U.S. patent application number 11/052427 was filed with the patent office on 2005-08-04 for thymic stromal lymphopoietin receptor molecules and uses thereof.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Baumann, Heinz, Farr, Andrew G., Leonard, Warren J., Levin, Steven D., Lodish, Harvey F., Ozaki, Katsutoshi, Pandey, Akhilesh.
Application Number | 20050170459 11/052427 |
Document ID | / |
Family ID | 22799949 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170459 |
Kind Code |
A1 |
Pandey, Akhilesh ; et
al. |
August 4, 2005 |
Thymic stromal lymphopoietin receptor molecules and uses
thereof
Abstract
The present invention provides Thymic Stromal Lymphopoietin
Receptor (TSLPR) polypeptides and nucleic acid molecules encoding
the same. The invention also provides selective binding agents,
vectors, host cells, and methods for producing TSLPR polypeptides.
The invention further provides pharmaceutical compositions and
methods for the diagnosis, treatment, amelioration, and/or
prevention of diseases, disorders, and conditions associated with
TSLPR polypeptides.
Inventors: |
Pandey, Akhilesh; (Malden,
MA) ; Ozaki, Katsutoshi; (Rockville, MD) ;
Baumann, Heinz; (Buffalo, NY) ; Levin, Steven D.;
(Seattle, WA) ; Farr, Andrew G.; (Seattle, WA)
; Leonard, Warren J.; (Bethesda, MD) ; Lodish,
Harvey F.; (Brookline, MA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
The Gov. of the U.S.A, as represented by The Secretary,
Department of Health & Human Services
The National Institutes of Health; and Health Research Inc.,
Roswell Park Division
|
Family ID: |
22799949 |
Appl. No.: |
11/052427 |
Filed: |
February 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11052427 |
Feb 7, 2005 |
|
|
|
09895593 |
Jun 28, 2001 |
|
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60214658 |
Jun 28, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/326; 530/388.1; 536/23.53 |
Current CPC
Class: |
C07K 14/7155 20130101;
A61K 38/00 20130101; C07K 2319/30 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/326; 530/388.1; 536/023.53 |
International
Class: |
C07H 021/04; C07K
016/18; C12N 005/06 |
Claims
What is claimed is:
1. A selective binding agent or fragment thereof that specifically
binds to a polypeptide produced by a process comprising culturing a
host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the polypeptide, and
optionally isolating the polypeptide from the culture, wherein the
nucleic acid molecule is selected from the group consisting of: (a)
the nucleotide sequence as set forth in SEQ ID NO: 1; (b) a
nucleotide sequence encoding the polypeptide as set forth in SEQ ID
NO: 2; (c) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of either (a) or
(b); and (d) a nucleotide sequence complementary to either (a) or
(b).
2. A selective binding agent or fragment thereof that specifically
binds to a polypeptide produced by a process comprising culturing a
host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the polypeptide, and
optionally isolating the polypeptide from the culture, wherein the
nucleic acid molecule is selected from the group consisting of: (a)
a nucleotide sequence encoding a polypeptide which is at least
about 70 percent identical to the polypeptide as set forth in SEQ
ID NO: 2, wherein the encoded polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (b) a nucleotide sequence
encoding an allelic variant or splice variant of the nucleotide
sequence as set forth in SEQ ID NO: 1 or (a); (c) a region of the
nucleotide sequence of SEQ ID NO: 1, (a), or (b) encoding a
polypeptide fragment of at least about 25 amino acid residues,
wherein the polypeptide fragment has an activity of the encoded
polypeptide as set forth in SEQ ID NO: 2, or is antigenic; (d) a
region of the nucleotide sequence of SEQ ID NO: 1, or any of
(a)-(c) comprising a fragment of at least about 16 nucleotides; (e)
a nucleotide sequence which hybridizes under moderately or highly
stringent conditions to the complement of any of (a)-(d); and (f) a
nucleotide sequence complementary to any of (a)-(d).
3. A selective binding agent or fragment thereof that specifically
binds to a polypeptide produced by a process comprising culturing a
host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the polypeptide, and
optionally isolating the polypeptide from the culture, wherein the
nucleic acid molecule is selected from the group consisting of: (a)
a nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 2 with at least one conservative amino acid substitution,
wherein the encoded polypeptide has an activity of the polypeptide
set forth in SEQ ID NO: 2; (b) a nucleotide sequence encoding a
polypeptide as set forth in SEQ ID NO: 2 with at least one amino
acid insertion, wherein the encoded polypeptide has an activity of
the polypeptide set forth in SEQ ID NO: 2; (c) a nucleotide
sequence encoding a polypeptide as set forth in SEQ ID NO: 2 with
at least one amino acid deletion, wherein the encoded polypeptide
has an activity of the polypeptide set forth in SEQ ID NO: 2; (d) a
nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 2 which has a C- and/or N-terminal truncation, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
SEQ ID NO: 2; (e) a nucleotide sequence encoding a polypeptide as
set forth in SEQ ID NO: 2 with at least one modification selected
from the group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, C-terminal truncation, and
N-terminal truncation, wherein the encoded polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2; (f) a
nucleotide sequence of any of (a)-(e) comprising a fragment of at
least about 16 nucleotides; (g) a nucleotide sequence which
hybridizes under moderately or highly stringent conditions to the
complement of any of (a)-(f); and (h) a nucleotide sequence
complementary to any of (a)-(e).
4. A selective binding agent or fragment thereof that specifically
binds to an isolated polypeptide comprising the amino acid sequence
as set forth in SEQ ID NO: 2.
5. A selective binding agent or fragment thereof that specifically
binds to an isolated polypeptide comprising the amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
as set forth in either SEQ ID NO: 3, optionally further comprising
an amino-terminal methionine; (b) an amino acid sequence for an
ortholog of SEQ ID NO: 2; (c) an amino acid sequence which is at
least about 70 percent identical to the amino acid sequence of SEQ
ID NO: 2, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (d) a fragment of the amino
acid sequence set forth in SEQ ID NO: 2 comprising at least about
25 amino acid residues, wherein the fragment has an activity of the
polypeptide set forth in SEQ ID NO: 2, or is antigenic; and (e) an
amino acid sequence for an allelic variant or splice variant of the
amino acid sequence as set forth in SEQ ID NO: 2, or any of
(a)-(c).
6. A selective binding agent or fragment thereof that specifically
binds to an isolated polypeptide comprising the amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
as set forth in SEQ ID NO: 2 with at least one conservative amino
acid substitution, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (b) the amino acid sequence
as set forth in SEQ ID NO: 2 with at least one amino acid
insertion, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (c) the amino acid sequence
as set forth in SEQ ID NO: 2 with at least one amino acid deletion,
wherein the polypeptide has an activity of the polypeptide set
forth in SEQ ID NO: 2; (d) the amino acid sequence as set forth in
SEQ ID NO: 2 which has a C- and/or N-terminal truncation, wherein
the polypeptide has an activity of the polypeptide set forth in SEQ
ID NO: 2; and (e) the amino acid sequence as set forth in SEQ ID
NO: 2 with at least one modification selected from the group
consisting of amino acid substitutions, amino acid insertions,
amino acid deletions, C-terminal truncation, and N-terminal
truncation, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2.
7. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising: (a) a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
(b) a second polypeptide produced by a process comprising culturing
a host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the second polypeptide, and
optionally isolating the second polypeptide from the culture,
wherein the nucleic acid molecule is selected from the group
consisting of: (i) the nucleotide sequence as set forth in SEQ ID
NO: 1; (ii) a nucleotide sequence encoding the polypeptide as set
forth in SEQ ID NO: 2; (iii) a nucleotide sequence which hybridizes
under moderately or highly stringent conditions to the complement
of either (i) or (ii); and (iv) a nucleotide sequence complementary
to either (i) or (ii), wherein the heterodimer complex is capable
of binding thymic stromal lymphopoietin.
8. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising: (a) a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
(b) a second polypeptide produced by a process comprising culturing
a host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the second polypeptide, and
optionally isolating the second polypeptide from the culture,
wherein the nucleic acid molecule is selected from the group
consisting of: (i) a nucleotide sequence encoding a polypeptide
which is at least about 70 percent identical to the polypeptide as
set forth in SEQ ID NO: 2, wherein the encoded polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2; (ii) a
nucleotide sequence encoding an allelic variant or splice variant
of the nucleotide sequence as set forth in SEQ ID NO: 1 or (i);
(iii) a region of the nucleotide sequence of SEQ ID NO: 1, (i), or
(ii) encoding a polypeptide fragment of at least about 25 amino
acid residues, wherein the polypeptide fragment has an activity of
the encoded polypeptide as set forth in SEQ ID NO: 2, or is
antigenic; (iv) a region of the nucleotide sequence of SEQ ID NO:
1, or any of (i)-(iii) comprising a fragment of at least about 16
nucleotides; (v) a nucleotide sequence which hybridizes under
moderately or highly stringent conditions to the complement of any
of (i)-(iv); and (vi) a nucleotide sequence complementary to any of
(i)-(iv), wherein the heterodimer complex is capable of binding
thymic stromal lymphopoietin.
9. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising: (a) a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
(b) a second polypeptide produced by a process comprising culturing
a host cell comprising a vector comprising a nucleic acid molecule
under suitable conditions to express the second polypeptide, and
optionally isolating the second polypeptide from the culture,
wherein the nucleic acid molecule is selected from the group
consisting of: (i) a nucleotide sequence encoding a polypeptide as
set forth in SEQ ID NO: 2 with at least one conservative amino acid
substitution, wherein the encoded polypeptide has an activity of
the polypeptide set forth in SEQ ID NO: 2; (ii) a nucleotide
sequence encoding a polypeptide as set forth in SEQ ID NO: 2 with
at least one amino acid insertion, wherein the encoded polypeptide
has an activity of the polypeptide set forth in SEQ ID NO: 2; (iii)
a nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 2 with at least one amino acid deletion, wherein the encoded
polypeptide has an activity of the polypeptide set forth in SEQ ID
NO: 2; (iv) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 2 which has a C- and/or N-terminal truncation,
wherein the encoded polypeptide has an activity of the polypeptide
set forth in SEQ ID NO: 2; (v) a nucleotide sequence encoding a
polypeptide as set forth in SEQ ID NO: 2 with at least one
modification selected from the group consisting of amino acid
substitutions, amino acid insertions, amino acid deletions,
C-terminal truncation, and N-terminal truncation, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
SEQ ID NO: 2; (vi) a nucleotide sequence of any of (i)-(v)
comprising a fragment of at least about 16 nucleotides; (vii) a
nucleotide sequence which hybridizes under moderately or highly
stringent conditions to the complement of any of (i)-(vi); and
(viii) a nucleotide sequence complementary to any of (i)-(v),
wherein the heterodimer complex is capable of binding thymic
stromal lymphopoietin.
10. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
a second polypeptide comprising the amino acid sequence as set
forth in SEQ ID NO: 2, wherein the heterodimer complex is capable
of binding thymic stromal lymphopoietin.
11. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising: (a) a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
(b) a second polypeptide comprising the amino acid sequence
selected from the group consisting of: (i) the amino acid sequence
as set forth in either SEQ ID NO: 3, optionally further comprising
an amino-terminal methionine; (ii) an amino acid sequence for an
ortholog of SEQ ID NO: 2; (iii) an amino acid sequence which is at
least about 70 percent identical to the amino acid sequence of SEQ
ID NO: 2, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (iv) a fragment of the amino
acid sequence set forth in SEQ ID NO: 2 comprising at least about
25 amino acid residues, wherein the fragment has an activity of the
polypeptide set forth in SEQ ID NO: 2, or is antigenic; and (v) an
amino acid sequence for an allelic variant or splice variant of the
amino acid sequence as set forth in SEQ ID NO: 2, or any of
(i)-(iii), wherein the heterodimer complex is capable of binding
thymic stromal lymphopoietin.
12. A selective binding agent or fragment thereof that specifically
binds to an isolated heterodimer complex comprising: (a) a first
polypeptide that is the alpha chain of interleukin 7 receptor, and
(b) a second polypeptide comprising the amino acid sequence
selected from the group consisting of: (i) the amino acid sequence
as set forth in SEQ ID NO: 2 with at least one conservative amino
acid substitution, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (ii) the amino acid sequence
as set forth in SEQ ID NO: 2 with at least one amino acid
insertion, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (iii) the amino acid
sequence as set forth in SEQ ID NO: 2 with at least one amino acid
deletion, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; (iv) the amino acid sequence
as set forth in SEQ ID NO: 2 which has a C- and/or N-terminal
truncation, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; and (v) the amino acid
sequence as set forth in SEQ ID NO: 2 with at least one
modification selected from the group consisting of amino acid
substitutions, amino acid insertions, amino acid deletions,
C-terminal truncation, and N-terminal truncation, wherein the
polypeptide has an activity of the polypeptide set forth in SEQ ID
NO: 2, wherein the heterodimer complex is capable of binding thymic
stromal lymphopoietin.
13. The selective binding agent or fragment thereof of claim 5 that
specifically binds the polypeptide comprising the amino acid
sequence set forth in SEQ ID NO: 2, or a fragment thereof.
14. The selective binding agent or fragment thereof of claim 11
wherein the second polypeptide comprises the amino acid sequence
set forth in SEQ ID NO: 2, or a fragment thereof.
15. The selective binding agent or fragment thereof of any of
claims 1-14 that is an antibody or fragment thereof.
16. The selective binding agent of claim 15 that is a humanized
antibody.
17. The selective binding agent of claim 15 that is a human
antibody or fragment thereof.
18. The selective binding agent of claim 15 that is a polyclonal
antibody or fragment thereof.
19. The selective binding agent claim 15 that is a monoclonal
antibody or fragment thereof.
20. The selective binding agent of claim 15 that is a chimeric
antibody or fragment thereof.
21. The selective binding agent of claim 15 that is a CDR-grafted
antibody or fragment thereof.
22. The selective binding agent of claim 15 that is an
antiidiotypic antibody or fragment thereof.
23. The selective binding agent of claim 15 that is a variable
region fragment.
24. The variable region fragment of claim 15 that is a Fab or a
Fab' fragment.
25. The selective binding agent of claim 15 that is bound to a
detectable label.
26. The selective binding agent of claim 15 that antagonizes TSLPR
polypeptide biological activity.
27. A method for treating, preventing, or ameliorating a TSLPR
polypeptide-related disease, condition, or disorder comprising
administering to a patient an effective amount of a selective
binding agent according to claim 15.
28. A selective binding agent produced by immunizing an animal with
a polypeptide comprising an amino acid sequence of SEQ ID NO:
2.
29. A selective binding agent produced by immunizing an animal with
a heterodimer complex comprising a first polypeptide that is the
alpha chain of interleukin 7 receptor a second polypeptide having
the amino acid sequence of SEQ ID NO: 2.
30. A hybridoma that produces a selective binding agent according
to any of claims 1-14.
31. A method of detecting or quantitating the amount of a TSLPR
polypeptide or TSLPR heterodimer complex using the antibody or
fragment therof of claim 15.
32. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
polypeptide having the amino acid sequence of SEQ ID NO: 2.
33. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
heterodimer complex comprising a first polypeptide that is the
alpha chain of interleukin 7 receptor and a second polypeptide
having the amino acid sequence of SEQ ID NO: 2.
Description
[0001] This application is a divisional application of U.S. patent
application Ser. No. 09/895,593, filed Jun. 28, 2001, the
disclosure of which is incorporated by reference herein, and which
claims the benefit of U.S. Provisional Patent Application No.
60/214,658, filed on Jun. 28, 2000, the disclosure of which is
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to Thymic Stromal
Lymphopoietin Receptor (TSLPR) polypeptides and nucleic acid
molecules encoding the same. The invention also relates to
selective binding agents, vectors, host cells, and methods for
producing TSLPR polypeptides. The invention further relates to
pharmaceutical compositions and methods for the diagnosis,
treatment, amelioration, and/or prevention of diseases, disorders,
and conditions associated with TSLPR polypeptides.
BACKGROUND OF THE INVENTION
[0003] Technical advances in the identification, cloning,
expression, and manipulation of nucleic acid molecules and the
deciphering of the human genome have greatly accelerated the
discovery of novel therapeutics. Rapid nucleic acid sequencing
techniques can now generate sequence information at unprecedented
rates and, coupled with computational analyses, allow the assembly
of overlapping sequences into partial and entire genomes and the
identification of polypeptide-encoding regions. A comparison of a
predicted amino acid sequence against a database compilation of
known amino acid sequences allows one to determine the extent of
homology to previously identified sequences and/or structural
landmarks. The cloning and expression of a polypeptide-encoding
region of a nucleic acid molecule provides a polypeptide product
for structural and functional analyses. The manipulation of nucleic
acid molecules and encoded polypeptides may confer advantageous
properties on a product for use as a therapeutic.
[0004] In spite of the significant technical advances in genome
research over the past decade, the potential for the development of
novel therapeutics based on the human genome is still largely
unrealized. Many genes encoding potentially beneficial polypeptide
therapeutics or those encoding polypeptides, which may act as
"targets" for therapeutic molecules, have still not been
identified. Accordingly, it is an object of the invention to
identify novel polypeptides, and nucleic acid molecules encoding
the same, which have diagnostic or therapeutic benefit.
[0005] Cytokines regulate a variety of cellular responses including
proliferation, differentiation, and survival. Among the different
classes of cytokines are the type I cytokines, which form four
.alpha.-helical bundle structures that exhibit an up-up-down-down
topology (Bazan, 1990, Immunol. Today 11: 350-54; Leonard and
O'Shea, 1998, Annu. Rev. Immunol. 16: 293-322; Leonard, Fundamental
Immunology 741-74 (Paul, ed., Lippincott Raven Publishers 4 ed.,
1999)). Type I cytokines include many interleukins, such as IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, and IL-15
as well as other hematologically-active molecules such as GM-CSF,
erythropoietin, thrombopoietin, and molecules such as growth
hormone and prolactin. Signaling by type I cytokines involves
interaction with homodimers, heterodimers, or higher order receptor
oligomers of the type I cytokine receptor superfamily. Ligand
binding induces dimerization or higher order oligomerization,
resulting in downstream signaling, in part involving the Jak-STAT
pathway (Bazan, supra; Leonard and O'Shea, supra; Leonard,
supra).
[0006] Thymic stromal lymphopoietin (TSLP) is a cytokine whose
biological activities overlap with those of IL-7. For example, both
TSLP and IL-7 induce tyrosine phosphorylation of the transcription
factor Stat5 (Isaksen et al., 1999, J. Immunol. 163: 5971-77). TSLP
activity was originally identified in the conditioned medium of a
thymic stromal cell line that supported the development of murine
IgM.sup.+ B-cells from fetal liver hematopoietic progenitor cells
(Friend et al., 1994 Exp. Hematol. 22: 321-28). Moreover, TSLP can
promote B-cell lymphopoiesis in long-term bone marrow cultures and
can co-stimulate both thymocytes and mature T-cells (Friend et al.,
supra; Levin et al., 1999, J. Immunol. 162: 677-83). TSLP may also
serve as an extrinsic signal to specifically rearrange the T-cell
receptor gamma locus (Candeias et al., 1997, Immunol. Lett. 57:
9-14). Thus, the isolation and characterization of the cytokine
receptor for TSLP would allow for the identification of compounds
useful in treating TSLP-related diseases or conditions, such as
those affecting B-cell development, T-cell development, T-cell
receptor gene rearrangement, or regulation of the Stat5
transcription factor.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel TSLPR nucleic acid
molecules and encoded polypeptides.
