAL is a scavenger of bacterial products at sites
of inflammation (Nielsen et al., 1996, Gut 38:414-420). It has also
been suggested that NGAL has an immunomodulatory function involving
the binding of lipophilic inflammatory mediators (Bundgaard et al.,
1994, Biochem. Biophys. Res. Commun. 202:1468-1475). NGAL is
synthesized constitutively at a particular developmental point
during the maturation of granulocyte precursors in the bone marrow
(Borregaard et al., 1995, Blood 85:812-817). In addition, NGAL
synthesis can be induced in epithelial cells under certain
conditions such as inflammation and malignancy (Neilsen et al.,
supra; Bartsch et al., 1995, FEBS Lett. 357:255-259; Bundgaard et
al., supra).
[0007] A full-length cDNA encoding human NGAL protein has been
cloned and sequenced (Bundgaard et al., supra). In addition, the
human NGAL gene, which includes seven exons and six introns, has
been cloned and sequenced, and its expression in various tissues
has been analyzed (Cowland et al., 1997, Genomics 45:17-23). The
human NGAL gene encodes a polypeptide of 197 amino acids, with a
19- or 20-amino acid signal sequence, and a mature NGAL polypeptide
containing 178 amino acids (Bundgaard, supra). The motifs Gly-X-Trp
(amino acids 48-50 in mature human NGAL) and Thr-Asp/Asn-Tyr (amino
acids 132-134 in mature human NGAL) are present in all known
lipocalins (Bundgaard et al., supra). On the basis of X-ray
crystallography, it has been suggested that these motifs are
important in the tertiary structure common to lipocalins, i.e., an
eight-stranded antiparallel .beta.-barrel surrounding a hydrophobic
core (Cowan et al., 1990, Proteins: Structure Function and Genetics
8:44-61). The cysteine residues 95 and 194 in the human NGAL
sequence are conserved, and have been reported to form an
intramolecular disulfide bridge (Bundgaard, supra; Cowan et al.,
1990, supra). Human NGAL contains a single N-glycosylation site (an
asparagine residue) at position 65 of the mature amino acid
sequence (approximately position 84 or 85 of the pre-NGAL
polypeptide).
SUMMARY
[0008] A two-stage, transcriptionally regulated apoptotic pathway
has been discovered. In the first stage, IL-3 withdrawal results in
transcriptional activation of the NGAL gene followed by synthesis
and secretion of NGAL protein. In the second stage, secreted NGAL
protein induces apoptosis in lymphoid cells by an autocrine
mechanism.
[0009] On the basis of this discovery, the invention provides a
method of inducing apoptosis in a lymphoid cell. The cell can be
from a mammal, e.g., a human. The method includes administering an
amount of an NGAL polypeptide or NGAL-like polypeptide effective to
ameliorate a symptom of a lymphoid disease, e.g., a leukemia or
autoimmune disorder. In some embodiments, the polypeptide contains
an amino acid sequence having at least 80% sequence identity with
amino acid 21 to C-terminal amino acid of the human, mouse, or rat
NGAL amino acid sequence in FIG. 9 (SEQ ID NOS:5, 6, and 7,
respectively). In some embodiments, the polypeptide contains an
amino acid sequence containing amino acid 21 to the C-terminal
amino acid of the human, mouse or rat NGAL amino acid sequence in
FIG. 9 (SEQ ID NOS:5, 6, and 7, respectively) with up to 30
conservative amino acid substitutions, and up to 20 amino acid
deletions or non-conservative amino acid substitutions. In some
embodiments, the polypeptide contains a consensus or composite
sequence alignable with amino acid 21 to the C-terminal amino acid
of the NGAL amino acid alignment in FIG. 9, wherein each position
in the consensus or composite sequence contains an amino acid or a
gap selected from the corresponding position in the alignment in
SEQ ID NOS: 1, 2, or 3. Specific examples of mature NGAL
polypeptides are amino acid 21 to the C-terminal amino acid of the
human NGAL amino acid sequence in FIG. 9 (SEQ ID NO:5); amino acid
21 to the C-terminal amino acid of the mouse NGAL amino acid
sequence in FIG. 9 (SEQ ID NO:6); and amino acid 21 to the
C-terminal amino acid of the rat NGAL amino acid sequence in FIG. 9
(SEQ ID NO:7).
[0010] The lymphoid cell in which apoptosis is induced can be a
cell in vivo, for example a T-lymphocyte or a B-lymphocyte, which
may or may not be leukemic. The NGAL or NGAL-like polypeptide can
be administered parenterally, e.g., intravenously.
[0011] The invention also features a method of treating a leukemia
in a mammal, e.g, a human. The method includes administering to the
mammal an amount of an NGAL polypeptide or NGAL-like polypeptide
effective to ameliorate a symptom of the leukemia.
[0012] The invention also features a method of treating an immune
disorder in a mammal, e.g., a human. The method includes
administering an amount of an NGAL polypeptide or NGAL-like
polypeptide effective to ameliorate a symptom of the immune
disorder. Immune disorders that can be treated with an NGAL
polypeptide or NGAL-like polypeptide include autoimmune disorders
such as autoimmunue lymphproliferative syndrome (ALPS).
[0013] As used herein, "NGAL polypeptide" means a glycosylated or
nonglycosylated polypeptide whose amino acid sequence is a
naturally occurring, mature NGAL amino acid sequence. An NGAL
polypeptide can be isolated from a natural source or it can be
produced by recombinant DNA methods. Examples of NGAL polypeptides
include polypeptides consisting of the amino acid sequences set
forth in FIGS. 9 and 10, excluding the N-terminal signal
sequences.
[0014] As used herein, "NGAL-like polypeptide" means a polypeptide
whose amino acid sequence meets at least one of the following
criteria:
[0015] (a) it contains an amino acid sequence that has at least 80%
sequence identity with amino acid 21 to the C-terminal amino acid
of the human, mouse, or rat NGAL amino acid sequence set forth in
FIG. 9;
[0016] (b) it contains an amino acid sequence consisting of amino
acid 21 to the C-terminal amino acid of the human, mouse, or rat
NGAL amino acid sequence set forth in FIG. 9, with up to 30
conservative amino acid substitutions; and up to 20 amino acid
deletions or non-conservative amino acid substitutions (in any
combination); or (c) it contains a consensus or composite sequence
alignable with amino acid 21 to the C-terminal amino acid of the
three-sequence, NGAL amino acid alignment in FIG. 9,
wherein each position in the consensus or composite sequence
contains an amino acid or a gap selected from the three entries at
the corresponding position in the alignment in FIG. 9.
[0017] As used herein, "mature NGAL amino acid sequence" means the
amino acid sequence of an NGAL gene product after removal of a
signal sequence in a eukaryotic cell secretion process.
[0018] As used herein, "conservative amino acid substitution" means
a substitution within an amino acid family. Families of amino acids
are recognized in the art and are based on physical and chemical
properties of the amino acid side chains. Families include the
following: amino acids with basic side chains (e.g. lysine,
arginine, and histidine); amino acids with acidic side chains
(e.g., aspartic acid and glutamic acid); amino acids with uncharged
polar side chains (e.g. glycine, asparagine, glutamine, serine,
threonine, tyrosine, and cysteine); amino acids with nonpolar side
chains (e.g. alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, and tryptophan); amino acids with
branched side chains (e.g., threonine, valine, and isoleucine); and
amino acids with aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, and histidine). An amino acid can belong
to more than one family.
[0019] As used herein, "therapeutically effective" amount or dose
refers to that amount of the compound sufficient to result in
amelioration of at least one symptom of a disease or disorder,
e.g., a leukemia or autoimmune disorder. Such symptoms are known in
the art (for example, see Berkow et al., The Merck Manual, Merck
Research Laboratories, N.J., 1992)
[0020] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present application, including definitions, will
control. All publications, patents, and other references mentioned
herein are incorporated by reference in their entirety.
[0021] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, useful methods and materials are described
below. The materials, methods and examples are illustrative only
and not intended to be limiting. Other features and advantages of
the invention will be apparent from the detailed description and
from the claims.
DESCRIPTION OF DRAWINGS
[0022] FIGS. 1A-1C are schematic diagrams illustrating the
apoptotic pathway by which NGAL induces apoptosis in lymphoid
cells.
[0023] FIG. 2 is graph summarizing data from experiments wherein
naive FL5.12 cells were exposed to culture medium from FL5.12 cells
cultured in the presence or absence of IL-3. The results
demonstrate that culture medium from IL-3-deprived FL5.12 cells
induces apoptosis in naive FL5.12 cells. Cell viability was
quantitated by trypan blue exclusion. "CM" designates culture
medium. Open squares, CM supplemented with 3 ng/ml IL-3; closed
diamonds, CM without IL-3 added.
[0024] FIG. 3 is a graph summarizing data from experiments wherein
naive FL5.12 cells were exposed to culture medium from COS-7 cells
transfected with an expression vector containing a 24p3 coding
sequence, or culture medium from COS-7 controls transfected with
the expression vector only. The results demonstrate that the
culture medium from COS-7 cells expressing 24p3 induce apoptosis.
Open squares, medium from COS-7 cells expressing 24p3; closed
diamonds, medium from COS-7 negative control cells.
[0025] FIG. 4 is a graph summarizing data from experiments wherein
the effect of purified 24p3 protein on cultured FL5.12 cells was
tested. The results demonstrate that purified 24p3 protein induces
apoptosis in FL5.12 cells. Viability was determined by trypan-blue
exclusion at the indicated times. Open squares, negative control
(no 24p3 protein added to medium); closed diamonds, purified 24p3
protein added to culture medium at a concentration of 10 ng/ml.
[0026] FIG. 5 is a graph summarizing data from experiments on the
time course of cell death following dexamethasone addition to
medium. Apoptosis was quantitated by Annexin V-FITC/PI staining.
Open squares, dexamethasone treatment; closed diamonds, negative
control.
[0027] FIG. 6 is a graph summarizing results of experiments to test
the ability of culture medium from cytokine-deprived IL-2 and
IL-7-dependent cell lines to induce apoptosis. The results
demonstrate that culture media from the cytokine-deprived IL-2 and
IL-7-dependent cell lines are unable to induce apoptosis. Cell
viability was quantitated by trypan blue exclusion. Open squares,
culture medium from cells grown in presence of IL-2 added; stars,
culture medium from cells grown in absence of IL-2 added; open
circles, culture medium from cells grown in presence of IL-7 added;
closed squares, culture medium from cells grown in absence of IL-7
added.
[0028] FIG. 7 is a histogram summarizing results from experiments
to test the effect of IGF-1 on 24p3-mediated apoptosis. The results
demonstrate that IGF-1 blocks apoptosis initiated by withdrawal of
IL-3 from FL5.12 cells. The results also demonstrate that IGF-1 has
no effect on apoptosis resulting from direct addition of 24p3
protein to the culture medium of FL5.12 cells. IGF-1 (Calbiochem)
was added to IL-3 deprived or 24p3-treated FL5.12 cells (final
concentration, 250 ng/ml) and cells analyzed for viability by
trypan-blue exclusion at 48 hours.
[0029] FIG. 8 is a graph summarizing results of experiments on the
effect of Bcl-X.sub.L expression. The results demonstrate that both
IL-3 withdrawal and 24p3 addition fail to induce cell death in
FL5.12 cells expressing BCl-X.sub.L. Cell viability was quantitated
by trypan-blue exclusion. Open squares, IL-3 present in medium;
closed diamonds, IL-3 absent from medium; open circles, culture
medium from 24p3 protein-expressing COS-7 cells added; closed
triangles, culture medium from COS-7 control cells added.
[0030] FIG. 9 is an alignment of complete amino acid sequences of
pre-NGAL polypeptides from human (SEQ ID NO:1), mouse (SEQ ID NO:2)
and rat (SEQ ID NO:3). SEQ ID NO:5 is human NGAL from amino acid 21
trough the COOH terminus. SEQ ID NO:6 is mouse NGAL (24p3) from
amino acid 21 through the COOH terminus. SEQ ID NO:7 is rat NGAL
from amino acid 21 trough the COOH terminus.
[0031] FIG. 10 is an alignment of complete amino acid sequences
from pre-NGAL polypeptides from mouse (SEQ ID NO:2 ) and human (SEQ
ID NO:1). A mouse/human consensus sequence is shown also (SEQ ID
NO:4).
DETAILED DESCRIPTION
[0032] Experimental results leading to the present invention
indicate that a major function of IL-3 in promoting cell viability
is to maintain the NGAL (e.g., human NGAL, marine 24p3) gene in a
transcriptionally repressed state. IGF-1 can substitute for IL-3 by
preventing apoptosis following cytokine deprivation (See
Rodriguez-Tarduchy et al., 1992, J. Immunol. 149:535-540). The
inventors have found that like IL-3, IGF-1 blocks NGAL
transcriptional activation, explaining how IGF-1 can prevent
apoptosis. When transcriptional repression of the NGAL gene is
artificially bypassed by addition of NGAL protein, IL-3 and IGF-1
fail to prevent apoptosis. Thus, the NGAL protein can be used
therapeutically to induce apoptosis specifically in lymphoid cells,
regardless of the presence of cytokines such as IL-3 and IGF-1.
While the inventors do not intend to be bound by theory, a
predicted apoptotic pathway is illustrated schematically in FIGS.
1A-1C.
[0033] Leukemias are a group of neoplastic diseases of
blood-forming organs. Leukemias are characterized by an abnormal
increase in the production of leukocytes, including lymphoid cells.
Because the invention provides for reducing a lymphoid cell
population through induction of apoptosis specifically in lymphoid
cells, the invention is useful in treating leukemias and other
diseases or disorders, e.g., immune disorders, that are
characterized by an abnormally high number of lymphoid cells.
NGAL and NGAL-Like Polypeptides
[0034] Preferably, the polypeptide used in methods of the invention
includes a mature human NGAL amino acid sequence. For example, a
suitable NGAL polypeptide consists of amino acid 21 to the terminal
amino acid of the human NGAL amino acid sequence set forth in FIG.
9 or FIG. 10. However, a polypeptide containing any of various NGAL
or NGAL-like amino acid sequences can also be used in the
invention. For example, those of skill in the art will recognize
that within a species, natural amino acid polymorphisms may occur
in the NGAL amino acid sequences found in different individuals.
Accordingly, the use of various naturally occurring forms of human
wild type NGAL polypeptides is within the scope of the
invention.
[0035] The use of natural NGAL polypeptides (or portions thereof)
from various mammalian species to induce apoptosis in lymphoid
cells is within the scope of the invention. The currently known
natural NGAL sequences, i.e., those from human, mouse, and rat,
have highly conserved amino acid sequences (FIG. 9), and
cross-species activity in specifically inducing apoptosis in
lymphoid cells (but not other cell types). This is demonstrated in
the Experimental Examples (below). The invention therefore
includes, for example, the use of a mouse or rat NGAL polypeptide
in a human patient, or the use of a human NGAL polypeptide in a
non-human animal undergoing veterinary treatment. In some
embodiments, a chimeric NGAL polypeptide is used, e.g., an
artificial NGAL-like polypeptide formed by replacing a portion of a
human NGAL amino acid sequence with a corresponding portion of an
NGAL amino acid sequence from another species.
[0036] A polypeptide containing a consensus (composite) sequence,
wherein each amino acid position represents an amino acid or a gap
from the alignment in FIG. 9 or FIG. 10, will induce apoptosis in
lymphoid cells, and thus be useful in methods of the invention. An
NGAL-like polypeptide containing at least 80% sequence identity,
e.g., 85%, 90%, 95%, 98% or 99%, with the mature human (SEQ ID
NO:5), mouse (SEQ ID NO:6), or rat (SEQ ID NO:7) NGAL amino acid
sequence in FIG. 9 will also be useful in methods of the invention.