[0008] The invention provides for an isolated nucleic acid molecule
comprising a nucleotide sequence selected from the group consisting
of:
[0009] (a) the nucleotide sequence as set forth in any of SEQ ID
NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO:
11;
[0010] (b) a nucleotide sequence encoding the polypeptide as set
forth in any of SEQ ID NO:2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0011] (c) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of either (b) or
(c); and
[0012] (d) a nucleotide sequence complementary to either (b) or
(c).
[0013] The invention also provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0014] (a) a nucleotide sequence encoding a polypeptide which is at
least about 70 percent identical to the polypeptide as set forth in
any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0015] (b) a nucleotide sequence encoding an allelic variant or
splice variant of the nucleotide sequence as set forth in any of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID
NO: 1, or (a); (c) a region of the nucleotide sequence of any of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID
NO: 11, (a), or (b) encoding a polypeptide fragment of at least
about 25 amino acid residues, wherein the polypeptide fragment has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 5, or SEQ ID NO: 8, or is antigenic;
[0016] (d) a region of the nucleotide sequence of any of SEQ ID NO:
1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 1, or
any of (a)-(c) comprising a fragment of at least about 16
nucleotides;
[0017] (e) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(d);
and
[0018] (f) a nucleotide sequence complementary to any of
(a)-(d).
[0019] The invention further provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0020] (a) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at
least one conservative amino acid substitution, wherein the encoded
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0021] (b) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at
least one amino acid insertion, wherein the encoded polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 5, or SEQ ID NO: 8;
[0022] (c) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at
least one amino acid deletion, wherein the encoded polypeptide has
an activity of the polypeptide set forth in any of SEQ ID NO: 2,
SEQ ID NO: 5, or SEQ ID NO: 8;
[0023] (d) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 which
has a C- and/or N-terminal truncation, wherein the encoded
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0024] (e) a nucleotide sequence encoding a polypeptide as set
forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at
least one modification selected from the group consisting of amino
acid substitutions, amino acid insertions, amino acid deletions,
C-terminal truncation, and N-terminal truncation, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0025] (e) a nucleotide sequence of any of (a)-(e) comprising a
fragment of at least about 16 nucleotides;
[0026] (g) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(f);
and
[0027] (h) a nucleotide sequence complementary to any of
(a)-(e).
[0028] The present invention provides for an isolated polypeptide
comprising the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.
[0029] The invention also provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0030] (a) the amino acid sequence as set forth in any of SEQ ID
NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, optionally further comprising
an amino-terminal methionine;
[0031] (b) an amino acid sequence for an ortholog of any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;
[0032] (c) an amino acid sequence which is at least about 70
percent identical to the amino acid sequence of any of SEQ ID NO:
2, SEQ ID NO: 5, or SEQ ID NO: 8, wherein the polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 5, or SEQ ID NO: 8;
[0033] (d) a fragment of the amino acid sequence set forth in any
of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 comprising at least
about 25 amino acid residues, wherein the fragment has an activity
of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5,
or SEQ ID NO: 8, or is antigenic; and
[0034] (e) an amino acid sequence for an allelic variant or splice
variant of the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or any of (a)-(c).
[0035] The invention further provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0036] (a) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least one conservative
amino acid substitution, wherein the polypeptide has an activity of
the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8;
[0037] (b) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least one amino acid
insertion, wherein the polypeptide has an activity of the
polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ
ID NO: 8;
[0038] (c) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least one amino acid
deletion, wherein the polypeptide has an activity of the
polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ
ID NO: 8;
[0039] (d) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 which has a C- and/or
N-terminal truncation, wherein the polypeptide has an activity of
the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8; and
[0040] (e) the amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 with at least one modification
selected from the group consisting of amino acid substitutions,
amino acid insertions, amino acid deletions, C-terminal truncation,
and N-terminal truncation, wherein the polypeptide has an activity
of the polypeptide set forth in any of SEQ ID NO: 2, SEQ ID NO: 5,
or SEQ ID NO: 8.
[0041] Also provided are fusion polypeptides comprising TSLPR amino
acid sequences.
[0042] The present invention also provides for an expression vector
comprising the isolated nucleic acid molecules as set forth herein,
recombinant host cells comprising the recombinant nucleic acid
molecules as set forth herein, and a method of producing a TSLPR
polypeptide comprising culturing the host cells and optionally
isolating the polypeptide so produced.
[0043] A transgenic non-human animal comprising a nucleic acid
molecule encoding a TSLPR polypeptide is also encompassed by the
invention. The TSLPR nucleic acid molecules are introduced into the
animal in a manner that allows expression and increased levels of a
TSLPR polypeptide, which may include increased circulating levels.
Alternatively, the TSLPR nucleic acid molecules are introduced into
the animal in a manner that prevents expression of endogenous TSLPR
polypeptide (i.e., generates a transgenic animal possessing a TSLPR
polypeptide gene knockout). The transgenic non-human animal is
preferably a mammal, and more preferably a rodent, such as a rat or
a mouse.
[0044] Also provided are derivatives of the TSLPR polypeptides of
the present invention.
[0045] Additionally provided are selective binding agents such as
antibodies and peptides capable of specifically binding the TSLPR
polypeptides of the invention. Such antibodies and peptides may be
agonistic or antagonistic.
[0046] Pharmaceutical compositions comprising the nucleotides,
polypeptides, or selective binding agents of the invention and one
or more pharmaceutically acceptable formulation agents are also
encompassed by the invention. The pharmaceutical compositions are
used to provide therapeutically effective amounts of the
nucleotides or polypeptides of the present invention. The invention
is also directed to methods of using the polypeptides, nucleic acid
molecules, and selective binding agents.
[0047] The TSLPR polypeptides and nucleic acid molecules of the
present invention may be used to treat, prevent, ameliorate, and/or
detect diseases and disorders, including those recited herein.
[0048] The present invention also provides a method of assaying
test molecules to identify a test molecule that binds to a TSLPR
polypeptide. The method comprises contacting a TSLPR polypeptide
with a test molecule to determine the extent of binding of the test
molecule to the polypeptide. The method further comprises
determining whether such test molecules are agonists or antagonists
of a TSLPR polypeptide. The present invention further provides a
method of testing the impact of molecules on the expression of
TSLPR polypeptide or on the activity of TSLPR polypeptide.
[0049] Methods of regulating expression and modulating (i.e.,
increasing or decreasing) levels of a TSLPR polypeptide are also
encompassed by the invention. One method comprises administering to
an animal a nucleic acid molecule encoding a TSLPR polypeptide. In
another method, a nucleic acid molecule comprising elements that
regulate or modulate the expression of a TSLPR polypeptide may be
administered. Examples of these methods include gene therapy, cell
therapy, and anti-sense therapy as further described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIGS. 1A-1B illustrate the nucleotide sequence of the murine
TSLPR gene (SEQ ID NO: 1) and deduced amino acid sequence of murine
TSLPR polypeptide (SEQ ID NO: 2). The predicted signal peptide
(underline) and transmembrane domain (double underline) are
indicated;
[0051] FIG. 2 illustrates an amino acid sequence alignment of
murine TSLPR polypeptide (upper sequence; SEQ ID NO: 2) and murine
common cytokine receptor .gamma. chain (.gamma..sub.c) (lower
sequence; SEQ ID NO: 12). Identical residues (boxed), potential
N-linked glycosylation sites (*), and predicted signal peptide and
transmembrane domain (underline) are indicated;
[0052] FIGS. 3A-3B illustrate the nucleotide sequence of the human
TSLPR gene (SEQ ID NO: 4) and the deduced amino acid sequence of
human TSLPR polypeptide (SEQ ID NO: 5). The predicted signal
peptide (underline) and transmembrane domain (double underline) are
indicated;
[0053] FIGS. 4A-4B illustrate the nucleotide sequence of human
TSLPR/FLAG (SEQ ID NO: 7) and the deduced amino acid sequence of
human TSLPR/FLAG polypeptide (SEQ ID NO: 8). The FLAG peptide
(dotted underline), predicted signal peptide (underline), and
predicted transmembrane domain (double underline) are
indicated;
[0054] FIG. 5 illustrates an amino acid sequence alignment of
murine TSLPR polypeptide (upper sequence; SEQ ID NO: 2) and human
TSLPR polypeptide (lower sequence; SEQ ID NO: 5);
[0055] FIGS. 6A-6C illustrate (A) in vitro translation of murine
TSLPR polypeptide, (B) immunoprecipitation of murine TSLPR
polypeptide from NAG 8/7 cells, and (C) northern blot analysis of
murine TSLPR mRNA expression.
[0056] FIG. 7 illustrates the results obtained in proliferation
assays using cells transfected with chimeric expression constructs
for c-Kit/.gamma..sub.c, c-Kit/TSLPR and c-Kit/.beta., or
c-Kit/.gamma..sub.c and c-Kit/.beta..
[0057] FIGS. 8A-8C illustrate the results obtained in affinity
labeling assays in which .sup.125I-TSLP was added to 293 cells
transfected with expression constructs for murine IL-7R.alpha.,
murine TSLPR, murine IL-7R.alpha. and murine TSLPR, or human
IL-7R.alpha. and murine TSLPR, and then cross-linked with DSS.
[0058] FIGS. 9A-9D illustrate the results obtained in displacement
binding assays.
[0059] FIG. 10 illustrates the results obtained in CAT assays in
which HepG2 cells were co-transfected with expression constructs
for IL-7R.alpha. and TSLPR, or .gamma..sub.c, and pHRRE-CAT.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All references cited in this application are
expressly incorporated by reference herein.
[0061] Definitions
[0062] The terms "TSLPR gene" or "TSLPR nucleic acid molecule" or
"TSLPR polynucleotide" refer to a nucleic acid molecule comprising
or consisting of a nucleotide sequence as set forth in any of SEQ
ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO:
1, a nucleotide sequence encoding the polypeptide as set forth in
any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, and nucleic
acid molecules as defined herein.
[0063] The term "TSLPR polypeptide allelic variant" refers to one
of several possible naturally occurring alternate forms of a gene
occupying a given locus on a chromosome of an organism or a
population of organisms.
[0064] The term "TSLPR polypeptide splice variant" refers to a
nucleic acid molecule, usually RNA, which is generated by
alternative processing of intron sequences in an RNA transcript of
TSLPR polypeptide amino acid sequence as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.
[0065] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the invention that (1) has been separated
from at least about 50 percent of proteins, lipids, carbohydrates,
or other materials with which it is naturally found when total
nucleic acid is isolated from the source cells, (2) is not linked
to all or a portion of a polynucleotide to which the "isolated
nucleic acid molecule" is linked in nature, (3) is operably linked
to a polynucleotide which it is not linked to in nature, or (4)
does not occur in nature as part of a larger polynucleotide
sequence. Preferably, the isolated nucleic acid molecule of the
present invention is substantially free from any other
contaminating nucleic acid molecule(s) or other contaminants that
are found in its natural environment that would interfere with its
use in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0066] The term "nucleic acid sequence" or "nucleic acid molecule"
refers to a DNA or RNA sequence. The term encompasses molecules
formed from any of the known base analogs of DNA and RNA such as,
but not limited to 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,
aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,
N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiou- racil,
beta-D-mannosylqueosine, 5'-methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0067] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
[0068] The term "expression vector" refers to a vector that is
suitable for transformation of a host cell and contains nucleic
acid sequences that direct and/or control the expression of
inserted heterologous nucleic acid sequences. Expression includes,
but is not limited to, processes such as transcription,
translation, and RNA splicing, if introns are present.
[0069] The term "operably linked" is used herein to refer to an
arrangement of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription
and/or translation of the coding sequence. For example, a coding
sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence,
so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0070] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0071] The term "TSLPR polypeptide" refers to a polypeptide
comprising the amino acid sequence of any of SEQ ID NO: 2, SEQ ID
NO: 5, or SEQ ID NO: 8 and related polypeptides. Related
polypeptides include TSLPR polypeptide fragments, TSLPR polypeptide
orthologs, TSLPR polypeptide variants, and TSLPR polypeptide
derivatives, which possess at least one activity of the polypeptide
as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.
TSLPR polypeptides may be mature polypeptides, as defined herein,
and may or may not have an amino-terminal methionine residue,
depending on the method by which they are prepared.
[0072] The term "TSLPR polypeptide fragment" refers to a
polypeptide that comprises a truncation at the amino-terminus (with
or without a leader sequence) and/or a truncation at the
carboxyl-terminus of the polypeptide as set forth in any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. The term "TSLPR polypeptide
fragment" also refers to amino-terminal and/or carboxyl-terminal
truncations of TSLPR polypeptide orthologs, TSLPR polypeptide
derivatives, or TSLPR polypeptide variants, or to amino-terminal
and/or carboxyl-terminal truncations of the polypeptides encoded by
TSLPR polypeptide allelic variants or TSLPR polypeptide splice
variants. TSLPR polypeptide fragments may result from alternative
RNA splicing or from in vivo protease activity. Membrane-bound
forms of a TSLPR polypeptide are also contemplated by the present
invention. In preferred embodiments, truncations and/or deletions
comprise about 10 amino acids, or about 20 amino acids, or about 50
amino acids, or about 75 amino acids, or about 100 amino acids, or
more than about 100 amino acids. The polypeptide fragments so
produced will comprise about 25 contiguous amino acids, or about 50
amino acids, or about 75 amino acids, or about 100 amino acids, or
about 150 amino acids, or about 200 amino acids.
[0073] Such TSLPR polypeptide fragments may optionally comprise an
amino-terminal methionine residue. It will be appreciated that such
fragments can be used, for example, to generate antibodies to TSLPR
polypeptides.
[0074] The term "TSLPR polypeptide ortholog" refers to a
polypeptide from another species that corresponds to TSLPR
polypeptide amino acid sequence as set forth in any of SEQ ID NO:
2, SEQ ID NO: 5, or SEQ ID NO: 8. For example, mouse and human
TSLPR polypeptides are considered orthologs of each other.
[0075] The term "TSLPR polypeptide variants" refers to TSLPR
polypeptides comprising amino acid sequences having one or more
amino acid sequence substitutions, deletions (such as internal
deletions and/or TSLPR polypeptide fragments), and/or additions
(such as internal additions and/or TSLPR fusion polypeptides) as
compared to the TSLPR polypeptide amino acid sequence set forth in
any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 (with or without
a leader sequence). Variants may be naturally occurring (e.g.,
TSLPR polypeptide allelic variants, TSLPR polypeptide orthologs,
and TSLPR polypeptide splice variants) or artificially constructed.
Such TSLPR polypeptide variants may be prepared from the
corresponding nucleic acid molecules having a DNA sequence that
varies accordingly from the DNA sequence as set forth in any of SEQ
ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO:
11. In preferred embodiments, the variants have from 1 to 3, or
from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or
from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100,
or more than 100 amino acid substitutions, insertions, additions
and/or deletions, wherein the substitutions may be conservative, or
non-conservative, or any combination thereof.
[0076] The term "TSLPR polypeptide derivatives" refers to the
polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8, TSLPR polypeptide fragments, TSLPR polypeptide
orthologs, or TSLPR polypeptide variants, as defined herein, that
have been chemically modified. The term "TSLPR polypeptide
derivatives" also refers to the polypeptides encoded by TSLPR
polypeptide allelic variants or TSLPR polypeptide splice variants,
as defined herein, that have been chemically modified.
[0077] The term "mature TSLPR polypeptide" refers to a TSLPR
polypeptide lacking a leader sequence. A mature TSLPR polypeptide
may also include other modifications such as proteolytic processing
of the amino-terminus (with or without a leader sequence) and/or
the carboxyl-terminus, cleavage of a smaller polypeptide from a
larger precursor, N-linked and/or O-linked glycosylation, and the
like. Exemplary mature TSLPR polypeptides are depicted by the amino
acid sequences as set forth in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ
ID NO: 9.
[0078] The term "TSLPR fusion polypeptide" refers to a fusion of
one or more amino acids (such as a heterologous protein or peptide)
at the amino- or carboxyl-terminus of the polypeptide as set forth
in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, TSLPR
polypeptide fragments, TSLPR polypeptide orthologs, TSLPR
polypeptide variants, or TSLPR derivatives, as defined herein. The
term "TSLPR fusion polypeptide" also refers to a fusion of one or
more amino acids at the amino- or carboxyl-terminus of the
polypeptide encoded by TSLPR polypeptide allelic variants or TSLPR
polypeptide splice variants, as defined herein.
[0079] The term "biologically active TSLPR polypeptides" refers to
TSLPR polypeptides having at least one activity characteristic of
the polypeptide comprising the amino acid sequence of any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In addition, a TSLPR
polypeptide may be active as an immunogen; that is, the TSLPR
polypeptide contains at least one epitope to which antibodies may
be raised.
[0080] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates, or
other materials with which it is naturally found when isolated from
the source cell, (2) is not linked (by covalent or noncovalent
interaction) to all or a portion of a polypeptide to which the
"isolated polypeptide" is linked in nature, (3) is operably linked
(by covalent or noncovalent interaction) to a polypeptide with
which it is not linked in nature, or (4) does not occur in nature.
Preferably, the isolated polypeptide is substantially free from any
other contaminating polypeptides or other contaminants that are
found in its natural environment that would interfere with its
therapeutic, diagnostic, prophylactic or research use.
[0081] The term "identity," as known in the art, refers to a
relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
polypeptides, as the case may be, as determined by the match
between strings of two or more nucleotide or two or more amino acid
sequences. "Identity" measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (i.e., "algorithms").
[0082] The term "similarity" is a related concept, but in contrast
to "identity," "similarity" refers to a measure of relatedness
which includes both identical matches and conservative substitution
matches. If two polypeptide sequences have, for example, 10/20
identical amino acids, and the remainder are all non-conservative
substitutions, then the percent identity and similarity would both
be 50%. If in the same example, there are five more positions where
there are conservative substitutions, then the percent identity
remains 50%, but the percent similarity would be 75% (15/20).
Therefore, in cases where there are conservative substitutions, the
percent similarity between two polypeptides will be higher than the
percent identity between those two polypeptides. The term
"naturally occurring" or "native" when used in connection with
biological materials such as nucleic acid molecules, polypeptides,
host cells, and the like, refers to materials which are found in
nature and are not manipulated by man. Similarly, "non-naturally
occurring" or "non-native" as used herein refers to a material that
is not found in nature or that has been structurally modified or
synthesized by man.
[0083] The terms "effective amount" and "therapeutically effective
amount" each refer to the amount of a TSLPR polypeptide or TSLPR
nucleic acid molecule used to support an observable level of one or
more biological activities of the TSLPR polypeptides as set forth
herein.
[0084] The term "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refers to one
or more formulation materials suitable for accomplishing or
enhancing the delivery of the TSLPR polypeptide, TSLPR nucleic acid
molecule, or TSLPR selective binding agent as a pharmaceutical
composition.