Further, an NGAL-like polypeptide containing amino acid 21 to the
C-terminal amino acid of the human (SEQ ID NO:5), mouse (SEQ ID
NO:6), or rat NGAL (SEQ ID NO:7) amino acid sequence in FIG. 9,
with up to 30, e.g., 1, 3, 5, 10, 15, 20, or 25, conservative amino
acid substitutions; and up to 20, e.g., 1, 3, 5, 10 or 15,
non-conservative amino acid substitutions or deletions (in any
combination, e.g., 10 deletions and 10 substitutions) will be
useful in methods of the invention as long as the resulting
polypeptide still induces apoptosis in a lymphoid cell. In the
preceding sentence, "deletion" refers one amino acid. Thus, "20
deletions" means deletion of a total of 20 amino acid residues,
which may or may not be consecutive.
[0037] The determination of percent identity between two amino acid
sequences is accomplished using the BLAST 2.0 program, which is
available to the public on the world wide web at
ncbi.nlm.nih.gov/BLAST. Sequence comparison is performed using an
ungapped alignment and using the default parameters (Blossom 62
matrix, gap existence cost of 11, per residue gap cost of 1, and a
lambda ratio of 0.85). The mathematical algorithm used in BLAST
programs is described in Altschul et al., 1997, Nucleic Acids
Research 25:3389-3402.
[0038] In certain embodiments, an NEAL polypeptide or NGAL-like
polypeptide used in the invention is glycosylated. For example, the
glycosyl moiety is N-linked to an amino acid residue, e.g., an
asparagine residue, whose position can be from residue 60 to
residue 70, e.g., residue 65, in a mature NGAL polypeptide. See
Rudd et al., 1999, Biochemistry 38:13937-13950. A glycosylated NGAL
polypeptide can be obtained by purification of a naturally
occurring NGAL polypeptide from a suitable source, e.g.,
neutrophils from humans, rabbits, mice, or rats. Recombinant,
glycosylated NGAL polypeptides or NGAL-like polypeptides can be
produced by conventional methods, using transformed eukaryotic
cells, e.g., yeast cells.
[0039] In some embodiments of the invention, an NGAL polypeptide or
NGAL-like polypeptide is modified by derivatization of amino acid
side chains, chemical conjugation, or fusion to non-NGAL peptide
moieties For example an NGAL amino acid sequence can be fused to an
N-terminal peptide moiety or C-terminal peptide moiety, to increase
in vivo serum half-life of the polypeptide. In some embodiments,
the NGAL polypeptide contains one or more modified amino acids,
e.g., D-amino acids. Modified amino acids are useful for purposes
such as increasing serum half-life of the polypeptide.
Production of NGAL and NGAL-Like Polypeptides
[0040] Polypeptides for use in the invention can be obtained by any
suitable method. One method of producing an NGAL polypeptide or
NGAL-like polypeptide is recombinant production, which involves
genetic transformation of a host cell with a recombinant nucleic
acid vector encoding the polypeptide or prepolypeptide, expression
of the recombinant nucleic acid in the transformed host cell, and
collection and purification of the NGAL or NGAL-like polypeptide.
Guidance and information concerning recombinant DNA methods and
materials for production of polypeptides can be found in numerous
treatises and reference manuals, e.g., Sambrook et al., 1989,
Molecular Cloning--A Laboratory Manual, 2.sup.nd d Ed., Cold Spring
Harbor Press; Ausubel et al., (eds.), 1994, Current Protocols in
Molecular Biology, John Wiley & Sons, Inc.; Innis et al., 1990,
PCR Protocols, Academic Press. Complete nucleotide sequences are
available publicly from GenBank: Accession No. 1171700 (human
NGAL); 266619 (rat NGAL); 112725 (mouse NGAL). For specific
guidance concerning cloning of human NGAL cDNA by PCR, and
recombinant production of human NGAL polypeptides, see Bundgaard et
al., 1994, Biochem. Biophys. Res. Commun. 202:1468-1475. See also
Bartsch et al., 1995, FEBS Lett. 357-255-259.
[0041] NGAL polypeptides useful in the invention also can be
isolated from natural sources. For example human NGAL can be
isolated from human neutrophils using methods and materials such as
those described in Kjeldsen et al., 1993, J. Biol. Chem.
268:10425-10432.
[0042] Alternatively, an NGAL polypeptide or NGAL-like polypeptide
can be obtained directly by chemical synthesis, e.g., using a
commercial peptide synthesizer according to the vendor's
instructions. Methods and materials for chemical synthesis of
polypeptides are well known in the art.
[0043] Techniques for purification of NGAL polypeptides from
biological material are known in the art. For specific guidance
concerning purification of NGAL polypeptides, see, e.g., Kjeldsen
et al., supra. In addition, techniques for production of anti-NGAL
antibodies and the use of the antibodies in purification and assay
of NGAL polypeptides are known in the art. See, e.g., Kjeldsen et
al., supra; Liu et al., 1997, Molecular Reproduction and
Development 46:507-514.
Effective Dose
[0044] Toxicity and therapeutic efficacy of the NGAL polypeptides
and NGAL-like polypeptides of the invention can be determined by
standard pharmaceutical procedures, using either cells in culture
or experimental animals to determine the LD50 (the dose lethal to
50% of the population) and the ED50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD50/ED50. Polypeptides that exhibit large
therapeutic indices are preferred. While polypeptides that exhibit
toxic side effects may be used, care should be taken to design a
delivery system that targets such compounds to the site of affected
tissue to minimize potential damage to non-target cells and,
thereby, reduce side effects.
[0045] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can also be calculated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (that is, the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
Formulation, Dosage and Administration
[0046] An NGAL polypeptide or NGAL-like polypeptide can be
administered according to the invention by any suitable method.
Preferably, the polypeptide is administered parenterally, to avoid
digestion in the stomach. Parenteral administration can be
systemic, e.g., by an intravenous route. In some embodiments of the
invention, the polypeptide is administered locally, e.g., into a
tumor or lymph node.
[0047] The present invention provides a pharmaceutical composition
for treating an individual in need of treatment for a lymphoid cell
disease (e.g., a leukemia or autoimmune disorder). The treatment
method entails administering a therapeutically effective amount of
an NGAL polypeptide or NGAL-like polypeptide that causes apoptosis
of a lymphoid cell and a pharmaceutically acceptable carrier,
diluent, excipient, or adjuvant.
[0048] The pharmaceutical compositions can be used for humans or
animals (e.g., mammals) and will typically include any one or more
of a pharmaceutically acceptable diluent, carrier, excipient, or
adjuvant. The choice of pharmaceutical carrier, excipient, and
diluent can be selected with regard to the intended route of
administration and standard pharmaceutical practice. The
pharmaceutical compositions can include as (or in addition to) the
carrier, excipient, or diluent, any suitable binder(s),
lubricant(s), suspending agent(s), coating agent(s), or
solubilizing agent(s).
[0049] The invention includes pharmaceutical formulations that
include a pharmaceutically acceptable excipient and an NGAL
polypeptide or NGAL-like polypeptide. Such pharmaceutical
formulations can be used in a method of treating a lymphoid cell
disease such that at least one symptom of the disease is
ameliorated. Such a method entails administering to the organism a
therapeutically effective amount of the pharmaceutical formulation,
i.e., an amount sufficient to ameliorate signs and/or symptoms of
the lymphoid cell disease. In particular, such pharmaceutical
formulations can be used to treat lymphoid cell disease in mammals
such as humans and domesticated mammals (e.g., cows, pigs, dogs,
and cats). The efficacy of such treatment can be estimated in an
animal model system well known to those of skill in the art as
discussed herein.
[0050] Treatment includes administering a pharmaceutically
effective amount of a composition containing an NGAL polypeptide or
NGAL-like polypeptide to a subject in need of such treatment,
thereby ameliorating symptoms of a lymphoid cell disorder in the
subject. Such a composition typically contains from about 0.1 to
90% by weight (such as 1 to 20% or 1 to 10%) of an NGAL polypeptide
or NGAL-like polypeptide of the invention in a pharmaceutically
acceptable carrier.
[0051] Injectable formulations of the compositions can contain
various carriers such as vegetable oils, dimethylacetamide,
dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl
myristate, ethanol, polyols (glycerol, propylene glycol, liquid
polyethylene glycol, and the like). For intravenous injections,
water soluble versions of the compounds can be administered by the
drip method, whereby a pharmaceutical formulation containing the
NGAL polypeptide or NGAL-like polypeptide and a physiologically
acceptable excipient is infused. Physiologically acceptable
excipients can include, for example, 5% dextrose, 0.9% saline,
Ringer's solution, or other suitable excipients. For intramuscular
preparations, a sterile formulation of a suitable soluble salt form
of the compounds can be dissolved and administered in a
pharmaceutical excipient such as Water-for-Injection, 0.9% saline,
or 5% glucose solution. A suitable insoluble form of the compound
can be prepared and administered as a suspension in an aqueous base
or a pharmaceutically acceptable oil base, such as an ester of a
long chain fatty acid, (e.g., ethyl oleate). Oral or topical
methods of delivery may be used. Such methods are known in the
art.
[0052] The optimal percentage of the NGAL polypeptide or NGAL-like
polypeptide in each pharmaceutical formulation varies according to
the formulation itself and the therapeutic effect desired in the
specific pathologies and correlated therapeutic regimens.
Appropriate dosages of the NGAL-polypeptide or NGAL-like
polypeptide can be determined by those of ordinary skill in the art
of medicine by monitoring the mammal for signs of disease
amelioration or inhibition, and increasing or decreasing the dosage
and/or frequency of treatment as desired. The optimal amount of the
NGAL polypeptide or NGAL-like polypeptide used for treatment of
lymphoid cell diseases depends upon the manner of administration,
the age and the body weight of the subject, and the condition of
the subject to be treated. Generally, the NGAL polypeptide or
NGAL-like polypeptide is administered at a dosage of 1 to 100 mg/kg
body weight, and typically at a dosage of 1 to 10 mg/kg body
weight. In treatment of a lymphoid cell disorder such as a leukemia
or immune disorder, dosage is adjusted so as to achieve an NGAL
polypeptide or NGAL-like polypeptide serum concentration in the
range of 0.1 ng/ml to 100 ng/ml, e.g., in the range of 1.0 ng/ml to
20 ng/ml, at least once every two weeks, e.g., once per week, once
every third day, once every second day, or once per day. Such
optimization is within ordinary skill in the art. The compound can
also be administered chronically. The skilled artisan will
appreciate that certain factors may influence the dosage and timing
required to effectively treat a subject, including but not limited
to the severity of the disease or disorder, previous treatments,
the general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a compound can include a single treatment or,
preferably, can include a series of treatments.
[0053] Natural NGAL is a secreted protein, and its target in the
induction of apoptosis in lymphoid cells is believed to be exposed
on the outside of the lymphoid cells, which occur in the blood and
lymphatic system. Therefore, in the practice of the invention the
NGAL polypeptide or NGAL-like polypeptide need not cross
cytoplasmic membranes or otherwise enter into cells, nor does it
need to penetrate solid tissues to be effective.
[0054] An NGAL polypeptide or NGAL-like polypeptide can be
formulated into a pharmaceutical composition by admixture with
pharmaceutically acceptable nontoxic excipients or carriers. Such
compositions can be prepared for use in parenteral administration,
particularly in the form of liquid solutions or suspensions. The
composition can be administered conveniently in unit dosage form.
Such methods are described, e.g., in Remington's Pharmaceutical
Sciences, Mack Pub. Co., Easton, Pa. In some embodiments, the
polypeptide is administered gradually, in a buffered saline
solution, by intravenous infusion.
Animal Models
[0055] Animal models can be used for testing NGAL polypeptides and
NGAL-like polypeptides, e.g., for their efficacy in treating a
disorder, estimating toxicity, and dosages. Methods for performing
such tests are known in the art. Suitable animal models include
animal models for leukemias and autoimmune disorders, e.g., an Fas
knockout mouse which exhibits an autoimmune lymphoproliferative
syndrome (APLS).
EXPERIMENTAL EXAMPLES
[0056] The invention is farther illustrated by the following
examples. The examples are provided for illustrative purposes only.
They are not to be construed as limiting the scope or content of
the invention in any way.
[0057] Throughout the examples, the term "24p3" is used to
designate the murine form of NGAL found in mice.
Example 1
Experimental Methods
Culture of Cell Lines and Primary Cells
[0058] For cell lines, all the culture media were supplemented with
10% heat-inactivated FBS. COS-7, NIH 3T3, HeLa, and WEHI 7.1.C.4
cells were maintained in Dulbecco's modified Eagle's medium (DMEM);
U20S cells were cultured in McCoy 5A medium; Jurkat and MT4 cells
were maintained in RPMI-1640 medium; and HL-60 cells were
maintained in Iscove's modified DMEM. FL5.12, LyD9, Baf/3 and 32D
cells were cultured in RPMI-1640 medium supplemented with 0.05 mM
2-mercaptoethanol and 3 ng/ml recombinant IL-3 (Pharmingen). HT-2
cells were cultured in RPMI-1640 medium supplemented with 0.05 mM
2-mercaptoethanol, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium
pyruvate, and 200 IU/ml recombinant IL-2 (Pharmingen). D1-F4 cells
were cultured in RPMI-1640 medium supplemented with 0.05 mM
2-mercaptoethanol and 50 ng/ml recombinant IL-7 (PeproTech).
[0059] Human monocytes obtained from healthy donors provided by Dr.
Mario Stevenson) were initially cultured in DMEM supplemented with
10% heat-inactivated human serum (Sigma), 2 mM L-glutamine, and 8
ng/ml MCSF (Sigma). After 3 days, MCSF was removed from the culture
medium. Primary thymocytes from four week-old mice were cultured in
DMEM supplemented with 10% heat-inactivated PBS and 1 mM sodium
pyruvate.
Transfections of Cell Lines
[0060] For establishment of a 24p3 ecdysone-inducible cell line,
the 24p3 cDNA containing a hemagglutinin (HA) tag at the C-terminus
was cloned into the ecdysone-inducible vector, pIND (Invitrogen).
FL5.12 cells were transfected with Superfect (Qiagen) according to
the manufacture's instructions. FL5.12 cells were first transfected
with pVGRXR expressing the subunits of the ecdysone receptor and
selected with 600 .mu.g/ml Zeocin (Invitrogen) and the resulting
clones were then transfected with pIND/24p3-HA and selected with
800 .mu.g/ml G418 (GIBCO-BRL). 24p3 expression was induced by
addition of 10 .mu.M ponasterone A (Invitrogen).
[0061] For construction of a stable cell line expressing 24p3, the
24p3 EDNA was PCR amplified and cloned into the EcoRI and BamHI
sites of pcDNA3 vector (Invitrogen). COS7 cells were transfected
with pcDNA3/24p3 and selected with G418 at 600 .mu.g/ml. G418
resistant colonies were isolated and screened for 24p3 expression
by Northern blotting and immunoblotting.
Measurements of Apoptosis
[0062] IL-3 withdrawal and cell viability determinations were
performed as described in Boise et al., 1993, Cell 74 597-608. For
cell death assays 2.times.10.sup.5 cells (lymphoid) or
8.times.10.sup.5 cells (fibroblast) in a 60 mm dish were incubated
with culture medium from IL-3 deprived FL5.12 cells or COS-7 cells
transfected with pcDNA3 or pcDNA3/24p3, supplemented with 3 ng/ml
IL-3. For fibroblasts both floating and adherent cells (after
trypsinization) were collected at the indicated time points and
analyzed by a trypan blue (Sigma) dye exclusion assay. A minimum of
400 cells was counted and all experiments were performed in
duplicate. Apoptosis was also assessed by staining with Annexin
V-FITC and propidium iodide (Calbiochem) according to the
manufacturer's instructions. Stained cells were analyzed in Beckton
Dickinson flow cytometer.