[0085] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0086] The term "selective binding agent" refers to a molecule or
molecules having specificity for a TSLPR polypeptide. As used
herein, the terms, "specific" and "specificity" refer to the
ability of the selective binding agents to bind to human TSLPR
polypeptides and not to bind to human non-TSLPR polypeptides. It
will be appreciated, however, that the selective binding agents may
also bind orthologs of the polypeptide as set forth in any of SEQ
ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, that is, interspecies
versions thereof, such as mouse and rat TSLPR polypeptides.
[0087] The term "transduction" is used to refer to the transfer of
genes from one bacterium to another, usually by a phage.
"Transduction" also refers to the acquisition and transfer of
eukaryotic cellular sequences by retroviruses.
[0088] The term "transfection" is used to refer to the uptake of
foreign or exogenous DNA by a cell, and a cell has been
"transfected" when the exogenous DNA has been introduced inside the
cell membrane. A number of transfection techniques are well known
in the art and are disclosed herein. See, e.g., Graham et al.,
1973, Virology 52: 456; Sambrook et al., Molecular Cloning, A
Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et
al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu
et al., 1981, Gene 13: 197. Such techniques can be used to
introduce one or more exogenous DNA moieties into suitable host
cells.
[0089] The term "transformation" as used herein refers to a change
in a cell's genetic characteristics, and a cell has been
transformed when it has been modified to contain a new DNA. For
example, a cell is transformed where it is genetically modified
from its native state. Following transfection or transduction, the
transforming DNA may recombine with that of the cell by physically
integrating into a chromosome of the cell, may be maintained
transiently as an episomal element without being replicated, or may
replicate independently as a plasmid. A cell is considered to have
been stably transformed when the DNA is replicated with the
division of the cell.
[0090] Relatedness of Nucleic Acid Molecules and/or
Polypeptides
[0091] It is understood that related nucleic acid molecules include
allelic or splice variants of the nucleic acid molecule of any of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID
NO: 11, and include sequences which are complementary to any of the
above nucleotide sequences. Related nucleic acid molecules also
include a nucleotide sequence encoding a polypeptide comprising or
consisting essentially of a substitution, modification, addition
and/or deletion of one or more amino acid residues compared to the
polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8. Such related TSLPR polypeptides may comprise, for
example, an addition and/or a deletion of one or more N-linked or
O-linked glycosylation sites or an addition and/or a deletion of
one or more cysteine residues.
[0092] Related nucleic acid molecules also include fragments of
TSLPR nucleic acid molecules which encode a polypeptide of at least
about 25 contiguous amino acids, or about 50 amino acids, or about
75 amino acids, or about 100 amino acids, or about 150 amino acids,
or about 200 amino acids, or more than 200 amino acid residues of
the TSLPR polypeptide of any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ
ID NO: 8.
[0093] In addition, related TSLPR nucleic acid molecules also
include those molecules which comprise nucleotide sequences which
hybridize under moderately or highly stringent conditions as
defined herein with the fully complementary sequence of the TSLPR
nucleic acid molecule of any of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID
NO: 7, SEQ ID NO: 10, or SEQ ID NO: 11, or of a molecule encoding a
polypeptide, which polypeptide comprises the amino acid sequence as
shown in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or of
a nucleic acid fragment as defined herein, or of a nucleic acid
fragment encoding a polypeptide as defined herein. Hybridization
probes may be prepared using the TSLPR sequences provided herein to
screen cDNA, genomic or synthetic DNA libraries for related
sequences. Regions of the DNA and/or amino acid sequence of TSLPR
polypeptide that exhibit significant identity to known sequences
are readily determined using sequence alignment algorithms as
described herein and those regions may be used to design probes for
screening.
[0094] The term "highly stringent conditions" refers to those
conditions that are designed to permit hybridization of DNA strands
whose sequences are highly complementary, and to exclude
hybridization of significantly mismatched DNAs. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of "highly stringent conditions" for
hybridization and washing are 0.015 M sodium chloride, 0.0015 M
sodium citrate at 65-68.degree. C. or 0.015 M sodium chloride,
0.0015 M sodium citrate, and 50% formamide at 42.degree. C. See
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et
al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL
Press Limited).
[0095] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used--however, the rate of hybridization will be affected.
Other agents may be included in the hybridization and washing
buffers for the purpose of reducing non-specific and/or background
hybridization. Examples are 0.1% bovine serum albumin, 0.1%
polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate, NaDodSO.sub.4, (SDS), ficoll, Denhardt's solution,
sonicated salmon sperm DNA (or another non-complementary DNA), and
dextran sulfate, although other suitable agents can also be used.
The concentration and types of these additives can be changed
without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH
6.8-7.4; however, at typical ionic strength conditions, the rate of
hybridization is nearly independent of pH. See Anderson et al.,
Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press
Limited).
[0096] Factors affecting the stability of DNA duplex include base
composition, length, and degree of base pair mismatch.
Hybridization conditions can be adjusted by one skilled in the art
in order to accommodate these variables and allow DNAs of different
sequence relatedness to form hybrids. The melting temperature of a
perfectly matched DNA duplex can be estimated by the following
equation:
T.sub.m(.degree. C.)=81.5+16.6(log[Na+])+0.41(% G+C)-600/N-0.72(%
formamide)
[0097] where N is the length of the duplex formed, [Na+] is the
molar concentration of the sodium ion in the hybridization or
washing solution, % G+C is the percentage of (guanine+cytosine)
bases in the hybrid. For imperfectly matched hybrids, the melting
temperature is reduced by approximately 1.degree. C. for each 1%
mismatch.
[0098] The term "moderately stringent conditions" refers to
conditions under which a DNA duplex with a greater degree of base
pair mismatching than could occur under "highly stringent
conditions" is able to form. Examples of typical "moderately
stringent conditions" are 0.015 M sodium chloride, 0.0015 M sodium
citrate at 50-65.degree. C. or 0.015 M sodium chloride, 0.0015 M
sodium citrate, and 20% formamide at 37-50.degree. C. By way of
example, "moderately stringent conditions" of 50.degree. C. in
0.015 M sodium ion will allow about a 21% mismatch.
[0099] It will be appreciated by those skilled in the art that
there is no absolute distinction between "highly stringent
conditions" and "moderately stringent conditions." For example, at
0.015 M sodium ion (no formamide), the melting temperature of
perfectly matched long DNA is about 71.degree. C. With a wash at
65.degree. C. (at the same ionic strength), this would allow for
approximately a 6% mismatch. To capture more distantly related
sequences, one skilled in the art can simply lower the temperature
or raise the ionic strength.
[0100] A good estimate of the melting temperature in 1M NaCl* for
oligonucleotide probes up to about 20 nt is given by:
T.sub.m=2.degree. C. per A-T base pair+4.degree. C. per G-C base
pair
[0101] *The sodium ion concentration in 6.times. salt sodium
citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using
Purified Genes 683 (Brown and Fox, eds., 1981).
[0102] High stringency washing conditions for oligonucleotides are
usually at a temperature of 0-5.degree. C. below the Tm of the
oligonucleotide in 6.times.SSC, 0.1% SDS.
[0103] In another embodiment, related nucleic acid molecules
comprise or consist of a nucleotide sequence that is at least about
70 percent identical to the nucleotide sequence as shown in any of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID
NO: 1, or comprise or consist essentially of a nucleotide sequence
encoding a polypeptide that is at least about 70 percent identical
to the polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO:
5, or SEQ ID NO: 8. In preferred embodiments, the nucleotide
sequences are about 75 percent, or about 80 percent, or about 85
percent, or about 90 percent, or about 95, 96, 97, 98, or 99
percent identical to the nucleotide sequence as shown in any of SEQ
ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO:
11, or the nucleotide sequences encode a polypeptide that is about
75 percent, or about 80 percent, or about 85 percent, or about 90
percent, or about 95, 96, 97, 98, or 99 percent identical to the
polypeptide sequence as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 5, or SEQ ID NO: 8. Related nucleic acid molecules encode
polypeptides possessing at least one activity of the polypeptide
set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:
8.
[0104] Differences in the nucleic acid sequence may result in
conservative and/or non-conservative modifications of the amino
acid sequence relative to the amino acid sequence of any of SEQ ID
NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.
[0105] Conservative modifications to the amino acid sequence of any
of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 (and the
corresponding modifications to the encoding nucleotides) will
produce a polypeptide having functional and chemical
characteristics similar to those of TSLPR polypeptides. In
contrast, substantial modifications in the functional and/or
chemical characteristics of TSLPR polypeptides may be accomplished
by selecting substitutions in the amino acid sequence of any of SEQ
ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 that differ significantly
in their effect on maintaining (a) the structure of the molecular
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
[0106] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis."
[0107] Conservative amino acid substitutions also encompass
non-naturally occurring amino acid residues that are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics, and other
reversed or inverted forms of amino acid moieties.
[0108] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0109] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0110] 2) neutral hydrophilic: Cys, Ser, Thr;
[0111] 3) acidic: Asp, Glu;
[0112] 4) basic: Asn, Gln, His, Lys, Arg;
[0113] 5) residues that influence chain orientation: Gly, Pro;
and
[0114] 6) aromatic: Trp, Tyr, Phe.
[0115] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human TSLPR polypeptide that are homologous with
non-human TSLPR polypeptides, or into the non-homologous regions of
the molecule.
[0116] In making such changes, the hydropathic index of amino acids
may be considered.
[0117] Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics. The
hydropathic indices 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).
[0118] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte et al., 1982, J. Mol. Biol.
157: 105-31). It is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score and still retain a similar biological activity. In
making changes based upon the hydropathic index, the substitution
of amino acids whose hydropathic indices are within .+-.2 is
preferred, those which are within .+-.1 are particularly preferred,
and those within .+-.0.5 are even more particularly preferred.
[0119] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended for use
in immunological embodiments, as in the present case. The greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein.
[0120] The following hydrophilicity values have been assigned to
these 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); and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within +2 is preferred, those which are
within +1 are particularly preferred, and those within +0.5 are
even more particularly preferred. One may also identify epitopes
from primary amino acid sequences on the basis of hydrophilicity.
These regions are also referred to as "epitopic core regions."
[0121] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
TSLPR polypeptide, or to increase or decrease the affinity of the
TSLPR polypeptides described herein. Exemplary amino acid
substitutions are set forth in Table I.
1TABLE I Amino Acid Substitutions Original Residues Exemplary
Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg
Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn
Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile
Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile
Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln, Asn
Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly
Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,
Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0122] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth in any of SEQ ID NO: 2,
SEQ ID NO: 5, or SEQ ID NO: 8 using well-known techniques. For
identifying suitable areas of the molecule that may be changed
without destroying biological activity, one skilled in the art may
target areas not believed to be important for activity. For
example, when similar polypeptides with similar activities from the
same species or from other species are known, one skilled in the
art may compare the amino acid sequence of a TSLPR polypeptide to
such similar polypeptides. With such a comparison, one can identify
residues and portions of the molecules that are conserved among
similar polypeptides. It will be appreciated that changes in areas
of the TSLPR molecule that are not conserved relative to such
similar polypeptides would be less likely to adversely affect the
biological activity and/or structure of a TSLPR polypeptide. One
skilled in the art would also know that, even in relatively
conserved regions, one may substitute chemically similar amino
acids for the naturally occurring residues while retaining activity
(conservative amino acid residue substitutions). Therefore, even
areas that may be important for biological activity or for
structure may be subject to conservative amino acid substitutions
without destroying the biological activity or without adversely
affecting the polypeptide structure.
[0123] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a TSLPR polypeptide that correspond to amino acid
residues that are important for activity or structure in similar
polypeptides. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues of TSLPR polypeptides.
[0124] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of TSLPR polypeptide with respect to its three
dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each amino acid residue. The variants could be
screened using activity assays known to those with skill in the
art. Such variants could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0125] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult, 1996, Curr. Opin.
Biotechnol. 7: 422-27; Chou et al., 1974, Biochemistry 13: 222-45;
Chou et al., 1974, Biochemistry 113: 211-22; Chou et al., 1978,
Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-48; Chou et al., 1978,
Ann. Rev. Biochem. 47: 251-276; and Chou et al., 1979, Biophys. J.
26: 367-84. Moreover, computer programs are currently available to
assist with predicting secondary structure. One method of
predicting secondary structure is based upon homology modeling. For
example, two polypeptides or proteins which have a sequence
identity of greater than 30%, or similarity greater than 40%, often
have similar structural topologies. The recent growth of the
protein structural database (PDB) has provided enhanced
predictability of secondary structure, including the potential
number of folds within the structure of a polypeptide or protein.
See Holm et al., 1999, Nucleic Acids Res. 27: 244-47. It has been
suggested that there are a limited number of folds in a given
polypeptide or protein and that once a critical number of
structures have been resolved, structural prediction will become
dramatically more accurate (Brenner et al., 1997, Curr. Opin.
Struct. Biol. 7: 369-76).
[0126] Additional methods of predicting secondary structure include
"threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87;
Sippl et al., 1996, Structure 4: 15-19), "profile analysis" (Bowie
et al., 1991, Science, 253: 164-70; Gribskov et al., 1990, Methods
Enzymol. 183: 146-59; Gribskov et al., 1987, Proc. Nat. Acad. Sci.
U.S.A. 84: 4355-58), and "evolutionary linkage" (See Holm et al.,
supra, and Brenner et al., supra).
[0127] Preferred TSLPR polypeptide variants include glycosylation
variants wherein the number and/or type of glycosylation sites have
been altered compared to the amino acid sequence set forth in any
of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In one embodiment,
TSLPR polypeptide variants comprise a greater or a lesser number of
N-linked glycosylation sites than the amino acid sequence set forth
in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or
Asn-X-Thr, wherein the amino acid residue designated as X may be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions that eliminate this sequence will remove an existing
N-linked carbohydrate chain. Also provided is a rearrangement of
N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring)
are eliminated and one or more new N-linked sites are created.
Additional preferred TSLPR variants include cysteine variants,
wherein one or more cysteine residues are deleted or substituted
with another amino acid (e.g., serine) as compared to the amino
acid sequence set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8. Cysteine variants are useful when TSLPR polypeptides
must be refolded into a biologically active conformation such as
after the isolation of insoluble inclusion bodies. Cysteine
variants generally have fewer cysteine residues than the native
protein, and typically have an even number to minimize interactions
resulting from unpaired cysteines.
[0128] In other embodiments, related nucleic acid molecules
comprise or consist of a nucleotide sequence encoding a polypeptide
as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8
with at least one amino acid insertion and wherein the polypeptide
has an activity of the polypeptide set forth in any of SEQ ID NO:
2, SEQ ID NO: 5, or SEQ ID NO: 8, or a nucleotide sequence encoding
a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8 with at least one amino acid deletion and wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Related nucleic acid
molecules also comprise or consist of a nucleotide sequence
encoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 5, or SEQ ID NO: 8 wherein the polypeptide has a carboxyl-
and/or amino-terminal truncation and further wherein the
polypeptide has an activity of the polypeptide set forth in any of
SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Related nucleic acid
molecules also comprise or consist of a nucleotide sequence
encoding a polypeptide as set forth in any of SEQ ID NO: 2, SEQ ID
NO: 5, or SEQ ID NO: 8 with at least one modification selected from
the group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, carboxyl-terminal truncations,
and amino-terminal truncations and wherein the polypeptide has an
activity of the polypeptide set forth in any of SEQ ID NO: 2, SEQ
ID NO: 5, or SEQ ID NO: 8. In addition, the polypeptide comprising
the amino acid sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, or
SEQ ID NO: 8, or other TSLPR polypeptide, may be fused to a
homologous polypeptide to form a homodimer or to a heterologous
polypeptide to form a heterodimer. Heterologous peptides and
polypeptides include, but are not limited to: an epitope to allow
for the detection and/or isolation of a TSLPR fusion polypeptide; a
transmembrane receptor protein or a portion thereof, such as an
extracellular domain or a transmembrane and intracellular domain; a
ligand or a portion thereof which binds to a transmembrane receptor
protein; an enzyme or portion thereof which is catalytically
active; a polypeptide or peptide which promotes oligomerization,
such as a leucine zipper domain; a polypeptide or peptide which
increases stability, such as an immunoglobulin constant region; and
a polypeptide which has a therapeutic activity different from the
polypeptide comprising the amino acid sequence as set forth in any
of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or other TSLPR
polypeptide.
[0129] Fusions can be made either at the amino-terminus or at the
carboxyl-terminus of the polypeptide comprising the amino acid
sequence set forth in any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID
NO: 8, or other TSLPR polypeptide. Fusions may be direct with no
linker or adapter molecule or may be through a linker or adapter
molecule. A linker or adapter molecule may be one or more amino
acid residues, typically from about 20 to about 50 amino acid
residues. A linker or adapter molecule may also be designed with a
cleavage site for a DNA restriction endonuclease or for a protease
to allow for the separation of the fused moieties. It will be
appreciated that once constructed, the fusion polypeptides can be
derivatized according to the methods described herein.
[0130] In a further embodiment of the invention, the polypeptide
comprising the amino acid sequence of any of SEQ ID NO: 2, SEQ ID
NO: 5, or SEQ ID NO: 8, or other TSLPR polypeptide, is fused to one
or more domains of an Fc region of human IgG. Antibodies comprise
two functionally independent parts, a variable domain known as
"Fab," that binds an antigen, and a constant domain known as "Fc,"
that is involved in effector functions such as complement
activation and attack by phagocytic cells. An Fc has a long serum
half-life, whereas an Fab is short-lived. Capon et al., 1989,
Nature 337: 525-31. When constructed together with a therapeutic
protein, an Fc domain can provide longer half-life or incorporate
such functions as Fc receptor binding, protein A binding,
complement fixation, and perhaps even placental transfer. Id. Table
II summarizes the use of certain Fc fusions known in the art.
2TABLE II Fc Fusion with Therapeutic Proteins Form of Fc Fusion
partner Therapeutic implications Reference IgG1 N-terminus of
Hodgkin's disease; U.S. Pat. No. CD30-L anaplastic lymphoma; T-
5,480,981 cell leukemia Murine Fc.gamma.2a IL-10 anti-inflammatory;
Zheng et al., 1995, J. transplant rejection Immunol. 154: 5590-600
IgG1 TNF receptor septic shock Fisher et al., 1996, N. Engl. J.
Med. 334: 1697-1702; Van Zee et al., 1996, J. Immunol. 156: 2221-30
IgG, IgA, IgM, TNF receptor inflammation, U.S. Pat. No. or IgE
autoimmune disorders 5,808,029 (excluding the first domain) IgG1
CD4 receptor AIDS Capon et al., 1989, Nature 337: 525-31 IgG1,
N-terminus anti-cancer, antiviral Harvill et al., 1995, IgG3 of
IL-2 Immunotech. 1: 95-105 IgG1 C-terminus of osteoarthritis; WO
97/23614 OPG bone density IgG1 N-terminus of anti-obesity PCT/US
97/23183, filed leptin Dec. 11, 1997 Human Ig C.gamma.1 CTLA-4
autoimmune disorders Linsley, 1991, J. Exp. Med., 174: 561-69
[0131] In one example, a human IgG hinge, CH2, and CH3 region may
be fused at either the amino-terminus or carboxyl-terminus of the
TSLPR polypeptides using methods known to the skilled artisan. In
another example, a human IgG hinge, CH2, and CH3 region may be
fused at either the amino-terminus or carboxyl-terminus of a TSLPR
polypeptide fragment (e.g., the predicted extracellular portion of
TSLPR polypeptide).