[0063] For DNA fragmentation analysis, DNA from 2.times.10.sup.6
cells was isolated by phenol extraction and analyzed on a 1%
agarose gel as described in Rodriguez-Tarduchy et al., 1990, EMBO
J. 9:2997-3002.
Transcription Profiling using DNA Microarrays
[0064] FL5.12 cells were cultured and subjected to ID3 deprivation
as described (see Boise, 1993, Cell 74:597-608). Poly(A)+mRNA was
isolated eight hours following IL-3 withdrawal using an Oligotex
Direct mRNA isolation kit from Qiagen. A cDNA library was generated
using Superscript choice system from GIBCO-BRL following the
manufacture's instructions. The cDNA library was transcribed in
vitro with biotinylated nucleotides (T7 Megascript kit from Ambion)
and the resulting cRNA was used to probe Affymetrix oligonucleotide
arrays representing 30,000 known genes or ESTs.
Northern Blot and Immunoblot Analysis
[0065] For Northern blotting, 2 .mu.g of poly(A)+RNA or 10 .mu.g of
total RNA was analyzed on denaturing formaldehyde agarose gels (See
Ausubel et al., Current Protocols in Molecular Biology, New York:
John Wiley & Sons, Inc., 1992). The blots were probed with the
indicated probes and washed under high stringency conditions.
[0066] For detection of 24p3 in culture medium, 2 ml of culture
medium of cells grown in the presence or absence of IL-3 was
concentrated in Centricon YM-10 filters (Millipore) and the
retentates were collected and analyzed on a 12% SDS-PAGE gel. The
membrane was incubated with 24p3 antibody (see Chu et al., 1996,
Biochem. J. 316:545-550) and developed with an ECL kit from
Amersham.
[0067] For analysis of Bad phosphorylation, FL5.12 cells were
treated with culture medium from Cos-7 cells transfected with
pcDNA3 or the 24p3 expression vector. Cells were lysed in 1% NP-40
lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM
MgCl.sub.2, 1 mM EDTA, 10 mM NaF, 0.2 mM Na.sub.3Vo.sub.4, 1 mM
Na.sub.3MoO.sub.4, and protease inhibitor tablets from
Boehringer-Mannheim. Cell lysates were incubated with 2 .mu.g of
Bad antibody (Transduction labs). Immune-complexes were resolved by
12% SDS-PAGE and transferred onto a PVDF membrane (Millipore).
Blots were incubated with either a phospho-specific Bad antibody
(New England Biolabs) or Bad antibody and developed with ECL kit
from Amersham.
Example 2
Transcriptional Activation Following IL-3 Deprivation
[0068] To identify genes that are activated by IL-3 withdrawal,
transcription profiling using high-density DNA microarrays was
performed. The mouse pro-B lymphocytic cell line FL5.12 was used
because it is dependent on IL-3 for growth and undergoes apoptosis
in the absence of cytokine (Boise et al., 1993, Cell 74:597-608),
which is first detectable approximately six hours following IL-3
deprivation (See McCubrey et al., 1989, Oncogene Res. 4, 97-109).
FL5.12 poly(A)+mRNA was isolated eight hours after IL-3 withdrawal
and used to interrogate Affymetrix DNA microarrays representing
approximately 30,000 genes. Transcription profiles of cells grown
in the presence or absence of IL-3 were compared. The genes for
which the most significant transcriptional changes occurred
following IL-3 withdrawal are listed in Table 1. The gene that
underwent the largest transcriptional activation was 24p3, which
encodes a member of the lipocalin family. A lower but substantial
level of activation was also observed for several other genes,
including mNip3 (See Chen et al., 1999, Biol. Chem. 274:7-10) and
p40Phox (see Endres et al., 1997, Immunity 7: 419-432). Conversely,
transcription of several genes decreased following IL-3 withdrawal,
the most dramatic of which was the transcription factor ATFx (see
Mishizawa et al., 1992, FEBS Lett. 299:36-38).
TABLE-US-00001 TABLE 1 Genes Transcriptionally Activated or
Repressed Following IL-3 Withdrawal from FL5.12 Cells Gene
(accession number) Fold Change 24p3 (X81627) +12.6 .alpha.-globin
(L75940) +12.3 Leukocystatin (AF031825) +7.7 P40 phox (U59488) +7.2
E1B interacting protein(AF041054) +7 Erythroid Krueppel-like
(M97200) +6.5 ATFx (AB012276) -50.5
[0069] Northern blot analysis confirmed that IL-3 withdrawal
resulted in transcriptional activation of 24p3 and repression of
ATFx, consistent with the DNA microarray analysis. A time course
experiment revealed that 24p3 transcriptional induction was first
detectable within two hours following IL-3 withdrawal. Finally, we
tested whether transcriptional activation of 24p3 following
cytoline deprivation was specific to FL5.12 cells. We found that
following IL-3 withdrawal, 24p3 ascription was also activated in
32D cells, another well-characterized IL-3 dependent cell line (See
Greenberger et al., Proc. Natl. Acad. Sci. USA 80:2931-2935).
Example 3
24v3 Induces Apoptosis
[0070] In immunoblot experiments we found that following IL-3
withdrawal, 24p3 was in the culture medium of FL5.12 cells. We
tested whether the culture medium from IL3-deprived cells could
induce apoptosis. Medium from cells cultured either in the presence
or absence of IL-3 was collected, supplemented with recombinant
IL-3, added to naive FL5.12 cells, and cell viability analyzed by a
trypan blue vital dye exclusion assay. Experimental results
summarized in FIG. 2 show that the medium from FL5.12 cells
cultured in the absence IL-3 induced death of other FL5.12 cells
even though IL-3 was present. In contrast, the medium from FL5.12
cells cultured in the presence of IL-3 had no effect (FIG. 2).
[0071] Experiments were performed to confirm that the cell death
was apoptotic. Cells treated with the culture medium from
IL-3-deprived cells and stained with annexin-V FITC/propidium
iodide (PD had fragmented DNA characteristic of apoptosis. These
experiments confirmed that the 24p3-containing medium from
IL-3-deprived FL5.12 cells was inducing apoptosis.
[0072] Three experimental approaches were used to confirm that 24p3
in the culture medium was responsible for the induction of
apoptosis. In the first approach, 24p3 was ectopically expressed in
COS-7 cells (which are resistant to 24p3-mediated apoptosis).
Expression and secretion of 24p3 was confirmed by Northern blot and
immunoblot analyses. We found that the culture medium from COS-7
cells expressing 24p3 induced apoptosis in FL5.12 cells as
evidenced by trypan blue exclusion (FIG. 3), Annexin-V FITC/PI
staining and DNA fragmentation analysis. In contrast, culture
medium from COS-7 cells transfected with only the expression vector
(negative controls) displayed no apoptotic activity.
[0073] In the second approach, we tested whether ectopic expression
of 24p3 in FL5.12 cells could induce apoptosis. A sequence encoding
pre24p3, i.e., with intact signal sequence, was placed under the
control of an ecdysone-inducible promoter and stably introduced
into FL5.12 cells. Addition of ecdysone induced expression and
secretion of 24p3 and apoptosis. Comparable expression of a 24p3
derivative lacking the N-terminal signal sequence failed to induce
cell death. This indicated that secretion of 24p3 is an essential
step of this apoptotic pathway.
[0074] In the third approach, we tested whether direct addition of
biochemically purified 24p3 polypeptide could induce apoptosis. We
found that addition of purified mouse 24p3 (Chu et al., 1996,
Biochem. J. 316:545-550) to FL5.12 cells led to apoptosis (FIG. 4).
Collectively, these results indicated that when present in the
culture medium, 24p3 promoted apoptosis of FL5.12 cells.
Example 4
Specificity
[0075] Several experiments were performed to assess the specificity
of apoptosis promoted by 24p3 and transcriptional activation of the
24p3 gene. First, the cell type specificity of the 24p3
pro-apoptotic activity was examined (Table 2). Based upon
trypan-blue exclusion and Annexin-V FITC/PI staining, 24p3 promoted
apoptosis in a variety of (but not all) lymphoid cell lines,
primary thymocytes, primary lymphocytes, and neutrophils. In
contrast, non-lymphoid cells and monocyte-derived macrophages were
resistant. Thus, the pro-apoptotic activity of 24p3 is highly cell
type specific.
TABLE-US-00002 TABLE 2 Cell Type Specificity of 24p3-Mediated
Apoptosis Cell type Susceptibility to 24p3 Lymphoid cell lines
Cytokine-dependent IL-3 FL5.12 Yes FL5.12/Bcl-x.sub.L No 32D Yes
BaF/3 Yes LyD9 Yes IL-2 HT-2 Yes IL-7 D1-F4 Yes
Cytokine-independent Jurkat No MT-4 Yes WEHI7.1.C.4 Yes
Non-lymphoid cell lines HeLa No COS-7 No NIH 3T3 No U20S No Primary
cells Murine primary Thymocytes Yes Murine primary Splenocytes Yes
Human primary Neutrophils Yes Human primary Macrophages No Human
peripheral blood lymphocytes Yes
[0076] Experiments were performed to assess the specificity of 24p3
transcriptional activation. Primary thymocytes are known to be
highly prone to apoptosis, which can be efficiently promoted by a
variety of agents, including 24p3 (Table 2) and corticosteroids
(see Wyllie, 1980, Nature 284:555-556). The 24p3 promoter was known
to have a glucocorticoid-response element (GRE) (See Garay et al.,
1996, Gene 170:173-180). Therefore, the ability of 24p3 to be
transcriptionally activated in primary thymocytes by the synthetic
glucocorticoid, dexamethasone was tested. Northern blot analysis
indicated that untreated primary thymocytes had a low level of 24p3
transcription, perhaps explaining their low spontaneous level of
apoptosis. Significantly, addition of dexamethasone substantially
increased 24p3 transcription and induced apoptosis with similar
kinetics (FIG. 5). Taken together, these observations suggested a
mechanism by which glucocorticoids induce apoptosis in primary
thymocytes.
[0077] Transcriptional activation of 24p3 by cytokine withdrawal in
cells dependent upon cytokines other than IL-3, transcription was
tested. The IL-2 dependent cell line, HT-2 (see Watson, 1980, J.
Exp. Med. 150:1510-1519), and the IL-7 dependent cell line, D1-F4
(see Khaled et al., 1999, Proc. Natl. Acad. Sci. USA
96:14476-14481) were analyzed (FIG. 6). Like FL5.12 cells, these
cell lines were dependent on their respective cytokines for growth
and undergo apoptosis upon cytokine withdrawal. It was found that
24p3 transcription was not activated in HT-2 cells grown in the
absence of IL-2 or in D1-F4 cells grown in the absence of IL-7.
Likewise, the medium from these cells cultured in the absence of
cytokine did not induce apoptosis.
[0078] A variety of mammalian cell lines were known to undergo
apoptosis when deprived of serum, similar to apoptosis in
cytokine-dependent cell lines following cytokine withdrawal (See
Barroso et al., 1997, J. Bioenerg. Biomembr. 29:259-267; and Pandey
et al., 1994, J. Cell Biochem. 58:135-150). It was found that 24p3
transcription was not activated following withdrawal of serum from
HL-60, Jurkat, or NIH 3T3 cells even though apoptosis occurred.
Collectively, our results indicated that transcriptional activation
of 24p3 following growth factor withdrawal was highly specific.
Example 5
IGF-1 Blocks 24R3 Transcriptional Activation
[0079] Insulin-like growth factor-1 (IGF-1) stimulates
proliferation and differentiation of a variety of cell types and
can inhibit apoptosis resulting from deprivation of serum or
cytokines (See Rodriguez-Tarduchy et al., 1992, J. Immunol.
149:535-540). These observations prompted us to investigate the
effect of IGF-1 on 24p3-mediated apoptosis. As expected, IGF-1
blocked apoptosis initiated by withdrawal of IL-3 from FL5.12
cells. Unexpectedly, however, IGF-1 had no effect on apoptosis
resulting from direct addition of 24p3 to FL5.12 cells (FIG.
7).
[0080] To investigate this observation, transcription of 24p3 under
these different conditions was analyzed. Northern blot analysis
revealed that IGF-1 blocked the transcriptional activation of 24p3
that normally occurred following IL-3 withdrawal. These results
indicated that IGF-1 promoted survival following IL-3 withdrawal by
inhibiting 24p3 transcriptional activation. Direct addition of 24p3
bypasses this transcriptional block, and therefore IGF-1 had no
effect.
Example 6
Role of Bcl-2 Family Members
[0081] Previous studies have shown that several apoptotic pathways
function by regulating phosphorylation of Bad, a pro-apoptotic
member of the Bcl-2 family (see, e.g., Zha et al., 1996, Cell
87:619-628; and Datta et al., 1997, Cell 91-231-241).
Phosphorylation of Bad blocks its pro-apoptotic activity, which is
promoted by IL-3 through a pathway involving PI3K and Akt.
Therefore, IL-3 deprivation results in dephosphorylation of Bad and
apoptosis.
[0082] The finding that 24p3 induced apoptosis even when IL-3 was
present, prompted us to analyze the effect of 24p3 on Bad
phosphorylation. It was found that Bad was phosphorylated when
cells were cultured in the presence of IL-3 and unphosphorylated
following IL-3 withdrawal. Significantly, addition of 24p3 also led
to dephosphorylation of Bad even though IL-3 was present. Thus,
24p3 appeared to be overriding the normal IL-3 signaling pathway
leading to dephosphorylation of Bad and apoptosis.
[0083] The Bcl-2 family member, Bcl-X.sub.L, was known to inhibit
apoptosis induced by a variety of stimuli including IL-3 withdrawal
(see Boise et al., 1993, Cell 74:597-608; Vanderheider et al., Mol.
Cell 3:159-167). The activity of 24p3 in FL5.12 cells expressing
BCl-X.sub.L (FL5.12/Bcl-X.sub.L cells) was analyzed (FIG. 8).
Culture media from IL-3 deprived FL5.12 cells and COS-7 cells
expressing 24p3 failed to induce apoptosis of FL5.12/Bcl-X.sub.L
cells. Thus, Bcl-X.sub.L blocked apoptosis induced both by IL-3
withdrawal and by 24p3 addition.
[0084] Apoptosis following 24p3 addition occurred more slowly than
apoptosis resulting from IL-3 withdrawal. One explanation for this
observation is that factors in addition to 24p3 may contribute to
the efficiency of apoptosis following IL-3 withdrawal. In this
regard, mNip3 and p40Phox, which have been previously implicated in
apoptosis (See Chen et al., 1999, J. Biol. Chem. 274:7-10; Endres
et al., 1997, Immunity 7:419-431), were also transcriptionally
activated (Table 1). The reduced rate of apoptosis following 24p3
addition probably also reflects the fact that in these experiments
the culture medium still contained IL-3, which promotes
proliferation and survival.
[0085] IL-3 promotes cell survival through a signal transduction
pathway involving PI3K and Akt.sup.2,14 and resulting in an
inactivating phosphorylation of the pro-apoptotic Bcl-2 family
member, Bad. 24p3 is secreted and induces apoptosis upon addition
to cells. It therefore seems likely that 24p3 functions through an
extracellular receptor and initiates a signal transduction pathway.
Addition of 24p3 also led to dephosphorylation of Bad even in the
presence of IL-3. Thus, Bad may be the ultimate target of the 24p3
signal transduction pathway. 24p3 could act by blocking the
PI3K/Akt pathway or in an independent pathway that promotes
dephosphorylation of Bad.
[0086] These results indicate that 24p3 may be involved in immune
system homeostasis, which requires that expanded cell populations
be rapidly eliminated after their functions are completed. IL-3 is
produced and secreted primarily by activated T cells and thus as
the immune response begins to terminate IL-3 levels decrease. It
has previously been recognized that declining IL-3 levels would
prevent maturation of certain hematopoietic precursors and lead to
apoptotic death of IL-3 dependent cells, The results presented
herein reveal that declining IL-3 levels also induce 24p3
expression and secretion, providing an independent mechanism to
facilitate termination of the immune response.