[0132] The resulting TSLPR fusion polypeptide may be purified by
use of a Protein A affinity column. Peptides and proteins fused to
an Fc region have been found to exhibit a substantially greater
half-life in vivo than the unfused counterpart. Also, a fusion to
an Fc region allows for dimerization/multimerization of the fusion
polypeptide. The Fc region may be a naturally occurring Fc region,
or may be altered to improve certain qualities, such as therapeutic
qualities, circulation time, or reduced aggregation.
[0133] Identity and similarity of related nucleic acid molecules
and polypeptides are readily calculated by known methods. Such
methods include, but are not limited to those described in
Computational Molecular Biology (A. M. Lesk, ed., Oxford University
Press 1988); Biocomputing: Informatics and Genome Projects (D. W.
Smith, ed., Academic Press 1993); Computer Analysis of Sequence
Data (Part 1, A. M. Griffin and H. G. Griffin, eds., Humana Press
1994); G. von Heinle, Sequence Analysis in Molecular Biology
(Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J.
Devereux, eds., M. Stockton Press 1991); and Carillo et al., 1988,
SIAM J. Applied Math., 48: 1073.
[0134] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., 1984, Nucleic Acids Res. 12: 387; Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215:
403-10). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, Md.);
Altschul et al., 1990, supra). The well-known Smith Waterman
algorithm may also be used to determine identity.
[0135] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least 50 contiguous amino acids of
the claimed polypeptide.
[0136] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span," as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
0.1.times. the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix is also used by the
algorithm (see Dayhoff et al., 5 Atlas of Protein Sequence and
Structure (Supp. 3 1978)(PAM250 comparison matrix); Henikoff et
al., 1992, Proc. Natl. Acad. Sci USA 89: 10915-19 (BLOSUM 62
comparison matrix)).
[0137] Preferred parameters for polypeptide sequence comparison
include the following:
[0138] Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol. 48:
443-53;
[0139] Comparison matrix: BLOSUM 62 (Henikoff et al., supra);
[0140] Gap Penalty: 12
[0141] Gap Length Penalty: 4
[0142] Threshold of Similarity: 0
[0143] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0144] Preferred parameters for nucleic acid molecule sequence
comparison include the following:
[0145] Algorithm: Needleman and Wunsch, supra;
[0146] Comparison matrix: matches=+10, mismatch=0
[0147] Gap Penalty: 50
[0148] Gap Length Penalty: 3
[0149] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0150] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, and thresholds of
similarity may be used, including those set forth in the Program
Manual, Wisconsin Package, Version 9, September, 1997. The
particular choices to be made will be apparent to those of skill in
the art and will depend on the specific comparison to be made, such
as DNA-to-DNA, protein-to-protein, protein-to-DNA; and
additionally, whether the comparison is between given pairs of
sequences (in which case GAP or BestFit are generally preferred) or
between one sequence and a large database of sequences (in which
case FASTA or BLASTA are preferred).
[0151] Nucleic Acid Molecules
[0152] The nucleic acid molecules encoding a polypeptide comprising
the amino acid sequence of a TSLPR polypeptide can readily be
obtained in a variety of ways including, without limitation,
chemical synthesis, cDNA or genomic library screening, expression
library screening, and/or PCR amplification of cDNA.
[0153] Recombinant DNA methods used herein are generally those set
forth in Sambrook et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 1989) and/or Current
Protocols in Molecular Biology (Ausubel et al., eds., Green
Publishers Inc. and Wiley and Sons 1994). The invention provides
for nucleic acid molecules as described herein and methods for
obtaining such molecules.
[0154] Where a gene encoding the amino acid sequence of a TSLPR
polypeptide has been identified from one species, all or a portion
of that gene may be used as a probe to identify orthologs or
related genes from the same species. The probes or primers may be
used to screen cDNA libraries from various tissue sources believed
to express the TSLPR polypeptide. In addition, part or all of a
nucleic acid molecule having the sequence as set forth in any of
SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID
NO: 11 may be used to screen a genomic library to identify and
isolate a gene encoding the amino acid sequence of a TSLPR
polypeptide. Typically, conditions of moderate or high stringency
will be employed for screening to minimize the number of false
positives obtained from the screening.
[0155] Nucleic acid molecules encoding the amino acid sequence of
TSLPR polypeptides may also be identified by expression cloning
which employs the detection of positive clones based upon a
property of the expressed protein. Typically, nucleic acid
libraries are screened by the binding an antibody or other binding
partner (e.g., receptor or ligand) to cloned proteins that are
expressed and displayed on a host cell surface. The antibody or
binding partner is modified with a detectable label to identify
those cells expressing the desired clone.
[0156] Recombinant expression techniques conducted in accordance
with the descriptions set forth below may be followed to produce
these polynucleotides and to express the encoded polypeptides. For
example, by inserting a nucleic acid sequence that encodes the
amino acid sequence of a TSLPR polypeptide into an appropriate
vector, one skilled in the art can readily produce large quantities
of the desired nucleotide sequence. The sequences can then be used
to generate detection probes or amplification primers.
Alternatively, a polynucleotide encoding the amino acid sequence of
a TSLPR polypeptide can be inserted into an expression vector. By
introducing the expression vector into an appropriate host, the
encoded TSLPR polypeptide may be produced in large amounts.
[0157] Another method for obtaining a suitable nucleic acid
sequence is the polymerase chain reaction (PCR). In this method,
cDNA is prepared from poly(A)+RNA or total RNA using the enzyme
reverse transcriptase. Two primers, typically complementary to two
separate regions of cDNA encoding the amino acid sequence of a
TSLPR polypeptide, are then added to the cDNA along with a
polymerase such as Taq polymerase, and the polymerase amplifies the
cDNA region between the two primers.
[0158] Another means of preparing a nucleic acid molecule encoding
the amino acid sequence of a TSLPR polypeptide is chemical
synthesis using methods well known to the skilled artisan such as
those described by Engels et al., 1989, Angew. Chem. Intl. Ed. 28:
716-34. These methods include, inter alia, the phosphotriester,
phosphoramidite, and H-phosphonate methods for nucleic acid
synthesis. A preferred method for such chemical synthesis is
polymer-supported synthesis using standard phosphoramidite
chemistry. Typically, the DNA encoding the amino acid sequence of a
TSLPR polypeptide will be several hundred nucleotides in length.
Nucleic acids larger than about 100 nucleotides can be synthesized
as several fragments using these methods. The fragments can then be
ligated together to form the full-length nucleotide sequence of a
TSLPR gene. Usually, the DNA fragment encoding the amino-terminus
of the polypeptide will have an ATG, which encodes a methionine
residue. This methionine may or may not be present on the mature
form of the TSLPR polypeptide, depending on whether the polypeptide
produced in the host cell is designed to be secreted from that
cell. Other methods known to the skilled artisan may be used as
well.
[0159] In certain embodiments, nucleic acid variants contain codons
which have been altered for optimal expression of a TSLPR
polypeptide in a given host cell. Particular codon alterations will
depend upon the TSLPR polypeptide and host cell selected for
expression. Such "codon optimization" can be carried out by a
variety of methods, for example, by selecting codons which are
preferred for use in highly expressed genes in a given host cell.
Computer algorithms which incorporate codon frequency tables such
as "Eco_high.Cod" for codon preference of highly expressed
bacterial genes may be used and are provided by the University of
Wisconsin Package Version 9.0 (Genetics Computer Group, Madison,
Wis.). Other useful codon frequency tables include
"Celegans_high.cod," "Celegans_low.cod," "Drosophila_high.cod,"
"Human_high.cod," "Maize_high.cod," and "Yeast_high.cod."
[0160] In some cases, it may be desirable to prepare nucleic acid
molecules encoding TSLPR polypeptide variants. Nucleic acid
molecules encoding variants may be produced using site directed
mutagenesis, PCR amplification, or other appropriate methods, where
the primer(s) have the desired point mutations (see Sambrook et
al., supra, and Ausubel et al., supra, for descriptions of
mutagenesis techniques). Chemical synthesis using methods described
by Engels et al., supra, may also be used to prepare such
variants.
[0161] Other methods known to the skilled artisan may be used as
well.
[0162] Vectors and Host Cells
[0163] A nucleic acid molecule encoding the amino acid sequence of
a TSLPR polypeptide is inserted into an appropriate expression
vector using standard ligation techniques. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery such
that amplification of the gene and/or expression of the gene can
occur). A nucleic acid molecule encoding the amino acid sequence of
a TSLPR polypeptide may be amplified/expressed in prokaryotic,
yeast, insect (baculovirus systems) and/or eukaryotic host cells.
Selection of the host cell will depend in part on whether a TSLPR
polypeptide is to be post-translationally modified (e.g.,
glycosylated and/or phosphorylated). If so, yeast, insect, or
mammalian host cells are preferable. For a review of expression
vectors, see Meth. Enz., vol. 185 (D. V. Goeddel, ed., Academic
Press 1990).
[0164] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0165] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the TSLPR polypeptide coding sequence; the oligonucleotide
sequence encodes polyHis (such as hexaHis), or another "tag" such
as FLAG, HA (hemaglutinin influenza virus), or myc for which
commercially available antibodies exist. This tag is typically
fused to the polypeptide upon expression of the polypeptide, and
can serve as a means for affinity purification of the TSLPR
polypeptide from the host cell. Affinity purification can be
accomplished, for example, by column chromatography using
antibodies against the tag as an affinity matrix. Optionally, the
tag can subsequently be removed from the purified TSLPR polypeptide
by various means such as using certain peptidases for cleavage.
[0166] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source), or
synthetic, or the flanking sequences may be native sequences which
normally function to regulate TSLPR polypeptide expression. As
such, the source of a flanking sequence may be any prokaryotic or
eukaryotic organism, any vertebrate or invertebrate organism, or
any plant, provided that the flanking sequence is functional in,
and can be activated by, the host cell machinery.
[0167] Flanking sequences useful in the vectors of this invention
may be obtained by any of several methods well known in the art.
Typically, flanking sequences useful herein--other than the TSLPR
gene flanking sequences--will have been previously identified by
mapping and/or by restriction endonuclease digestion and can thus
be isolated from the proper tissue source using the appropriate
restriction endonucleases. In some cases, the full nucleotide
sequence of a flanking sequence may be known. Here, the flanking
sequence may be synthesized using the methods described herein for
nucleic acid synthesis or cloning.
[0168] Where all or only a portion of the flanking sequence is
known, it may be obtained using PCR and/or by screening a genomic
library with a suitable oligonucleotide and/or flanking sequence
fragment from the same or another species. Where the flanking
sequence is not known, a fragment of DNA containing a flanking
sequence may be isolated from a larger piece of DNA that may
contain, for example, a coding sequence or even another gene or
genes. Isolation may be accomplished by restriction endonuclease
digestion to produce the proper DNA fragment followed by isolation
using agarose gel purification, Qiagen.RTM. column chromatography
(Chatsworth, Calif.), or other methods known to the skilled
artisan. The selection of suitable enzymes to accomplish this
purpose will be readily apparent to one of ordinary skill in the
art.
[0169] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell.
Amplification of the vector to a certain copy number can, in some
cases, be important for the optimal expression of a TSLPR
polypeptide. If the vector of choice does not contain an origin of
replication site, one may be chemically synthesized based on a
known sequence, and ligated into the vector. For example, the
origin of replication from the plasmid pBR322 (New England Biolabs,
Beverly, Mass.) is suitable for most gram-negative bacteria and
various origins (e.g., SV40, polyoma, adenovirus, vesicular
stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication component is not needed for mammalian
expression vectors (for example, the SV40 origin is often used only
because it contains the early promoter).
[0170] A transcription termination sequence is typically located 3'
of the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly-T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0171] A selectable marker gene element encodes a protein necessary
for the survival and growth of a host cell grown in a selective
culture medium. Typical selection marker genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, tetracycline, or kanamycin for prokaryotic host cells;
(b) complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex media. Preferred
selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
[0172] Other selection genes may be used to amplify the gene that
will be expressed. Amplification is the process wherein genes that
are in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and thymidine kinase. The mammalian cell
transformants are placed under selection pressure wherein only the
transformants are uniquely adapted to survive by virtue of the
selection gene present in the vector. Selection pressure is imposed
by culturing the transformed cells under conditions in which the
concentration of selection agent in the medium is successively
changed, thereby leading to the amplification of both the selection
gene and the DNA that encodes a TSLPR polypeptide. As a result,
increased quantities of TSLPR polypeptide are synthesized from the
amplified DNA.
[0173] A ribosome binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of a TSLPR polypeptide to be expressed. The
Shine-Dalgamo sequence is varied but is typically a polypurine
(i.e., having a high A-G content). Many Shine-Dalgarno sequences
have been identified, each of which can be readily synthesized
using methods set forth herein and used in a prokaryotic
vector.
[0174] A leader, or signal, sequence may be used to direct a TSLPR
polypeptide out of the host cell. Typically, a nucleotide sequence
encoding the signal sequence is positioned in the coding region of
a TSLPR nucleic acid molecule, or directly at the 5' end of a TSLPR
polypeptide coding region. Many signal sequences have been
identified, and any of those that are functional in the selected
host cell may be used in conjunction with a TSLPR nucleic acid
molecule. Therefore, a signal sequence may be homologous (naturally
occurring) or heterologous to the TSLPR nucleic acid molecule.
Additionally, a signal sequence may be chemically synthesized using
methods described herein. In most cases, the secretion of a TSLPR
polypeptide from the host cell via the presence of a signal peptide
will result in the removal of the signal peptide from the secreted
TSLPR polypeptide. The signal sequence may be a component of the
vector, or it may be a part of a TSLPR nucleic acid molecule that
is inserted into the vector.
[0175] Included within the scope of this invention is the use of
either a nucleotide sequence encoding a native TSLPR polypeptide
signal sequence joined to a TSLPR polypeptide coding region or a
nucleotide sequence encoding a heterologous signal sequence joined
to a TSLPR polypeptide coding region. The heterologous signal
sequence selected should be one that is recognized and processed,
i.e., cleaved by a signal peptidase, by the host cell. For
prokaryotic host cells that do not recognize and process the native
TSLPR polypeptide signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, or
heat-stable enterotoxin II leaders. For yeast secretion, the native
TSLPR polypeptide signal sequence may be substituted by the yeast
invertase, alpha factor, or acid phosphatase leaders. In mammalian
cell expression the native signal sequence is satisfactory,
although other mammalian signal sequences may be suitable.
[0176] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various presequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add pro-sequences, which also may affect
glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein)
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the amino-terminus.
Alternatively, use of some enzyme cleavage sites may result in a
slightly truncated form of the desired TSLPR polypeptide, if the
enzyme cuts at such area within the mature polypeptide.
[0177] In many cases, transcription of a nucleic acid molecule is
increased by the presence of one or more introns in the vector;
this is particularly true where a polypeptide is produced in
eukaryotic host cells, especially mammalian host cells. The introns
used may be naturally occurring within the TSLPR gene especially
where the gene used is a full-length genomic sequence or a fragment
thereof. Where the intron is not naturally occurring within the
gene (as for most cDNAs), the intron may be obtained from another
source. The position of the intron with respect to flanking
sequences and the TSLPR gene is generally important, as the intron
must be transcribed to be effective. Thus, when a TSLPR cDNA
molecule is being transcribed, the preferred position for the
intron is 3' to the transcription start site and 5' to the poly-A
transcription termination sequence.
[0178] Preferably, the intron or introns will be located on one
side or the other (i.e., 5' or 3') of the cDNA such that it does
not interrupt the coding sequence. Any intron from any source,
including viral, prokaryotic and eukaryotic (plant or animal)
organisms, may be used to practice this invention, provided that it
is compatible with the host cell into which it is inserted. Also
included herein are synthetic introns. Optionally, more than one
intron may be used in the vector.
[0179] The expression and cloning vectors of the present invention
will typically contain a promoter that is recognized by the host
organism and operably linked to the molecule encoding the TSLPR
polypeptide. Promoters are untranscribed sequences located upstream
(i.e., 5') to the start codon of a structural gene (generally
within about 100 to 1000 bp) that control the transcription of the
structural gene. Promoters are conventionally grouped into one of
two classes: inducible promoters and constitutive promoters.
Inducible promoters initiate increased levels of transcription from
DNA under their control in response to some change in culture
conditions, such as the presence or absence of a nutrient or a
change in temperature. Constitutive promoters, on the other hand,
initiate continual gene product production; that is, there is
little or no control over gene expression. A large number of
promoters, recognized by a variety of potential host cells, are
well known. A suitable promoter is operably linked to the DNA
encoding TSLPR polypeptide by removing the promoter from the source
DNA by restriction enzyme digestion and inserting the desired
promoter sequence into the vector. The native TSLPR promoter
sequence may be used to direct amplification and/or expression of a
TSLPR nucleic acid molecule. A heterologous promoter is preferred,
however, if it permits greater transcription and higher yields of
the expressed protein as compared to the native promoter, and if it
is compatible with the host cell system that has been selected for
use.
[0180] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase; a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their sequences have been published, thereby
enabling one skilled in the art to ligate them to the desired DNA
sequence, using linkers or adapters as needed to supply any useful
restriction sites.
[0181] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B
virus and most preferably Simian Virus 40 (SV40). Other suitable
mammalian promoters include heterologous mammalian promoters, for
example, heat-shock promoters and the actin promoter.
[0182] Additional promoters which may be of interest in controlling
TSLPR gene expression include, but are not limited to: the SV40
early promoter region (Bemoist and Chambon, 1981, Nature 290:
304-10); the CMV promoter; the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell
22: 787-97); the herpes thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); the regulatory
sequences of the metallothionine gene (Brinster et al., 1982,
Nature 296: 39-42); prokaryotic expression vectors such as the
beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.
Acad. Sci. U.S.A., 75: 3727-31); or the tac promoter (DeBoer et
al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80: 21-25). Also of
interest are the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: the elastase I gene control region which is
active in pancreatic acinar cells (Swift et al., 1984, Cell 38:
639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
50: 399-409 (1986); MacDonald, 1987, Hepatology 7: 425-515); the
insulin gene control region which is active in pancreatic beta
cells (Hanahan, 1985, Nature 315: 115-22); the immunoglobulin gene
control region which is active in lymphoid cells (Grosschedl et
al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature 318:
533-38; Alexander et al., 1987, Mol. Cell. Biol., 7: 1436-44); the
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45: 485-95); the albumin gene control region which is active
in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-76); the
alpha-feto-protein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol., 5: 1639-48; Hammer et
al., 1987, Science 235: 53-58); the alpha 1-antitrypsin gene
control region which is active in the liver (Kelsey et al., 1987,
Genes and Devel. 1: 161-71); the beta-globin gene control region
which is active in myeloid cells (Mogram et al., 1985, Nature 315:
338-40; Kollias et al., 1986, Cell 46: 89-94); the myelin basic
protein gene control region which is active in oligodendrocyte
cells in the brain (Readhead et al., 1987, Cell 48: 703-12); the
myosin light chain-2 gene control region which is active in
skeletal muscle (Sani, 1985, Nature 314: 283-86); and the
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986, Science 234: 1372-78).