Example 7
Requirement of 24p3 Expression for Apoptosis Induced by IL-3
Deprivation
[0087] The requirement for 24p3 expression for apoptosis induced by
IL-3 deprivation of FL5-12 cells was investigated using antisense
experiments. Two phosphorothioate antisense (AS) oligonucleotides
were used and a phosphorothioate sense oligonucleotide was used as
a control.
[0088] The phosphorothioate oligonucleotides were purchased from
Genosys (The Woodlands, Texas). The sense and antisense 1
oligonucleotides spanned -12 to +5. Antisense 2 oligonucleotide
spanned +585 to +593 of 24p3 mRNA (=1, translation start site).
F15.12 cells were transfected with 2 .mu.M of each oligonucleotide
using Lipofectamine (Gibco-BRL). After 24 hours, cells were washed
with RPMI medium plus 10% fetal calf serum (FCS) and again
transfected with 2 .mu.M of oligonucleotide. Cells were incubated
for 24 hours after the second transfection then IL-3 was
withdrawn,
[0089] Both 24p3 AS oligonucleotides substantially reduced 24p3
levels, whereas the sense oligonucleotide had no effect. The AS-1
nucleotide was particularly effective, preventing apoptosis in
approximately 75% of the cells. Moreover, after treatment with 24p3
AS oligonucleotides, the conditioned medium from IL-3-deprived
FL5.12 cells was no longer pro-apoptotic.
[0090] The ability of an antibody raised against 24p3 to block
apoptosis was tested. In these experiments, 0.5 .mu.g of affinity
purified antibody against 24p3 (see Chu et al., Biochem. J. 316:545
(1996)) or 2 .mu.g of preimmune serum was added after IL-3
withdrawal from IL-3 dependent primary bone marrow cells. Cell
viability was then determined by annexin V-FITC/PI staining.
Antibody against 24p3 blocked apoptosis in these cells, confirming
that 24p3 is required to promote apoptosis.
[0091] The ability of 24p3 to promote apoptosis is cell type
specific. Using trypan blue exclusion and annexin V-FITC/PI
staining, 24p3 promoted apoptosis in many but not all leukocytic
cell lines, primary thymocytes, primary lymphocytes, and
neutrophils. In contrast, nonhematopoietic cells and
monocyte-derived macrophages were resistant. These data demonstrate
that 24p3-mediated apoptosis is cell type specific. Leukemias or
other disorders characterized by the presence of cell types that
are susceptible to 24p3/NGAL-induced apoptosis can be treated by
administration of NGAL or NGAL-like polypeptides.
OTHER EMBODIMENTS
[0092] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. For example, novel, human NGAL polypeptides
occurring as a result of natural polymorphism may be found, and can
be employed in the invention. Accordingly, other embodiments are
within the scope of the following claims.
Sequence CWU 1
1
91198PRTHomo sapiens 1Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala
Leu Leu Gly Ala Leu 1 5 10 15His Ala Gln Ala Gln Asp Ser Thr Ser
Asp Leu Ile Pro Ala Pro Pro 20 25 30Leu Ser Lys Val Pro Leu Gln Gln
Asn Phe Gln Asp Asn Gln Phe Gln 35 40 45Gly Lys Trp Tyr Val Val Gly
Leu Ala Gly Asn Ala Ile Leu Arg Glu 50 55 60Asp Lys Asp Pro Gln Lys
Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu 65 70 75 80Asp Lys Ser Tyr
Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys 85 90 95Asp Tyr Trp
Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe 100 105 110Thr
Leu Gly Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val 115 120
125Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys
130 135 140Lys Val Ser Gln Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr
Gly Arg145 150 155 160Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn
Phe Ile Arg Phe Ser 165 170 175Lys Ser Leu Gly Leu Pro Glu Asn His
Ile Val Phe Pro Val Pro Ile 180 185 190Asp Gln Cys Ile Asp Gly
1952200PRTMus musculus 2Met Ala Leu Ser Val Met Cys Leu Gly Leu Ala
Leu Leu Gly Val Leu 1 5 10 15Gln Ser Gln Ala Gln Asp Ser Thr Gln
Asn Leu Ile Pro Ala Pro Ser 20 25 30Leu Leu Thr Val Pro Leu Gln Pro
Asp Phe Arg Ser Asp Gln Phe Arg 35 40 45Gly Arg Trp Tyr Val Val Gly
Leu Ala Gly Asn Ala Val Gln Lys Lys 50 55 60Thr Glu Gly Ser Phe Thr
Met Tyr Ser Thr Ile Tyr Glu Leu Gln Glu 65 70 75 80Asn Asn Ser Tyr
Asn Val Thr Ser Ile Leu Val Arg Asp Gln Asp Gln 85 90 95Gly Cys Arg
Tyr Trp Ile Arg Thr Phe Val Pro Ser Ser Arg Ala Gly 100 105 110Gln
Phe Thr Leu Gly Asn Met His Arg Tyr Pro Gln Val Gln Ser Tyr 115 120
125Asn Val Gln Val Ala Thr Thr Asp Tyr Asn Gln Phe Ala Met Val Phe
130 135 140Phe Arg Lys Thr Ser Glu Asn Lys Gln Tyr Phe Lys Ile Thr
Leu Tyr145 150 155 160Gly Arg Thr Lys Glu Leu Ser Pro Glu Leu Lys
Glu Arg Phe Thr Arg 165 170 175Phe Ala Lys Ser Leu Gly Leu Lys Asp
Asp Asn Ile Ile Phe Ser Val 180 185 190Pro Thr Asp Gln Cys Ile Asp
Asn 195 2003198PRTRattus rattus 3Met Gly Leu Gly Val Leu Cys Leu
Ala Leu Val Leu Leu Gly Val Leu 1 5 10 15Gln Arg Gln Ala Gln Asp
Ser Thr Gln Asn Leu Ile Pro Ala Pro Pro 20 25 30Leu Ile Ser Val Pro
Leu Gln Pro Gly Phe Trp Thr Glu Arg Phe Gln 35 40 45Gly Arg Trp Phe
Val Val Gly Leu Ala Ala Asn Ala Val Gln Lys Glu 50 55 60Arg Gln Ser
Arg Phe Thr Met Tyr Ser Thr Ile Tyr Glu Leu Gln Glu 65 70 75 80Asp
Asn Ser Tyr Asn Val Thr Ser Ile Leu Val Arg Gly Gln Gly Cys 85 90
95Arg Tyr Trp Ile Arg Thr Phe Val Pro Ser Ser Arg Pro Gly Gln Phe
100 105 110Thr Leu Gly Asn Ile His Ser Tyr Pro Gln Ile Gln Ser Tyr
Asp Val 115 120 125Gln Val Ala Asp Thr Asp Tyr Asp Gln Phe Ala Met
Val Phe Phe Gln 130 135 140Lys Thr Ser Glu Asn Lys Gln Tyr Phe Lys
Val Thr Leu Tyr Gly Arg145 150 155 160Thr Lys Gly Leu Ser Asp Glu
Leu Lys Glu Arg Phe Val Ser Phe Ala 165 170 175Lys Ser Leu Gly Leu