[0183] An enhancer sequence may be inserted into the vector to
increase the transcription of a DNA encoding a TSLPR polypeptide of
the present invention by higher eukaryotes. Enhancers are
cis-acting elements of DNA, usually about 10-300 bp in length, that
act on the promoter to increase transcription. Enhancers are
relatively orientation and position independent. They have been
found 5' and 3' to the transcription unit. Several enhancer
sequences available from mammalian genes are known (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin). Typically,
however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and adenovirus enhancers are exemplary enhancing elements for the
activation of eukaryotic promoters. While an enhancer may be
spliced into the vector at a position 5' or 3' to a TSLPR nucleic
acid molecule, it is typically located at a site 5' from the
promoter.
[0184] Expression vectors of the invention may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the flanking sequences described
herein are not already present in the vector, they may be
individually obtained and ligated into the vector. Methods used for
obtaining each of the flanking sequences are well known to one
skilled in the art.
[0185] Preferred vectors for practicing this invention are those
which are compatible with bacterial, insect, and mammalian host
cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1
(Invitrogen, San Diego, Calif.), pBSII (Stratagene, La Jolla,
Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,
Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL
(BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO 90/14363) and
pFastBacDual (Gibco-BRL, Grand Island, N.Y.).
[0186] Additional suitable vectors include, but are not limited to,
cosmids, plasmids, or modified viruses, but it will be appreciated
that the vector system must be compatible with the selected host
cell. Such vectors include, but are not limited to plasmids such as
Bluescript.RTM. plasmid derivatives (a high copy number ColE1-based
phagemid; Stratagene Cloning Systems, La Jolla Calif.), PCR cloning
plasmids designed for cloning Taq-amplified PCR products (e.g.,
TOPO.TM. TA Cloning.RTM. Kit and PCR2.1.RTM. plasmid derivatives;
Invitrogen), and mammalian, yeast or virus vectors such as a
baculovirus expression system (pBacPAK plasmid derivatives;
Clontech).
[0187] After the vector has been constructed and a nucleic acid
molecule encoding a TSLPR polypeptide has been inserted into the
proper site of the vector, the completed vector may be inserted
into a suitable host cell for amplification and/or polypeptide
expression. The transformation of an expression vector for a TSLPR
polypeptide into a selected host cell may be accomplished by well
known methods including methods such as transfection, infection,
calcium chloride, electroporation, microinjection, lipofection,
DEAE-dextran method, or other known techniques. The method selected
will in part be a function of the type of host cell to be used.
These methods and other suitable methods are well known to the
skilled artisan, and are set forth, for example, in Sambrook et
al., supra.
[0188] Host cells may be prokaryotic host cells (such as E. coli)
or eukaryotic host cells (such as a yeast, insect, or vertebrate
cell). The host cell, when cultured under appropriate conditions,
synthesizes a TSLPR polypeptide which can subsequently be collected
from the culture medium (if the host cell secretes it into the
medium) or directly from the host cell producing it (if it is not
secreted). The selection of an appropriate host cell will depend
upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
[0189] A number of suitable host cells are known in the art and
many are available from the American Type Culture Collection
(ATCC), Manassas, Va. Examples include, but are not limited to,
mammalian cells, such as Chinese hamster ovary cells (CHO), CHO
DHFR(-) cells (Urlaub et al., 1980, Proc. Natl. Acad. Sci. U.S.A.
97: 4216-20), human embryonic kidney (HEK) 293 or 293T cells, or
3T3 cells. The selection of suitable mammalian host cells and
methods for transformation, culture, amplification, screening,
product production, and purification are known in the art. Other
suitable mammalian cell lines, are the monkey COS-1 and COS-7 cell
lines, and the CV-1 cell line. Further exemplary mammalian host
cells include primate cell lines and rodent cell lines, including
transformed cell lines. Normal diploid cells, cell strains derived
from in vitro culture of primary tissue, as well as primary
explants, are also suitable. Candidate cells may be genotypically
deficient in the selection gene, or may contain a dominantly acting
selection gene. Other suitable mammalian cell lines include but are
not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929
cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK
hamster cell lines. Each of these cell lines is known by and
available to those skilled in the art of protein expression.
[0190] Similarly useful as host cells suitable for the present
invention are bacterial cells.
[0191] For example, the various strains of E. coli (e.g., HB101,
DH5.alpha., DH10, and MC1061) are well-known as host cells in the
field of biotechnology. Various strains of B. subtilis, Pseudomonas
spp., other Bacillus spp., Streptomyces spp., and the like may also
be employed in this method.
[0192] Many strains of yeast cells known to those skilled in the
art are also available as host cells for the expression of the
polypeptides of the present invention. Preferred yeast cells
include, for example, Saccharomyces cerivisae and Pichia
pastoris.
[0193] Additionally, where desired, insect cell systems may be
utilized in the methods of the present invention. Such systems are
described, for example, in Kitts et al., 1993, Biotechniques, 14:
810-17; Lucklow, 1993, Curr. Opin. Biotechnol. 4: 564-72; and
Lucklow et al., 1993, J. Virol., 67: 4566-79. Preferred insect
cells are Sf-9 and Hi5 (Invitrogen).
[0194] One may also use transgenic animals to express glycosylated
TSLPR polypeptides. For example, one may use a transgenic
milk-producing animal (a cow or goat, for example) and obtain the
present glycosylated polypeptide in the animal milk.
[0195] One may also use plants to produce TSLPR polypeptides,
however, in general, the glycosylation occurring in plants is
different from that produced in mammalian cells, and may result in
a glycosylated product which is not suitable for human therapeutic
use.
[0196] Polypeptide Production
[0197] Host cells comprising a TSLPR polypeptide expression vector
may be cultured using standard media well known to the skilled
artisan. The media will usually contain all nutrients necessary for
the growth and survival of the cells. Suitable media for culturing
E. coli cells include, for example, Luria Broth (LB) and/or
Terrific Broth (TB). Suitable media for culturing eukaryotic cells
include Roswell Park Memorial Institute medium 1640 (RPMI 1640),
Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle
Medium (DMEM), all of which may be supplemented with serum and/or
growth factors as necessary for the particular cell line being
cultured. A suitable medium for insect cultures is Grace's medium
supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal
calf serum as necessary.
[0198] Typically, an antibiotic or other compound useful for
selective growth of transfected or transformed cells is added as a
supplement to the media. The compound to be used will be dictated
by the selectable marker element present on the plasmid with which
the host cell was transformed. For example, where the selectable
marker element is kanamycin resistance, the compound added to the
culture medium will be kanamycin. Other compounds for selective
growth include ampicillin, tetracycline, and neomycin.
[0199] The amount of a TSLPR polypeptide produced by a host cell
can be evaluated using standard methods known in the art. Such
methods include, without limitation, Western blot analysis,
SDS-polyacrylamide gel electrophoresis, non-denaturing gel
electrophoresis, High Performance Liquid Chromatography (HPLC)
separation, immunoprecipitation, and/or activity assays such as DNA
binding gel shift assays.
[0200] If a TSLPR polypeptide has been designed to be secreted from
the host cells, the majority of polypeptide may be found in the
cell culture medium. If however, the TSLPR polypeptide is not
secreted from the host cells, it will be present in the cytoplasm
and/or the nucleus (for eukaryotic host cells) or in the cytosol
(for gram-negative bacteria host cells).
[0201] For a TSLPR polypeptide situated in the host cell cytoplasm
and/or nucleus (for eukaryotic host cells) or in the cytosol (for
bacterial host cells), the intracellular material (including
inclusion bodies for gram-negative bacteria) can be extracted from
the host cell using any standard technique known to the skilled
artisan. For example, the host cells can be lysed to release the
contents of the periplasm/cytoplasm by French press,
homogenization, and/or sonication followed by centrifugation.
[0202] If a TSLPR polypeptide has formed inclusion bodies in the
cytosol, the inclusion bodies can often bind to the inner and/or
outer cellular membranes and thus will be found primarily in the
pellet material after centrifugation. The pellet material can then
be treated at pH extremes or with a chaotropic agent such as a
detergent, guanidine, guanidine derivatives, urea, or urea
derivatives in the presence of a reducing agent such as
dithiothreitol at alkaline pH or tris carboxyethyl phosphine at
acid pH to release, break apart, and solubilize the inclusion
bodies. The solubilized TSLPR polypeptide can then be analyzed
using gel electrophoresis, immunoprecipitation, or the like. If it
is desired to isolate the TSLPR polypeptide, isolation may be
accomplished using standard methods such as those described herein
and in Marston et al., 1990, Meth. Enz., 182: 264-75.
[0203] In some cases, a TSLPR polypeptide may not be biologically
active upon isolation. Various methods for "refolding" or
converting the polypeptide to its tertiary structure and generating
disulfide linkages can be used to restore biological activity.
[0204] Such methods include exposing the solubilized polypeptide to
a pH usually above 7 and in the presence of a particular
concentration of a chaotrope. The selection of chaotrope is very
similar to the choices used for inclusion body solubilization, but
usually the chaotrope is used at a lower concentration and is not
necessarily the same as chaotropes used for the solubilization. In
most cases the refolding/oxidation solution will also contain a
reducing agent or the reducing agent plus its oxidized form in a
specific ratio to generate a particular redox potential allowing
for disulfide shuffling to occur in the formation of the protein's
cysteine bridges. Some of the commonly used redox couples include
cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric
chloride, dithiothreitol(DTT)/dithiane DTT, and
2-2-mercaptoethanol(bME)/- dithio-b(ME). In many instances, a
cosolvent may be used or may be needed to increase the efficiency
of the refolding, and the more common reagents used for this
purpose include glycerol, polyethylene glycol of various molecular
weights, arginine and the like.
[0205] If inclusion bodies are not formed to a significant degree
upon expression of a TSLPR polypeptide, then the polypeptide will
be found primarily in the supernatant after centrifugation of the
cell homogenate. The polypeptide may be further isolated from the
supernatant using methods such as those described herein.
[0206] The purification of a TSLPR polypeptide from solution can be
accomplished using a variety of techniques. If the polypeptide has
been synthesized such that it contains a tag such as Hexahistidine
(TSLPR polypeptide/hexaHis) or other small peptide such as FLAG
(Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at either
its carboxyl- or amino-terminus, it may be purified in a one-step
process by passing the solution through an affinity column where
the column matrix has a high affinity for the tag.
[0207] For example, polyhistidine binds with great affinity and
specificity to nickel. Thus, an affinity column of nickel (such as
the Qiagen.RTM. nickel columns) can be used for purification of
TSLPR polypeptide/polyHis. See, e.g., Current Protocols in
Molecular Biology .sctn. 10.11.8 (Ausubel et al., eds., Green
Publishers Inc. and Wiley and Sons 1993).
[0208] Additionally, TSLPR polypeptides may be purified through the
use of a monoclonal antibody that is capable of specifically
recognizing and binding to a TSLPR polypeptide.
[0209] Other suitable procedures for purification include, without
limitation, affinity chromatography, immunoaffinity chromatography,
ion exchange chromatography, molecular sieve chromatography, HPLC,
electrophoresis (including native gel electrophoresis) followed by
gel elution, and preparative isoelectric focusing ("Isoprime"
machine/technique, Hoefer Scientific, San Francisco, Calif.). In
some cases, two or more purification techniques may be combined to
achieve increased purity.
[0210] TSLPR polypeptides may also be prepared by chemical
synthesis methods (such as solid phase peptide synthesis) using
techniques known in the art such as those set forth by Merrifield
et al., 1963, J. Am. Chem. Soc. 85: 2149; Houghten et al., 1985,
Proc Natl Acad. Sci. USA 82: 5132; and Stewart and Young, Solid
Phase Peptide Synthesis (Pierce Chemical Co. 1984). Such
polypeptides may be synthesized with or without a methionine on the
amino-terminus. Chemically synthesized TSLPR polypeptides may be
oxidized using methods set forth in these references to form
disulfide bridges. Chemically synthesized TSLPR polypeptides are
expected to have comparable biological activity to the
corresponding TSLPR polypeptides produced recombinantly or purified
from natural sources, and thus may be used interchangeably with a
recombinant or natural TSLPR polypeptide.
[0211] Another means of obtaining TSLPR polypeptide is via
purification from biological samples such as source tissues and/or
fluids in which the TSLPR polypeptide is naturally found. Such
purification can be conducted using methods for protein
purification as described herein. The presence of the TSLPR
polypeptide during purification may be monitored, for example,
using an antibody prepared against recombinantly produced TSLPR
polypeptide or peptide fragments thereof.
[0212] A number of additional methods for producing nucleic acids
and polypeptides are known in the art, and the methods can be used
to produce polypeptides having specificity for TSLPR polypeptide.
See, e.g., Roberts et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:
12297-303, which describes the production of fusion proteins
between an mRNA and its encoded peptide. See also, Roberts, 1999,
Curr. Opin. Chem. Biol. 3: 268-73.
[0213] Additionally, U.S. Pat. No. 5,824,469 describes methods for
obtaining oligonucleotides capable of carrying out a specific
biological function. The procedure involves generating a
heterogeneous pool of oligonucleotides, each having a 5' randomized
sequence, a central preselected sequence, and a 3' randomized
sequence. The resulting heterogeneous pool is introduced into a
population of cells that do not exhibit the desired biological
function. Subpopulations of the cells are then screened for those
that exhibit a predetermined biological function. From that
subpopulation, oligonucleotides capable of carrying out the desired
biological function are isolated.
[0214] U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and
5,817,483 describe processes for producing peptides or
polypeptides. This is done by producing stochastic genes or
fragments thereof, and then introducing these genes into host cells
which produce one or more proteins encoded by the stochastic genes.
The host cells are then screened to identify those clones producing
peptides or polypeptides having the desired activity.
[0215] Another method for producing peptides or polypeptides is
described in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc.
Known as "Random Activation of Gene Expression for Gene Discovery"
(RAGE-GD), the process involves the activation of endogenous gene
expression or over-expression of a gene by in situ recombination
methods. For example, expression of an endogenous gene is activated
or increased by integrating a regulatory sequence into the target
cell which is capable of activating expression of the gene by
non-homologous or illegitimate recombination. The target DNA is
first subjected to radiation, and a genetic promoter inserted. The
promoter eventually locates a break at the front of a gene,
initiating transcription of the gene. This results in expression of
the desired peptide or polypeptide.
[0216] It will be appreciated that these methods can also be used
to create comprehensive TSLPR polypeptide expression libraries,
which can subsequently be used for high throughput phenotypic
screening in a variety of assays, such as biochemical assays,
cellular assays, and whole organism assays (e.g., plant, mouse,
etc.).
[0217] Synthesis
[0218] It will be appreciated by those skilled in the art that the
nucleic acid and polypeptide molecules described herein may be
produced by recombinant and other means.
[0219] Selective Binding Agents
[0220] The term "selective binding agent" refers to a molecule that
has specificity for one or more TSLPR polypeptides. Suitable
selective binding agents include, but are not limited to,
antibodies and derivatives thereof, polypeptides, and small
molecules. Suitable selective binding agents may be prepared using
methods known in the art. An exemplary TSLPR polypeptide selective
binding agent of the present invention is capable of binding a
certain portion of the TSLPR polypeptide thereby inhibiting the
binding of the polypeptide to a TSLPR polypeptide receptor.
[0221] Selective binding agents such as antibodies and antibody
fragments that bind TSLPR polypeptides are within the scope of the
present invention. The antibodies may be polyclonal including
monospecific polyclonal; monoclonal (MAbs); recombinant; chimeric;
humanized, such as complementarity-determining region
(CDR)-grafted; human; single chain; and/or bispecific; as well as
fragments; variants; or derivatives thereof. Antibody fragments
include those portions of the antibody that bind to an epitope on
the TSLPR polypeptide. Examples of such fragments include Fab and
F(ab') fragments generated by enzymatic cleavage of full-length
antibodies. Other binding fragments include those generated by
recombinant DNA techniques, such as the expression of recombinant
plasmids containing nucleic acid sequences encoding antibody
variable regions.
[0222] Polyclonal antibodies directed toward a TSLPR polypeptide
generally are produced in animals (e.g., rabbits or mice) by means
of multiple subcutaneous or intraperitoneal injections of TSLPR
polypeptide and an adjuvant. It may be useful to conjugate a TSLPR
polypeptide to a carrier protein that is immunogenic in the species
to be immunized, such as keyhole limpet hemocyanin, serum, albumin,
bovine thyroglobulin, or soybean trypsin inhibitor. Also,
aggregating agents such as alum are used to enhance the immune
response. After immunization, the animals are bled and the serum is
assayed for anti-TSLPR antibody titer.
[0223] Monoclonal antibodies directed toward TSLPR polypeptides are
produced using any method that provides for the production of
antibody molecules by continuous cell lines in culture. Examples of
suitable methods for preparing monoclonal antibodies include the
hybridoma methods of Kohler et al., 1975, Nature 256: 495-97 and
the human B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:
3001; Brodeur et al., Monoclonal Antibody Production Techniques and
Applications 51-63 (Marcel Dekker, Inc., 1987). Also provided by
the invention are hybridoma cell lines that produce monoclonal
antibodies reactive with TSLPR polypeptides.
[0224] Monoclonal antibodies of the invention may be modified for
use as therapeutics. One embodiment is a "chimeric" antibody in
which a portion of the heavy (H) and/or light (L) chain is
identical with or homologous to a corresponding sequence in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with or homologous to a corresponding
sequence in antibodies derived from another species or belonging to
another antibody class or subclass. Also included are fragments of
such antibodies, so long as they exhibit the desired biological
activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc.
Natl. Acad. Sci. 81: 6851-55.
[0225] In another embodiment, a monoclonal antibody of the
invention is a "humanized" antibody. Methods for humanizing
non-human antibodies are well known in the art. See U.S. Pat. Nos.
5,585,089 and 5,693,762. Generally, a humanized antibody has one or
more amino acid residues introduced into it from a source that is
non-human. Humanization can be performed, for example, using
methods described in the art (Jones et al., 1986, Nature 321:
522-25; Riechmann et al., 1998, Nature 332: 323-27; Verhoeyen et
al., 1988, Science 239: 1534-36), by substituting at least a
portion of a rodent complementarity-determining region for the
corresponding regions of a human antibody.
[0226] Also encompassed by the invention are human antibodies that
bind TSLPR polypeptides. Using transgenic animals (e.g., mice) that
are capable of producing a repertoire of human antibodies in the
absence of endogenous immunoglobulin production such antibodies are
produced by immunization with a TSLPR polypeptide antigen (i.e.,
having at least 6 contiguous amino acids), optionally conjugated to
a carrier. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad.
Sci. 90: 2551-55; Jakobovits et al., 1993, Nature 362: 255-58;
Bruggermann et al., 1993, Year in Immuno. 7: 33. In one method,
such transgenic animals are produced by incapacitating the
endogenous loci encoding the heavy and light immunoglobulin chains
therein, and inserting loci encoding human heavy and light chain
proteins into the genome thereof. Partially modified animals, that
is those having less than the full complement of modifications, are
then cross-bred to obtain an animal having all of the desired
immune system modifications. When administered an immunogen, these
transgenic animals produce antibodies with human (rather than,
e.g., murine) amino acid sequences, including variable regions
which are immunospecific for these antigens. See PCT App. Nos.
PCT/US96/05928 and PCT/US93/06926. Additional methods are described
in U.S. Pat. No. 5,545,807, PCT App. Nos. PCT/US91/245 and
PCT/GB89/01207, and in European Patent Nos. 546073B1 and 546073A1.
Human antibodies can also be produced by the expression of
recombinant DNA in host cells or by expression in hybridoma cells
as described herein.
[0227] In an alternative embodiment, human antibodies can also be
produced from phage-display libraries (Hoogenboom et al., 1991, J.
Mol. Biol. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581).
These processes mimic immune selection through the display of
antibody repertoires on the surface of filamentous bacteriophage,
and subsequent selection of phage by their binding to an antigen of
choice. One such technique is described in PCT App. No.
PCT/US98/17364, which describes the isolation of high affinity and
functional agonistic antibodies for MPL- and msk-receptors using
such an approach.
[0228] Chimeric, CDR grafted, and humanized antibodies are
typically produced by recombinant methods. Nucleic acids encoding
the antibodies are introduced into host cells and expressed using
materials and procedures described herein. In a preferred
embodiment, the antibodies are produced in mammalian host cells,
such as CHO cells. Monoclonal (e.g., human) antibodies may be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells as described herein.
[0229] The anti-TSLPR antibodies of the invention may be employed
in any known assay method, such as competitive binding assays,
direct and indirect sandwich assays, and immunoprecipitation assays
(Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC
Press, Inc., 1987)) for the detection and quantitation of TSLPR
polypeptides. The antibodies will bind TSLPR polypeptides with an
affinity that is appropriate for the assay method being
employed.
[0230] For diagnostic applications, in certain embodiments,
anti-TSLPR antibodies may be labeled with a detectable moiety. The
detectable moiety can be any one that is capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, .sup.35S, .sup.125I, .sup.99Tc, .sup.111In, or
.sup.67Ga; a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme,
such as alkaline phosphatase, .beta.-galactosidase, or horseradish
peroxidase (Bayer, et al., 1990, Meth. Enz. 184: 138-63).
[0231] Competitive binding assays rely on the ability of a labeled
standard (e.g., a TSLPR polypeptide, or an immunologically reactive
portion thereof) to compete with the test sample analyte (an TSLPR
polypeptide) for binding with a limited amount of anti-TSLPR
antibody. The amount of a TSLPR polypeptide in the test sample is
inversely proportional to the amount of standard that becomes bound
to the antibodies. To facilitate determining the amount of standard
that becomes bound, the antibodies typically are insolubilized
before or after the competition, so that the standard and analyte
that are bound to the antibodies may conveniently be separated from
the standard and analyte which remain unbound.
[0232] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody which is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety
(direct sandwich assays) or may be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0233] The selective binding agents, including anti-TSLPR
antibodies, are also useful for in vivo imaging. An antibody
labeled with a detectable moiety may be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host assayed. The antibody may be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0234] Selective binding agents of the invention, including
antibodies, may be used as therapeutics. These therapeutic agents
are generally agonists or antagonists, in that they either enhance
or reduce, respectively, at least one of the biological activities
of a TSLPR polypeptide. In one embodiment, antagonist antibodies of
the invention are antibodies or binding fragments thereof which are
capable of specifically binding to a TSLPR polypeptide and which
are capable of inhibiting or eliminating the functional activity of
a TSLPR polypeptide in vivo or in vitro. In preferred embodiments,
the selective binding agent, e.g., an antagonist antibody, will
inhibit the functional activity of a TSLPR polypeptide by at least
about 50%, and preferably by at least about 80%. In another
embodiment, the selective binding agent may be an anti-TSLPR
polypeptide antibody that is capable of interacting with a TSLPR
polypeptide binding partner (a ligand or receptor) thereby
inhibiting or eliminating TSLPR polypeptide activity in vitro or in
vivo.
[0235] Selective binding agents, including agonist and antagonist
anti-TSLPR polypeptide antibodies, are identified by screening
assays that are well known in the art.
[0236] The invention also relates to a kit comprising TSLPR
selective binding agents (such as antibodies) and other reagents
useful for detecting TSLPR polypeptide levels in biological
samples. Such reagents may include a detectable label, blocking
serum, positive and negative control samples, and detection
reagents.
[0237] Microarrays
[0238] It will be appreciated that DNA microarray technology can be
utilized in accordance with the present invention. DNA microarrays
are miniature, high-density arrays of nucleic acids positioned on a
solid support, such as glass. Each cell or element within the array
contains numerous copies of a single nucleic acid species that acts
as a target for hybridization with a complementary nucleic acid
sequence (e.g., mRNA). In expression profiling using DNA microarray
technology, mRNA is first extracted from a cell or tissue sample
and then converted enzymatically to fluorescently labeled cDNA.
This material is hybridized to the microarray and unbound cDNA is
removed by washing. The expression of discrete genes represented on
the array is then visualized by quantitating the amount of labeled
cDNA that is specifically bound to each target nucleic acid
molecule. In this way, the expression of thousands of genes can be
quantitated in a high throughput, parallel manner from a single
sample of biological material.
[0239] This high throughput expression profiling has a broad range
of applications with respect to the TSLPR molecules of the
invention, including, but not limited to: the identification and
validation of TSLPR disease-related genes as targets for
therapeutics; molecular toxicology of related TSLPR molecules and
inhibitors thereof; stratification of populations and generation of
surrogate markers for clinical trials; and enhancing related TSLPR
polypeptide small molecule drug discovery by aiding in the
identification of selective compounds in high throughput
screens.
[0240] Chemical Derivatives
[0241] Chemically modified derivatives of TSLPR polypeptides may be
prepared by one skilled in the art, given the disclosures described
herein. TSLPR polypeptide derivatives are modified in a manner that
is different--either in the type or location of the molecules
naturally attached to the polypeptide. Derivatives may include
molecules formed by the deletion of one or more naturally-attached
chemical groups. The polypeptide comprising the amino acid sequence
of any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or other
TSLPR polypeptide, may be modified by the covalent attachment of
one or more polymers. For example, the polymer selected is
typically water-soluble so that the protein to which it is attached
does not precipitate in an aqueous environment, such as a
physiological environment. Included within the scope of suitable
polymers is a mixture of polymers. Preferably, for therapeutic use
of the end-product preparation, the polymer will be
pharmaceutically acceptable.
[0242] The polymers each may be of any molecular weight and may be
branched or unbranched. The polymers each typically have an average
molecular weight of between about 2 kDa to about 100 kDa (the term
"about" indicating that in preparations of a water-soluble polymer,
some molecules will weigh more, some less, than the stated
molecular weight). The average molecular weight of each polymer is
preferably between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between
about 20 kDa and about 35 kDa.
[0243] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C.sub.1-C.sub.10), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran of, for example, about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.,
glycerol), and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules which may be used
to prepare covalently attached TSLPR polypeptide multimers.
[0244] In general, chemical derivatization may be performed under
any suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemical derivatives of
polypeptides will generally comprise the steps of: (a) reacting the
polypeptide with the activated polymer molecule (such as a reactive
ester or aldehyde derivative of the polymer molecule) under
conditions whereby the polypeptide comprising the amino acid
sequence of any of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or
other TSLPR polypeptide, becomes attached to one or more polymer
molecules, and (b) obtaining the reaction products. The optimal
reaction conditions will be determined based on known parameters
and the desired result. For example, the larger the ratio of
polymer molecules to protein, the greater the percentage of
attached polymer molecule. In one embodiment, the TSLPR polypeptide
derivative may have a single polymer molecule moiety at the
amino-terminus. See, e.g., U.S. Pat. No. 5,234,784.
[0245] The pegylation of a polypeptide may be specifically carried
out using any of the pegylation reactions known in the art. Such
reactions are described, for example, in the following references:
Francis et al., 1992, Focus on Growth Factors 3: 4-10; European
Patent Nos. 0154316 and 0401384; and U.S. Pat. No. 4,179,337. For
example, pegylation may be carried out via an acylation reaction or
an alkylation reaction with a reactive polyethylene glycol molecule
(or an analogous reactive water-soluble polymer) as described
herein. For the acylation reactions, a selected polymer should have
a single reactive ester group. For reductive alkylation, a selected
polymer should have a single reactive aldehyde group. A reactive
aldehyde is, for example, polyethylene glycol propionaldehyde,
which is water stable, or mono C.sub.1-C.sub.10 alkoxy or aryloxy
derivatives thereof (see U.S. Pat. No. 5,252,714).
[0246] In another embodiment, TSLPR polypeptides may be chemically
coupled to biotin. The biotin/TSLPR polypeptide molecules are then
allowed to bind to avidin, resulting in tetravalent
avidin/biotin/TSLPR polypeptide molecules. TSLPR polypeptides may
also be covalently coupled to dinitrophenol (DNP) or trinitrophenol
(TNP) and the resulting conjugates precipitated with anti-DNP or
anti-TNP-IgM to form decameric conjugates with a valency of 10.
[0247] Generally, conditions that may be alleviated or modulated by
the administration of the present TSLPR polypeptide derivatives
include those described herein for TSLPR polypeptides. However, the
TSLPR polypeptide derivatives disclosed herein may have additional
activities, enhanced or reduced biological activity, or other
characteristics, such as increased or decreased half-life, as
compared to the non-derivatized molecules.
[0248] Genetically Engineered Non-Human Animals
[0249] Additionally included within the scope of the present
invention are non-human animals such as mice, rats, or other
rodents; rabbits, goats, sheep, or other farm animals, in which the
genes encoding native TSLPR polypeptide have been disrupted (i.e.,
"knocked out") such that the level of expression of TSLPR
polypeptide is significantly decreased or completely abolished.
Such animals may be prepared using techniques and methods such as
those described in U.S. Pat. No. 5,557,032.
[0250] The present invention further includes non-human animals
such as mice, rats, or other rodents; rabbits, goats, sheep, or
other farm animals, in which either the native form of a TSLPR gene
for that animal or a heterologous TSLPR gene is over-expressed by
the animal, thereby creating a "transgenic" animal. Such transgenic
animals may be prepared using well known methods such as those
described in U.S. Pat. No. 5,489,743 and PCT Pub. No. WO
94/28122.
[0251] The present invention further includes non-human animals in
which the promoter for one or more of the TSLPR polypeptides of the
present invention is either activated or inactivated (e.g., by
using homologous recombination methods) to alter the level of
expression of one or more of the native TSLPR polypeptides.
[0252] These non-human animals may be used for drug candidate
screening. In such screening, the impact of a drug candidate on the
animal may be measured. For example, drug candidates may decrease
or increase the expression of the TSLPR gene. In certain
embodiments, the amount of TSLPR polypeptide that is produced may
be measured after the exposure of the animal to the drug candidate.
Additionally, in certain embodiments, one may detect the actual
impact of the drug candidate on the animal. For example,
over-expression of a particular gene may result in, or be
associated with, a disease or pathological condition. In such
cases, one may test a drug candidate's ability to decrease
expression of the gene or its ability to prevent or inhibit a
pathological condition. In other examples, the production of a
particular metabolic product such as a fragment of a polypeptide,
may result in, or be associated with, a disease or pathological
condition. In such cases, one may test a drug candidate's ability
to decrease the production of such a metabolic product or its
ability to prevent or inhibit a pathological condition.
[0253] Assaying for Other Modulators of TSLPR Polypeptide
Activity
[0254] In some situations, it may be desirable to identify
molecules that are modulators, i.e., agonists or antagonists, of
the activity of TSLPR polypeptide. Natural or synthetic molecules
that modulate TSLPR polypeptide may be identified using one or more
screening assays, such as those described herein. Such molecules
may be administered either in an ex vivo manner or in an in vivo
manner by injection, or by oral delivery, implantation device, or
the like.
[0255] "Test molecule" refers to a molecule that is under
evaluation for the ability to modulate (i.e., increase or decrease)
the activity of a TSLPR polypeptide. Most commonly, a test molecule
will interact directly with a TSLPR polypeptide. However, it is
also contemplated that a test molecule may also modulate TSLPR
polypeptide activity indirectly, such as by affecting TSLPR gene
expression, or by binding to a TSLPR polypeptide binding partner
(e.g., receptor or ligand). In one embodiment, a test molecule will
bind to a TSLPR polypeptide with an affinity constant of at least
about 10.sup.-6 M, preferably about 10.sup.-8 M, more preferably
about 10.sup.-9 M, and even more preferably about 10.sup.-10 M.
[0256] Methods for identifying compounds that interact with TSLPR
polypeptides are encompassed by the present invention. In certain
embodiments, a TSLPR polypeptide is incubated with a test molecule
under conditions that permit the interaction of the test molecule
with a TSLPR polypeptide, and the extent of the interaction is
measured. The test molecule can be screened in a substantially
purified form or in a crude mixture.
[0257] In certain embodiments, a TSLPR polypeptide agonist or
antagonist may be a protein, peptide, carbohydrate, lipid, or small
molecular weight molecule that interacts with TSLPR polypeptide to
regulate its activity. Molecules which regulate TSLPR polypeptide
expression include nucleic acids which are complementary to nucleic
acids encoding a TSLPR polypeptide, or are complementary to nucleic
acids sequences which direct or control the expression of TSLPR
polypeptide, and which act as anti-sense regulators of
expression.
[0258] Once a test molecule has been identified as interacting with
a TSLPR polypeptide, the molecule may be further evaluated for its
ability to increase or decrease TSLPR polypeptide activity. The
measurement of the interaction of a test molecule with TSLPR
polypeptide may be carried out in several formats, including
cell-based binding assays, membrane binding assays, solution-phase
assays, and immunoassays. In general, a test molecule is incubated
with a TSLPR polypeptide for a specified period of time, and TSLPR
polypeptide activity is determined by one or more assays for
measuring biological activity.
[0259] The interaction of test molecules with TSLPR polypeptides
may also be assayed directly using polyclonal or monoclonal
antibodies in an immunoassay. Alternatively, modified forms of
TSLPR polypeptides containing epitope tags as described herein may
be used in solution and immunoassays.
[0260] In the event that TSLPR polypeptides display biological
activity through an interaction with a binding partner (e.g., a
receptor or a ligand), a variety of in vitro assays may be used to
measure the binding of a TSLPR polypeptide to the corresponding
binding partner (such as a selective binding agent, receptor, or
ligand). These assays may be used to screen test molecules for
their ability to increase or decrease the rate and/or the extent of
binding of a TSLPR polypeptide to its binding partner. In one
assay, a TSLPR polypeptide is immobilized in the wells of a
microtiter plate. Radiolabeled TSLPR polypeptide binding partner
(for example, iodinated TSLPR polypeptide binding partner) and a
test molecule can then be added either one at a time (in either
order) or simultaneously to the wells. After incubation, the wells
can be washed and counted for radioactivity, using a scintillation
counter, to determine the extent to which the binding partner bound
to the TSLPR polypeptide. Typically, a molecule will be tested over
a range of concentrations, and a series of control wells lacking
one or more elements of the test assays can be used for accuracy in
the evaluation of the results. An alternative to this method
involves reversing the "positions" of the proteins, i.e.,
immobilizing TSLPR polypeptide binding partner to the microtiter
plate wells, incubating with the test molecule and radiolabeled
TSLPR polypeptide, and determining the extent of TSLPR polypeptide
binding. See, e.g., Current Protocols in Molecular Biology, chap.
18 (Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons
1995).
[0261] As an alternative to radiolabeling, a TSLPR polypeptide or
its binding partner may be conjugated to biotin, and the presence
of biotinylated protein can then be detected using streptavidin
linked to an enzyme, such as horse radish peroxidase (HRP) or
alkaline phosphatase (AP), which can be detected colorometrically,
or by fluorescent tagging of streptavidin. An antibody directed to
a TSLPR polypeptide or to a TSLPR polypeptide binding partner, and
which is conjugated to biotin, may also be used for purposes of
detection following incubation of the complex with enzyme-linked
streptavidin linked to AP or HRP.
[0262] A TSLPR polypeptide or a TSLPR polypeptide binding partner
can also be immobilized by attachment to agarose beads, acrylic
beads, or other types of such inert solid phase substrates. The
substrate-protein complex can be placed in a solution containing
the complementary protein and the test compound. After incubation,
the beads can be precipitated by centrifugation, and the amount of
binding between a TSLPR polypeptide and its binding partner can be
assessed using the methods described herein. Alternatively, the
substrate-protein complex can be immobilized in a column with the
test molecule and complementary protein passing through the column.
The formation of a complex between a TSLPR polypeptide and its
binding partner can then be assessed using any of the techniques
described herein (e.g., radiolabelling or antibody binding).
[0263] Another in vitro assay that is useful for identifying a test
molecule that increases or decreases the formation of a complex
between a TSLPR polypeptide binding protein and a TSLPR polypeptide
binding partner is a surface plasmon resonance detector system such
as the BIAcore assay system (Pharmacia, Piscataway, N.J.). The
BIAcore system is utilized as specified by the manufacturer. This
assay essentially involves the covalent binding of either TSLPR
polypeptide or a TSLPR polypeptide binding partner to a
dextran-coated sensor chip that is located in a detector. The test
compound and the other complementary protein can then be injected,
either simultaneously or sequentially, into the chamber containing
the sensor chip. The amount of complementary protein that binds can
be assessed based on the change in molecular mass that is
physically associated with the dextran-coated side of the sensor
chip, with the change in molecular mass being measured by the
detector system.
[0264] In some cases, it may be desirable to evaluate two or more
test compounds together for their ability to increase or decrease
the formation of a complex between a TSLPR polypeptide and a TSLPR
polypeptide binding partner. In these cases, the assays set forth
herein can be readily modified by adding such additional test
compound(s) either simultaneously with, or subsequent to, the first
test compound. The remainder of the steps in the assay are as set
forth herein.
[0265] In vitro assays such as those described herein may be used
advantageously to screen large numbers of compounds for an effect
on the formation of a complex between a TSLPR polypeptide and TSLPR
polypeptide binding partner. The assays may be automated to screen
compounds generated in phage display, synthetic peptide, and
chemical synthesis libraries.
[0266] Compounds which increase or decrease the formation of a
complex between a TSLPR polypeptide and a TSLPR polypeptide binding
partner may also be screened in cell culture using cells and cell
lines expressing either TSLPR polypeptide or TSLPR polypeptide
binding partner. Cells and cell lines may be obtained from any
mammal, but preferably will be from human or other primate, canine,
or rodent sources. The binding of a TSLPR polypeptide to cells
expressing TSLPR polypeptide binding partner at the surface is
evaluated in the presence or absence of test molecules, and the
extent of binding may be determined by, for example, flow cytometry
using a biotinylated antibody to a TSLPR polypeptide binding
partner. Cell culture assays can be used advantageously to further
evaluate compounds that score positive in protein binding assays
described herein.
[0267] Cell cultures can also be used to screen the impact of a
drug candidate. For example, drug candidates may decrease or
increase the expression of the TSLPR gene. In certain embodiments,
the amount of TSLPR polypeptide or a TSLPR polypeptide fragment
that is produced may be measured after exposure of the cell culture
to the drug candidate. In certain embodiments, one may detect the
actual impact of the drug candidate on the cell culture. For
example, the over-expression of a particular gene may have a
particular impact on the cell culture. In such cases, one may test
a drug candidate's ability to increase or decrease the expression
of the gene or its ability to prevent or inhibit a particular
impact on the cell culture. In other examples, the production of a
particular metabolic product such as a fragment of a polypeptide,
may result in, or be associated with, a disease or pathological
condition. In such cases, one may test a drug candidate's ability
to decrease the production of such a metabolic product in a cell
culture.