Lys Asp Asn Asn Ile Val Phe Ser Val Pro Thr 180 185 190Asp Gln Cys
Ile Asp Asn 1954200PRTArtificial SequenceConsensus sequence 4Met
Xaa Leu Xaa Leu Leu Xaa Leu Gly Leu Ala Leu Leu Gly Xaa Leu 1 5 10
15Xaa Xaa Gln Ala Gln Asp Ser Thr Xaa Xaa Leu Ile Pro Ala Pro Xaa
20 25 30Leu Xaa Xaa Val Pro Leu Gln Xaa Xaa Phe Xaa Xaa Xaa Gln Phe
Xaa 35 40 45Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Ala Ile Xaa
Arg Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Met Tyr Xaa Thr Ile Tyr Glu
Leu Xaa Glu 65 70 75 80Xaa Xaa Ser Tyr Asn Val Thr Ser Val Leu Xaa
Arg Xaa Xaa Xaa Gln 85 90 95Gly Cys Xaa Tyr Trp Ile Arg Thr Phe Val
Pro Xaa Xaa Xaa Xaa Gly 100 105 110Xaa Phe Thr Leu Gly Asn Ile Lys
Xaa Tyr Pro Xaa Leu Xaa Ser Tyr 115 120 125Xaa Val Xaa Val Xaa Ser
Thr Xaa Tyr Asn Gln Xaa Ala Met Val Phe 130 135 140Phe Lys Lys Xaa
Ser Xaa Asn Arg Xaa Tyr Phe Lys Ile Thr Leu Tyr145 150 155 160Gly
Arg Thr Lys Glu Leu Thr Xaa Glu Leu Lys Glu Xaa Phe Xaa Arg 165 170
175Phe Xaa Lys Ser Leu Gly Leu Xaa Glu Xaa Xaa Ile Val Phe Xaa Val
180 185 190Pro Xaa Asp Gln Cys Ile Asp Xaa 195 2005178PRTHomo
sapiens 5Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser
Lys Val 1 5 10 15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln
Gly Lys Trp Tyr 20 25 30Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg
Glu Asp Lys Asp Pro 35 40 45Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu
Lys Glu Asp Lys Ser Tyr 50 55 60Asn Val Thr Ser Val Leu Phe Arg Lys
Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80Arg Thr Phe Val Pro Gly Cys
Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95Ile Lys Ser Tyr Pro Gly
Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn
Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125Asn Arg
Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135
140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu
Gly145 150 155 160Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile
Asp Gln Cys Ile 165 170 175Asp Gly6180PRTMus musculus 6Gln Asp Ser
Thr Gln Asn Leu Ile Pro Ala Pro Ser Leu Leu Thr Val 1 5 10 15Pro
Leu Gln Pro Asp Phe Arg Ser Asp Gln Phe Arg Gly Arg Trp Tyr 20 25
30Val Val Gly Leu Ala Gly Asn Ala Val Gln Lys Lys Thr Glu Gly Ser
35 40 45Phe Thr Met Tyr Ser Thr Ile Tyr Glu Leu Gln Glu Asn Asn Ser
Tyr 50 55 60Asn Val Thr Ser Ile Leu Val Arg Asp Gln Asp Gln Gly Cys
Arg Tyr 65 70 75 80Trp Ile Arg Thr Phe Val Pro Ser Ser Arg Ala Gly
Gln Phe Thr Leu 85 90 95Gly Asn Met His Arg Tyr Pro Gln Val Gln Ser
Tyr Asn Val Gln Val 100 105 110Ala Thr Thr Asp Tyr Asn Gln Phe Ala
Met Val Phe Phe Arg Lys Thr 115 120 125Ser Glu Asn Lys Gln Tyr Phe
Lys Ile Thr Leu Tyr Gly Arg Thr Lys 130 135 140Glu Leu Ser Pro Glu
Leu Lys Glu Arg Phe Thr Arg Phe Ala Lys Ser145 150 155 160Leu Gly
Leu Lys Asp Asp Asn Ile Ile Phe Ser Val Pro Thr Asp Gln 165 170
175Cys Ile Asp Asn 1807178PRTRattus rattus 7Gln Asp Ser Thr Gln Asn
Leu Ile Pro Ala Pro Pro Leu Ile Ser Val 1 5 10 15Pro Leu Gln Pro
Gly Phe Trp Thr Glu Arg Phe Gln Gly Arg Trp Phe 20 25 30Val Val Gly
Leu Ala Ala Asn Ala Val Gln Lys Glu Arg Gln Ser Arg 35 40 45Phe Thr
Met Tyr Ser Thr Ile Tyr Glu Leu Gln Glu Asp Asn Ser Tyr 50 55 60Asn
Val Thr Ser Ile Leu Val Arg Gly Gln Gly Cys Arg Tyr Trp Ile 65 70
75 80Arg Thr Phe Val Pro Ser Ser Arg Pro Gly Gln Phe Thr Leu Gly
Asn 85 90 95Ile His Ser Tyr Pro Gln Ile Gln Ser Tyr Asp Val Gln Val
Ala Asp 100 105 110Thr Asp Tyr Asp Gln Phe Ala Met Val Phe Phe Gln
Lys Thr Ser Glu 115 120 125Asn Lys Gln Tyr Phe Lys Val Thr Leu Tyr
Gly Arg Thr Lys Gly Leu 130 135 140Ser Asp Glu Leu Lys Glu Arg Phe
Val Ser Phe Ala Lys Ser Leu Gly145 150 155 160Leu Lys Asp Asn Asn
Ile Val Phe Ser Val Pro Thr Asp Gln Cys Ile 165 170 175Asp
Asn8200PRTArtificial SequenceConsensus sequence 8Met Xaa Leu Xaa
Xaa Xaa Xaa Leu Gly Leu Ala Leu Leu Gly Xaa Leu 1 5 10 15Xaa Xaa
Gln Ala Gln Asp Ser Thr Xaa Xaa Leu Ile Pro Ala Pro Xaa 20 25 30Leu
Xaa Xaa Val Pro Leu Gln Xaa Xaa Phe Xaa Xaa Xaa Gln Phe Xaa 35 40
45Gly Xaa Trp Tyr Val Val Gly Leu Ala Gly Asn Ala Xaa Xaa Xaa Xaa
50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Met Tyr Xaa Thr Ile Tyr Glu Leu Xaa
Glu65 70 75 80Xaa Xaa Ser Tyr Asn Val Thr Ser Xaa Leu Xaa Arg Xaa
Xaa Xaa Xaa 85 90 95Xaa Cys Xaa Tyr Trp Ile Arg Thr Phe Val Pro Xaa
Xaa Xaa Xaa Gly 100 105 110Xaa Phe Thr Leu Gly Asn Xaa Lys Xaa Tyr
Pro Xaa Xaa Xaa Ser Tyr 115 120 125Xaa Val Xaa Val Xaa Xaa Thr Xaa
Tyr Asn Gln Xaa Ala Met Val Phe 130 135 140Phe Xaa Lys Xaa Ser Xaa
Asn Xaa Xaa Tyr Phe Lys Ile Thr Leu Tyr145 150 155 160Gly Arg Thr
Lys Glu Leu Thr Xaa Glu Leu Lys Glu Xaa Phe Xaa Arg 165 170 175Phe
Xaa Lys Ser Leu Gly Leu Xaa Xaa Xaa Xaa Ile Xaa Phe Xaa Val 180 185
190Pro Xaa Asp Gln Cys Ile Asp Xaa 195 2009180PRTArtificial
SequenceConsensus sequence 9Gln Asp Ser Thr Xaa Xaa Leu Ile Pro Ala
Pro Xaa Leu Xaa Xaa Val 1 5 10 15Pro Leu Gln Xaa Xaa Phe Xaa Xaa
Xaa Xaa Phe Xaa Gly Xaa Trp Xaa 20 25 30Val Val Gly Leu Ala Xaa Asn
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Met Tyr Xaa Thr
Ile Tyr Glu Leu Xaa Glu Xaa Xaa Ser Tyr 50 55 60Asn Val Thr Ser Xaa
Leu Xaa Arg Xaa Xaa Xaa Xaa Xaa Cys Xaa Tyr65 70 75 80Trp Ile Arg
Thr Phe Val Pro Xaa Xaa Xaa Xaa Gly Xaa Phe Thr Leu 85 90 95Gly Asn
Xaa Xaa Xaa Tyr Pro Xaa Xaa Xaa Ser Tyr Xaa Val Xaa Val 100 105
110Xaa Xaa Thr Xaa Tyr Xaa Gln Xaa Ala Met Val Phe Phe Xaa Lys Xaa
115 120 125Ser Xaa Asn Xaa Xaa Tyr Phe Lys Xaa Thr Leu Tyr Gly Arg
Thr Lys 130 135 140Xaa Leu Xaa Xaa Glu Leu Lys Glu Xaa Phe Xaa Xaa
Phe Xaa Lys Ser145 150 155 160Leu Gly Leu Xaa Xaa Xaa Xaa Ile Xaa
Phe Xaa Val Pro Xaa Asp Gln 165 170 175Cys Ile Asp Xaa 180
* * * * *