[0268] Internalizing Proteins
[0269] The tat protein sequence (from HIV) can be used to
internalize proteins into a cell. See, e.g., Falwell et al., 1994,
Proc. Natl. Acad. Sci. U.S.A. 91: 664-68. For example, an 11 amino
acid sequence (Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 13) of the HIV tat
protein (termed the "protein transduction domain," or TAT PDT) has
been described as mediating delivery across the cytoplasmic
membrane and the nuclear membrane of a cell. See Schwarze et al.,
1999, Science 285: 1569-72; and Nagahara et al., 1998, Nat. Med. 4:
1449-52. In these procedures, FITC-constructs (FITC-labeled
G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 14), which penetrate
tissues following intraperitoneal administration, are prepared, and
the binding of such constructs to cells is detected by
fluorescence-activated cell sorting (FACS) analysis. Cells treated
with a tat-.beta.-gal fusion protein will demonstrate .beta.-gal
activity. Following injection, expression of such a construct can
be detected in a number of tissues, including liver, kidney, lung,
heart, and brain tissue. It is believed that such constructs
undergo some degree of unfolding in order to enter the cell, and as
such, may require a refolding following entry into the cell.
[0270] It will thus be appreciated that the tat protein sequence
may be used to internalize a desired polypeptide into a cell. For
example, using the tat protein sequence, a TSLPR antagonist (such
as an anti-TSLPR selective binding agent, small molecule, soluble
receptor, or antisense oligonucleotide) can be administered
intracellularly to inhibit the activity of a TSLPR molecule. As
used herein, the term "TSLPR molecule" refers to both TSLPR nucleic
acid molecules and TSLPR polypeptides as defined herein. Where
desired, the TSLPR protein itself may also be internally
administered to a cell using these procedures. See also, Straus,
1999, Science 285: 1466-67.
[0271] Cell Source Identification Using TSLPR Polypeptide
[0272] In accordance with certain embodiments of the invention, it
may be useful to be able to determine the source of a certain cell
type associated with a TSLPR polypeptide. For example, it may be
useful to determine the origin of a disease or pathological
condition as an aid in selecting an appropriate therapy. In certain
embodiments, nucleic acids encoding a TSLPR polypeptide can be used
as a probe to identify cells described herein by screening the
nucleic acids of the cells with such a probe. In other embodiments,
one may use anti-TSLPR polypeptide antibodies to test for the
presence of TSLPR polypeptide in cells, and thus, determine if such
cells are of the types described herein.
[0273] TSLPR Polypeptide Compositions and Administration
[0274] Therapeutic compositions are within the scope of the present
invention. Such TSLPR polypeptide pharmaceutical compositions may
comprise a therapeutically effective amount of a TSLPR polypeptide
or a TSLPR nucleic acid molecule in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration.
Pharmaceutical compositions may comprise a therapeutically
effective amount of one or more TSLPR polypeptide selective binding
agents in admixture with a pharmaceutically or physiologically
acceptable formulation agent selected for suitability with the mode
of administration.
[0275] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0276] The pharmaceutical composition may contain formulation
materials for modifying, maintaining, or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine, or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite, or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, or other organic acids), bulking agents (such
as mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid, or hydrogen peroxide), solvents (such as glycerin,
propylene glycol, or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics; PEG; sorbitan esters; polysorbates such
as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as
alkali metal halides--preferably sodium or potassium chloride--or
mannitol sorbitol), delivery vehicles, diluents, excipients and/or
pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences
(18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990.
[0277] The optimal pharmaceutical composition will be determined by
a skilled artisan depending upon, for example, the intended route
of administration, delivery format, and desired dosage. See, e.g.,
Remington's Pharmaceutical Sciences, supra. Such compositions may
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the TSLPR molecule.
[0278] The primary vehicle or carrier in a pharmaceutical
composition may be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier for injection may be water,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute. In one
embodiment of the present invention, TSLPR polypeptide compositions
may be prepared for storage by mixing the selected composition
having the desired degree of purity with optional formulation
agents (Remington's Pharmaceutical Sciences, supra) in the form of
a lyophilized cake or an aqueous solution. Further, the TSLPR
polypeptide product may be formulated as a lyophilizate using
appropriate excipients such as sucrose.
[0279] The TSLPR polypeptide pharmaceutical compositions can be
selected for parenteral delivery. Alternatively, the compositions
may be selected for inhalation or for delivery through the
digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0280] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at a slightly lower pH, typically within a pH range of from about 5
to about 8.
[0281] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable, aqueous solution
comprising the desired TSLPR molecule in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a TSLPR molecule is
formulated as a sterile, isotonic solution, properly preserved. Yet
another preparation can involve the formulation of the desired
molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (such as polylactic
acid or polyglycolic acid), beads, or liposomes, that provides for
the controlled or sustained release of the product which may then
be delivered via a depot injection. Hyaluronic acid may also be
used, and this may have the effect of promoting sustained duration
in the circulation. Other suitable means for the introduction of
the desired molecule include implantable drug delivery devices.
[0282] In one embodiment, a pharmaceutical composition may be
formulated for inhalation. For example, TSLPR polypeptide may be
formulated as a dry powder for inhalation. TSLPR polypeptide or
nucleic acid molecule inhalation solutions may also be formulated
with a propellant for aerosol delivery. In yet another embodiment,
solutions may be nebulized. Pulmonary administration is further
described in PCT Pub. No. WO 94/20069, which describes the
pulmonary delivery of chemically modified proteins.
[0283] It is also contemplated that certain formulations may be
administered orally. In one embodiment of the present invention,
TSLPR polypeptides that are administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For
example, a capsule may be designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate
absorption of the TSLPR polypeptide. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders may also be employed.
[0284] Another pharmaceutical composition may involve an effective
quantity of TSLPR polypeptides in a mixture with non-toxic
excipients that are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions can be prepared in unit-dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0285] Additional TSLPR polypeptide pharmaceutical compositions
will be evident to those skilled in the art, including formulations
involving TSLPR polypeptides in sustained- or controlled-delivery
formulations. Techniques for formulating a variety of other
sustained- or controlled-delivery means, such as liposome carriers,
bio-erodible microparticles or porous beads and depot injections,
are also known to those skilled in the art. See, e.g.,
PCT/US93/00829, which describes the controlled release of porous
polymeric microparticles for the delivery of pharmaceutical
compositions.
[0286] Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g. films, or microcapsules. Sustained release matrices
may include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919 and European Patent No. 058481), copolymers of L-glutamic
acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers
22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al.,
1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem.
Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (European Patent No. 133988).
Sustained-release compositions may also include liposomes, which
can be prepared by any of several methods known in the art. See,
e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:
3688-92; and European Patent Nos. 036676, 088046, and 143949.
[0287] The TSLPR pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished
by filtration through sterile filtration membranes. Where the
composition is lyophilized, sterilization using this method may be
conducted either prior to, or following, lyophilization and
reconstitution. The composition for parenteral administration may
be stored in lyophilized form or in a solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0288] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0289] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0290] The effective amount of a TSLPR pharmaceutical composition
to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
thus vary depending, in part, upon the molecule delivered, the
indication for which the TSLPR molecule is being used, the route of
administration, and the size (body weight, body surface, or organ
size) and condition (the age and general health) of the
patient.
[0291] Accordingly, the clinician may titer the dosage and modify
the route of administration to obtain the optimal therapeutic
effect. A typical dosage may range from about 0.1 .mu.g/kg to up to
about 100 mg/kg or more, depending on the factors mentioned above.
In other embodiments, the dosage may range from 0.1 .mu.g/kg up to
about 100 mg/kg; or 1 .mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg
up to about 100 mg/kg.
[0292] The frequency of dosing will depend upon the pharmacokinetic
parameters of the TSLPR molecule in the formulation being used.
Typically, a clinician will administer the composition until a
dosage is reached that achieves the desired effect. The composition
may therefore be administered as a single dose, as two or more
doses (which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages may be ascertained through use of
appropriate dose-response data.
[0293] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally; through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems; or by implantation devices. Where
desired, the compositions may be administered by bolus injection or
continuously by infusion, or by implantation device.
[0294] Alternatively or additionally, the composition may be
administered locally via implantation of a membrane, sponge, or
other appropriate material onto which the desired molecule has been
absorbed or encapsulated. Where an implantation device is used, the
device may be implanted into any suitable tissue or organ, and
delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0295] In some cases, it may be desirable to use TSLPR polypeptide
pharmaceutical compositions in an ex vivo manner. In such
instances, cells, tissues, or organs that have been removed from
the patient are exposed to TSLPR polypeptide pharmaceutical
compositions after which the cells, tissues, or organs are
subsequently implanted back into the patient.
[0296] In other cases, a TSLPR polypeptide can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the TSLPR polypeptide. Such cells may be animal or human
cells, and may be autologous, heterologous, or xenogeneic.
Optionally, the cells may be immortalized. In order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. The encapsulation
materials are typically biocompatible, semi-permeable polymeric
enclosures or membranes that allow the release of the protein
product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
[0297] As discussed herein, it may be desirable to treat isolated
cell populations (such as stem cells, lymphocytes, red blood cells,
chondrocytes, neurons, and the like) with one or more TSLPR
polypeptides. This can be accomplished by exposing the isolated
cells to the polypeptide directly, where it is in a form that is
permeable to the cell membrane. Additional embodiments of the
present invention relate to cells and methods (e.g., homologous
recombination and/or other recombinant production methods) for both
the in vitro production of therapeutic polypeptides and for the
production and delivery of therapeutic polypeptides by gene therapy
or cell therapy. Homologous and other recombination methods may be
used to modify a cell that contains a normally
transcriptionally-silent TSLPR gene, or an under-expressed gene,
and thereby produce a cell which expresses therapeutically
efficacious amounts of TSLPR polypeptides.
[0298] Homologous recombination is a technique originally developed
for targeting genes to induce or correct mutations in
transcriptionally active genes. Kucherlapati, 1989, Prog. in Nucl.
Acid Res. & Mol. Biol. 36: 301. The basic technique was
developed as a method for introducing specific mutations into
specific regions of the mammalian genome (Thomas et al., 1986, Cell
44: 419-28; Thomas and Capecchi, 1987, Cell 51: 503-12; Doetschman
et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 8583-87) or to
correct specific mutations within defective genes (Doetschman et
al., 1987, Nature 330: 576-78). Exemplary homologous recombination
techniques are described in U.S. Pat. No. 5,272,071; European
Patent Nos. 9193051 and 505500; PCT/US90/07642, and PCT Pub No. WO
91/09955).
[0299] Through homologous recombination, the DNA sequence to be
inserted into the genome can be directed to a specific region of
the gene of interest by attaching it to targeting DNA. The
targeting DNA is a nucleotide sequence that is complementary
(homologous) to a region of the genomic DNA. Small pieces of
targeting DNA that are complementary to a specific region of the
genome are put in contact with the parental strand during the DNA
replication process. It is a general property of DNA that has been
inserted into a cell to hybridize, and therefore, recombine with
other pieces of endogenous DNA through shared homologous regions.
If this complementary strand is attached to an oligonucleotide that
contains a mutation or a different sequence or an additional
nucleotide, it too is incorporated into the newly synthesized
strand as a result of the recombination. As a result of the
proofreading function, it is possible for the new sequence of DNA
to serve as the template. Thus, the transferred DNA is incorporated
into the genome.
[0300] Attached to these pieces of targeting DNA are regions of DNA
that may interact with or control the expression of a TSLPR
polypeptide, e.g., flanking sequences. For example, a
promoter/enhancer element, a suppressor, or an exogenous
transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to
influence the transcription of DNA encoding the desired TSLPR
polypeptide. The control element controls a portion of the DNA
present in the host cell genome. Thus, the expression of the
desired TSLPR polypeptide may be achieved not by transfection of
DNA that encodes the TSLPR gene itself, but rather by the use of
targeting DNA (containing regions of homology with the endogenous
gene of interest) coupled with DNA regulatory segments that provide
the endogenous gene sequence with recognizable signals for
transcription of a TSLPR gene.
[0301] In an exemplary method, the expression of a desired targeted
gene in a cell (i.e., a desired endogenous cellular gene) is
altered via homologous recombination into the cellular genome at a
preselected site, by the introduction of DNA which includes at
least a regulatory sequence, an exon, and a splice donor site.
These components are introduced into the chromosomal (genomic) DNA
in such a manner that this, in effect, results in the production of
a new transcription unit (in which the regulatory sequence, the
exon, and the splice donor site present in the DNA construct are
operatively linked to the endogenous gene). As a result of the
introduction of these components into the chromosomal DNA, the
expression of the desired endogenous gene is altered.
[0302] Altered gene expression, as described herein, encompasses
activating (or causing to be expressed) a gene which is normally
silent (unexpressed) in the cell as obtained, as well as increasing
the expression of a gene which is not expressed at physiologically
significant levels in the cell as obtained. The embodiments further
encompass changing the pattern of regulation or induction such that
it is different from the pattern of regulation or induction that
occurs in the cell as obtained, and reducing (including
eliminating) the expression of a gene which is expressed in the
cell as obtained. One method by which homologous recombination can
be used to increase, or cause, TSLPR polypeptide production from a
cell's endogenous TSLPR gene involves first using homologous
recombination to place a recombination sequence from a
site-specific recombination system (e.g., Cre/loxP, FLP/FRT)
(Sauer, 1994, Curr. Opin. Biotechnol., 5: 521-27; Sauer, 1993,
Methods Enzymol., 225: 890-900) upstream of (i.e., 5' to) the
cell's endogenous genomic TSLPR polypeptide coding region. A
plasmid containing a recombination site homologous to the site that
was placed just upstream of the genomic TSLPR polypeptide coding
region is introduced into the modified cell line along with the
appropriate recombinase enzyme. This recombinase causes the plasmid
to integrate, via the plasmid's recombination site, into the
recombination site located just upstream of the genomic TSLPR
polypeptide coding region in the cell line (Baubonis and Sauer,
1993, Nucleic Acids Res. 21: 2025-29; O'Gorman et al., 1991,
Science 251: 1351-55). Any flanking sequences known to increase
transcription (e.g., enhancer/promoter, intron, translational
enhancer), if properly positioned in this plasmid, would integrate
in such a manner as to create a new or modified transcriptional
unit resulting in de novo or increased TSLPR polypeptide production
from the cell's endogenous TSLPR gene.
[0303] A further method to use the cell line in which the site
specific recombination sequence had been placed just upstream of
the cell's endogenous genomic TSLPR polypeptide coding region is to
use homologous recombination to introduce a second recombination
site elsewhere in the cell line's genome. The appropriate
recombinase enzyme is then introduced into the
two-recombination-site cell line, causing a recombination event
(deletion, inversion, and translocation) (Sauer, 1994, Curr. Opin.
Biotechnol., 5: 521-27; Sauer, 1993, Methods Enzymol., 225:
890-900) that would create a new or modified transcriptional unit
resulting in de novo or increased TSLPR polypeptide production from
the cell's endogenous TSLPR gene.
[0304] An additional approach for increasing, or causing, the
expression of TSLPR polypeptide from a cell's endogenous TSLPR gene
involves increasing, or causing, the expression of a gene or genes
(e.g., transcription factors) and/or decreasing the expression of a
gene or genes (e.g., transcriptional repressors) in a manner which
results in de novo or increased TSLPR polypeptide production from
the cell's endogenous TSLPR gene. This method includes the
introduction of a non-naturally occurring polypeptide (e.g., a
polypeptide comprising a site specific DNA binding domain fused to
a transcriptional factor domain) into the cell such that de novo or
increased TSLPR polypeptide production from the cell's endogenous
TSLPR gene results.
[0305] The present invention further relates to DNA constructs
useful in the method of altering expression of a target gene. In
certain embodiments, the exemplary DNA constructs comprise: (a) one
or more targeting sequences, (b) a regulatory sequence, (c) an
exon, and (d) an unpaired splice-donor site. The targeting sequence
in the DNA construct directs the integration of elements (a)-(d)
into a target gene in a cell such that the elements (b)-(d) are
operatively linked to sequences of the endogenous target gene.
[0306] In another embodiment, the DNA constructs comprise: (a) one
or more targeting sequences, (b) a regulatory sequence, (c) an
exon, (d) a splice-donor site, (e) an intron, and (f) a
splice-acceptor site, wherein the targeting sequence directs the
integration of elements (a)-(f) such that the elements of (b)-(f)
are operatively linked to the endogenous gene. The targeting
sequence is homologous to the preselected site in the cellular
chromosomal DNA with which homologous recombination is to occur. In
the construct, the exon is generally 3' of the regulatory sequence
and the splice-donor site is 3' of the exon.
[0307] If the sequence of a particular gene is known, such as the
nucleic acid sequence of TSLPR polypeptide presented herein, a
piece of DNA that is complementary to a selected region of the gene
can be synthesized or otherwise obtained, such as by appropriate
restriction of the native DNA at specific recognition sites
bounding the region of interest. This piece serves as a targeting
sequence upon insertion into the cell and will hybridize to its
homologous region within the genome. If this hybridization occurs
during DNA replication, this piece of DNA, and any additional
sequence attached thereto, will act as an Okazaki fragment and will
be incorporated into the newly synthesized daughter strand of DNA.
The present invention, therefore, includes nucleotides encoding a
TSLPR polypeptide, which nucleotides may be used as targeting
sequences.
[0308] TSLPR polypeptide cell therapy, e.g., the implantation of
cells producing TSLPR polypeptides, is also contemplated. This
embodiment involves implanting cells capable of synthesizing and
secreting a biologically active form of TSLPR polypeptide. Such
TSLPR polypeptide-producing cells can be cells that are natural
producers of TSLPR polypeptides or may be recombinant cells whose
ability to produce TSLPR polypeptides has been augmented by
transformation with a gene encoding the desired TSLPR polypeptide
or with a gene augmenting the expression of TSLPR polypeptide. Such
a modification may be accomplished by means of a vector suitable
for delivering the gene as well as promoting its expression and
secretion. In order to minimize a potential immunological reaction
in patients being administered a TSLPR polypeptide, as may occur
with the administration of a polypeptide of a foreign species, it
is preferred that the natural cells producing TSLPR polypeptide be
of human origin and produce human TSLPR polypeptide. Likewise, it
is preferred that the recombinant cells producing TSLPR polypeptide
be transformed with an expression vector containing a gene encoding
a human TSLPR polypeptide.
[0309] Implanted cells may be encapsulated to avoid the
infiltration of surrounding tissue. Human or non-human animal cells
may be implanted in patients in biocompatible, semipermeable
polymeric enclosures or membranes that allow the release of TSLPR
polypeptide, but that prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissue. Alternatively, the patient's own cells,
transformed to produce TSLPR polypeptides ex vivo, may be implanted
directly into the patient without such encapsulation.
[0310] Techniques for the encapsulation of living cells are known
in the art, and the preparation of the encapsulated cells and their
implantation in patients may be routinely accomplished. For
example, Baetge et al. (PCT Pub. No. WO 95/05452 and
PCT/US94/09299) describe membrane capsules containing genetically
engineered cells for the effective delivery of biologically active
molecules. The capsules are biocompatible and are easily
retrievable. The capsules encapsulate cells transfected with
recombinant DNA molecules comprising DNA sequences coding for
biologically active molecules operatively linked to promoters that
are not subject to down-regulation in vivo upon implantation into a
mammalian host. The devices provide for the delivery of the
molecules from living cells to specific sites within a recipient.
In addition, see U.S. Pat. Nos. 4,892,538; 5,011,472; and
5,106,627. A system for encapsulating living cells is described in
PCT Pub. No. WO 91/10425 (Aebischer et al.). See also, PCT Pub. No.
WO 91/10470 (Aebischer et al.); Winn et al., 1991, Exper. Neurol.
113: 322-29; Aebischer et al., 1991, Exper. Neurol. 111: 269-75;
and Tresco et al., 1992, ASAIO 38: 17-23.
[0311] In vivo and in vitro gene therapy delivery of TSLPR
polypeptides is also envisioned. One example of a gene therapy
technique is to use the TSLPR gene (either genomic DNA, cDNA,
and/or synthetic DNA) encoding a TSLPR polypeptide which may be
operably linked to a constitutive or inducible promoter to form a
"gene therapy DNA construct." The promoter may be homologous or
heterologous to the endogenous TSLPR gene, provided that it is
active in the cell or tissue type into which the construct will be
inserted. Other components of the gene therapy DNA construct may
optionally include DNA molecules designed for site-specific
integration (e.g., endogenous sequences useful for homologous
recombination), tissue-specific promoters, enhancers or silencers,
DNA molecules capable of providing a selective advantage over the
parent cell, DNA molecules useful as labels to identify transformed
cells, negative selection systems, cell specific binding agents
(as, for example, for cell targeting), cell-specific
internalization factors, transcription factors enhancing expression
from a vector, and factors enabling vector production.
[0312] A gene therapy DNA construct can then be introduced into
cells (either ex vivo or in vivo) using viral or non-viral vectors.
One means for introducing the gene therapy DNA construct is by
means of viral vectors as described herein. Certain vectors, such
as retroviral vectors, will deliver the DNA construct to the
chromosomal DNA of the cells, and the gene can integrate into the
chromosomal DNA. Other vectors will function as episomes, and the
gene therapy DNA construct will remain in the cytoplasm.
[0313] In yet other embodiments, regulatory elements can be
included for the controlled expression of the TSLPR gene in the
target cell. Such elements are turned on in response to an
appropriate effector. In this way, a therapeutic polypeptide can be
expressed when desired. One conventional control means involves the
use of small molecule dimerizers or rapalogs to dimerize chimeric
proteins which contain a small molecule-binding domain and a domain
capable of initiating a biological process, such as a DNA-binding
protein or transcriptional activation protein (see PCT Pub. Nos. WO
96/41865, WO 97/31898, and WO 97/31899). The dimerization of the
proteins can be used to initiate transcription of the
transgene.
[0314] An alternative regulation technology uses a method of
storing proteins expressed from the gene of interest inside the
cell as an aggregate or cluster. The gene of interest is expressed
as a fusion protein that includes a conditional aggregation domain
that results in the retention of the aggregated protein in the
endoplasmic reticulum. The stored proteins are stable and inactive
inside the cell. The proteins can be released, however, by
administering a drug (e.g., small molecule ligand) that removes the
conditional aggregation domain and thereby specifically breaks
apart the aggregates or clusters so that the proteins may be
secreted from the cell. See Aridor et al., 2000, Science 287:
816-17 and Rivera et al., 2000, Science 287: 826-30.
[0315] Other suitable control means or gene switches include, but
are not limited to, the systems described herein. Mifepristone
(RU486) is used as a progesterone antagonist.
[0316] The binding of a modified progesterone receptor
ligand-binding domain to the progesterone antagonist activates
transcription by forming a dimer of two transcription factors that
then pass into the nucleus to bind DNA. The ligand-binding domain
is modified to eliminate the ability of the receptor to bind to the
natural ligand. The modified steroid hormone receptor system is
further described in U.S. Pat. No. 5,364,791 and PCT Pub. Nos. WO
96/40911 and WO 97/10337.
[0317] Yet another control system uses ecdysone (a fruit fly
steroid hormone) which binds to and activates an ecdysone receptor
(cytoplasmic receptor). The receptor then translocates to the
nucleus to bind a specific DNA response element (promoter from
ecdysone-responsive gene). The ecdysone receptor includes a
transactivation domain, DNA-binding domain, and ligand-binding
domain to initiate transcription. The ecdysone system is further
described in U.S. Pat. No. 5,514,578 and PCT Pub. Nos. WO 97/38117,
WO 96/37609, and WO 93/03162.
[0318] Another control means uses a positive
tetracycline-controllable transactivator. This system involves a
mutated tet repressor protein DNA-binding domain (mutated tet R-4
amino acid changes which resulted in a reverse
tetracycline-regulated transactivator protein, i.e., it binds to a
tet operator in the presence of tetracycline) linked to a
polypeptide which activates transcription. Such systems are
described in U.S. Pat. Nos. 5,464,758, 5,650,298, and
5,654,168.
[0319] Additional expression control systems and nucleic acid
constructs are described in U.S. Pat. Nos. 5,741,679 and 5,834,186,
to Innovir Laboratories Inc.
[0320] In vivo gene therapy may be accomplished by introducing the
gene encoding TSLPR polypeptide into cells via local injection of a
TSLPR nucleic acid molecule or by other appropriate viral or
non-viral delivery vectors. Hefti 1994, Neurobiology 25: 1418-35.
For example, a nucleic acid molecule encoding a TSLPR polypeptide
may be contained in an adeno-associated virus (AAV) vector for
delivery to the targeted cells (see, e.g., Johnson, PCT Pub. No. WO
95/34670; PCT App. No. PCT/US95/07178). The recombinant AAV genome
typically contains AAV inverted terminal repeats flanking a DNA
sequence encoding a TSLPR polypeptide operably linked to functional
promoter and polyadenylation sequences.
[0321] Alternative suitable viral vectors include, but are not
limited to, retrovirus, adenovirus, herpes simplex virus,
lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus,
alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma
virus vectors. U.S. Pat. No. 5,672,344 describes an in vivo
viral-mediated gene transfer system involving a recombinant
neurotrophic HSV-1 vector. U.S. Pat. No. 5,399,346 provides
examples of a process for providing a patient with a therapeutic
protein by the delivery of human cells which have been treated in
vitro to insert a DNA segment encoding a therapeutic protein.
Additional methods and materials for the practice of gene therapy
techniques are described in U.S. Pat. Nos. 5,631,236 (involving
adenoviral vectors), 5,672,510 (involving retroviral vectors),
5,635,399 (involving retroviral vectors expressing cytokines).
[0322] Nonviral delivery methods include, but are not limited to,
liposome-mediated transfer, naked DNA delivery (direct injection),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation, and microparticle bombardment
(e.g., gene gun). Gene therapy materials and methods may also
include inducible promoters, tissue-specific enhancer-promoters,
DNA sequences designed for site-specific integration, DNA sequences
capable of providing a selective advantage over the parent cell,
labels to identify transformed cells, negative selection systems
and expression control systems (safety measures), cell-specific
binding agents (for cell targeting), cell-specific internalization
factors, and transcription factors to enhance expression by a
vector as well as methods of vector manufacture. Such additional
methods and materials for the practice of gene therapy techniques
are described in U.S. Pat. Nos. 4,970,154 (involving
electroporation techniques), 5,679,559 (describing a
lipoprotein-containing system for gene delivery), 5,676,954
(involving liposome carriers), 5,593,875 (describing methods for
calcium phosphate transfection), and 4,945,050 (describing a
process wherein biologically active particles are propelled at
cells at a speed whereby the particles penetrate the surface of the
cells and become incorporated into the interior of the cells), and
PCT Pub. No. WO 96/40958 (involving nuclear ligands).
[0323] It is also contemplated that TSLPR gene therapy or cell
therapy can further include the delivery of one or more additional
polypeptide(s) in the same or a different cell(s). Such cells may
be separately introduced into the patient, or the cells may be
contained in a single implantable device, such as the encapsulating
membrane described above, or the cells may be separately modified
by means of viral vectors.
[0324] A means to increase endogenous TSLPR polypeptide expression
in a cell via gene therapy is to insert one or more enhancer
elements into the TSLPR polypeptide promoter, where the enhancer
elements can serve to increase transcriptional activity of the
TSLPR gene. The enhancer elements used will be selected based on
the tissue in which one desires to activate the gene--enhancer
elements known to confer promoter activation in that tissue will be
selected. For example, if a gene encoding a TSLPR polypeptide is to
be "turned on" in T-cells, the Ick promoter enhancer element may be
used. Here, the functional portion of the transcriptional element
to be added may be inserted into a fragment of DNA containing the
TSLPR polypeptide promoter (and optionally, inserted into a vector
and/or 5' and/or 3' flanking sequences) using standard cloning
techniques. This construct, known as a "homologous recombination
construct," can then be introduced into the desired cells either ex
vivo or in vivo.
[0325] Gene therapy also can be used to decrease TSLPR polypeptide
expression by modifying the nucleotide sequence of the endogenous
promoter. Such modification is typically accomplished via
homologous recombination methods. For example, a DNA molecule
containing all or a portion of the promoter of the TSLPR gene
selected for inactivation can be engineered to remove and/or
replace pieces of the promoter that regulate transcription. For
example, the TATA box and/or the binding site of a transcriptional
activator of the promoter may be deleted using standard molecular
biology techniques; such deletion can inhibit promoter activity
thereby repressing the transcription of the corresponding TSLPR
gene. The deletion of the TATA box or the transcription activator
binding site in the promoter may be accomplished by generating a
DNA construct comprising all or the relevant portion of the TSLPR
polypeptide promoter (from the same or a related species as the
TSLPR gene to be regulated) in which one or more of the TATA box
and/or transcriptional activator binding site nucleotides are
mutated via substitution, deletion and/or insertion of one or more
nucleotides. As a result, the TATA box and/or activator binding
site has decreased activity or is rendered completely inactive.
This construct, which also will typically contain at least about
500 bases of DNA that correspond to the native (endogenous) 5' and
3' DNA sequences adjacent to the promoter segment that has been
modified, may be introduced into the appropriate cells (either ex
vivo or in vivo) either directly or via a viral vector as described
herein. Typically, the integration of the construct into the
genomic DNA of the cells will be via homologous recombination,
where the 5' and 3' DNA sequences in the promoter construct can
serve to help integrate the modified promoter region via
hybridization to the endogenous chromosomal DNA.
[0326] Therapeutic Uses
[0327] TSLPR nucleic acid molecules, polypeptides, and agonists and
antagonists thereof can be used to treat, diagnose, ameliorate, or
prevent a number of diseases, disorders, or conditions, including
TSLP-related diseases, disorders, or conditions. TSLP-related
diseases, disorders, or conditions may be related to B-cell
development, T-cell development, T-cell receptor gene
rearrangement, or regulation of the Stat5 transcription factor.
Diseases caused by or mediated by undesirable levels of TSLP are
encompassed within the scope of the invention. Undesirable levels
include excessive levels of TSLP and sub-normal levels of TSLP.
[0328] TSLPR polypeptide agonists and antagonists include those
molecules that regulate TSLPR polypeptide activity and either
increase or decrease at least one activity of the mature form of
the TSLPR polypeptide. Agonists or antagonists may be co-factors,
such as a protein, peptide, carbohydrate, lipid, or small molecular
weight molecule, which interact with TSLPR polypeptide and thereby
regulate its activity. Potential polypeptide agonists or
antagonists include antibodies that react with either soluble or
membrane-bound forms of TSLPR polypeptides that comprise part or
all of the extracellular domains of the said proteins. Molecules
that regulate TSLPR polypeptide expression typically include
nucleic acids encoding TSLPR polypeptide that can act as anti-sense
regulators of expression.
[0329] TSLPR nucleic acid molecules, polypeptides, and agonists and
antagonists thereof may be used (simultaneously or sequentially) in
combination with one or more cytokines, growth factors,
antibiotics, anti-inflammatories, and/or chemotherapeutic agents as
is appropriate for the condition being treated.
[0330] Other diseases or disorders caused by or mediated by
undesirable levels of TSLPR polypeptides are encompassed within the
scope of the invention. Undesirable levels include excessive levels
of TSLPR polypeptides and sub-normal levels of TSLPR
polypeptides.
[0331] Uses of TSLPR Nucleic Acids and Polypeptides
[0332] Nucleic acid molecules of the invention (including those
that do not themselves encode biologically active polypeptides) may
be used to map the locations of the TSLPR gene and related genes on
chromosomes. Mapping may be done by techniques known in the art,
such as PCR amplification and in situ hybridization.
[0333] TSLPR nucleic acid molecules (including those that do not
themselves encode biologically active polypeptides) may be useful
as hybridization probes in diagnostic assays to test, either
qualitatively or quantitatively, for the presence of a TSLPR
nucleic acid molecule in mammalian tissue or bodily fluid
samples.
[0334] Other methods may also be employed where it is desirable to
inhibit the activity of one or more TSLPR polypeptides. Such
inhibition may be effected by nucleic acid molecules that are
complementary to and hybridize to expression control sequences
(triple helix formation) or to TSLPR mRNA. For example, antisense
DNA or RNA molecules, which have a sequence that is complementary
to at least a portion of a TSLPR gene can be introduced into the
cell. Anti-sense probes may be designed by available techniques
using the sequence of the TSLPR gene disclosed herein. Typically,
each such antisense molecule will be complementary to the start
site (5' end) of each selected TSLPR gene. When the antisense
molecule then hybridizes to the corresponding TSLPR mRNA,
translation of this mRNA is prevented or reduced. Anti-sense
inhibitors provide information relating to the decrease or absence
of a TSLPR polypeptide in a cell or organism.
[0335] Alternatively, gene therapy may be employed to create a
dominant-negative inhibitor of one or more TSLPR polypeptides. In
this situation, the DNA encoding a mutant polypeptide of each
selected TSLPR polypeptide can be prepared and introduced into the
cells of a patient using either viral or non-viral methods as
described herein.
[0336] Each such mutant is typically designed to compete with
endogenous polypeptide in its biological role.
[0337] In addition, a TSLPR polypeptide, whether biologically
active or not, may be used as an immunogen, that is, the
polypeptide contains at least one epitope to which antibodies may
be raised. Selective binding agents that bind to a TSLPR
polypeptide (as described herein) may be used for in vivo and in
vitro diagnostic purposes, including, but not limited to, use in
labeled form to detect the presence of TSLPR polypeptide in a body
fluid or cell sample. The antibodies may also be used to prevent,
treat, or diagnose a number of diseases and disorders, including
those recited herein. The antibodies may bind to a TSLPR
polypeptide so as to diminish or block at least one activity
characteristic of a TSLPR polypeptide, or may bind to a polypeptide
to increase at least one activity characteristic of a TSLPR
polypeptide (including by increasing the pharmacokinetics of the
TSLPR polypeptide).
[0338] The murine and human TSLPR nucleic acids of the present
invention are also useful tools for isolating the corresponding
chromosomal TSLPR polypeptide genes. For example, mouse chromosomal
DNA containing TSLPR sequences can be used to construct knockout
mice, thereby permitting an examination of the in vivo role for
TSLPR polypeptide. The human TSLPR genomic DNA can be used to
identify heritable tissue-degenerating diseases.
[0339] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
EXAMPLE 1
Cloning of the Murine and Human TSLPR Polypeptide Genes
[0340] Generally, materials and methods as described in Sambrook et
al., supra were used to clone and analyze the genes encoding murine
and human TSLPR polypeptides.
[0341] Sequences encoding the murine TSLPR polypeptide were
identified in a BLAST search of an EST database using sequences
corresponding to the cytoplasmic domain of the erythropoietin
receptor. Several overlapping murine ESTs, which encode a novel
type I cytokine receptor molecule, were obtained in the BLAST
search. The cytoplasmic domain of the cytokine receptor encoded by
these sequences was found to share significant similarity to that
of the common cytokine receptor .gamma. chain (.gamma..sub.c), the
erythropoietin receptor, and the IL-9 receptor .alpha. chain.
[0342] The common cytokine receptor .gamma. chain is an essential
subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15
(Noguchi et al., 1993, Science 262: 1877-80; Kondo et al., 1994,
Science 263: 1453-54; Kondo et al., 1993, Science 262: 1874-77;
Russell et al., 1994, Science 266: 1042-45; Takeshita et al., 1992,
Science 257: 379-82; Russell et al., 1993, Science 262: 1880-83;
Giri et al., 1994, EMBO J. 13: 2822-30; Kimura et al., 1995, Int.
Immunol. 7: 115-20). The mutation of .gamma..sub.c in humans can
result in X-linked severe combined immunodeficiency (Noguchi et
al., 1993, Cell 73: 147-57; Leonard et al., 1995, Immunol. Rev.
148: 97-114).
[0343] Since none of the ESTs sequences identified in the BLAST
search contained the entire open reading frame for TSLPR
polypeptide, a mouse embryo library was screened to obtain a
full-length cDNA. The positive colony containing the longest insert
was used to prepare plasmid DNA by standard methods. The cDNA
insert from this colony was 2 kb in length. DNA sequence analysis
confirmed that the clone contained the entire reading frame for
TSLPR polypeptide.
[0344] Sequence analysis of the full-length cDNA for murine TSLPR
polypeptide indicated that the gene comprises a 1110 bp open
reading frame encoding a protein of 370 amino acids and possessing
a potential signal peptide of 17 amino acids in length at its
amino-terminus (FIGS. 1A-1B; predicted signal peptide indicated by
underline).
[0345] The open reading frame was found to encode a type I
transmembrane protein having two potential N-linked glycosylation
sites and a cytoplasmic domain of 104 amino acids containing a
single tyrosine residue.
[0346] In contrast, murine .gamma..sub.c comprises 369 amino acids
a has a cytoplasmic domain of 86 amino acids containing two
tyrosine residues (Kumaki et al., 1993, Biochem. Biophys. Res.
Commun. 193: 356-63; Cao et al., 1993, Proc. Natl. Acad. Sci.
U.S.A. 90: 8464-68; Kobayash et al., 1993, Gene 130: 303-04). FIG.
2 illustrates an amino acid sequence alignment of murine TSLPR
polypeptide (upper sequence) and murine .gamma..sub.c (lower
sequence). Murine TSLPR polypeptide was found to share 26% sequence
identity and 47% sequence similarity with .gamma..sub.c at the
amino acid level. The sequence of murine TSLPR polypeptide is
somewhat atypical for type I cytokine receptors in that only one
pair of cysteines is conserved and the W-S-X-W-S (SEQ ID NO: 15)
motif is replaced by a W-T-A-V-T (SEQ ID NO: 16) motif. The
predicted molecular weight of murine TSLPR polypeptide is 37
kD.
[0347] Sequences encoding the human TSLPR polypeptide were
identified in a BLAST search of a proprietary database of cDNA
sequences (Amgen, Thousand Oaks, Calif.) using the murine TSLPR
nucleic acid sequence as a query sequence. Two clones containing
human cDNA sequences and sharing the greatest homology with the
murine TSLPR nucleic acid sequence were identified in this search:
9604927 (SEQ ID NO: 10) and 9508990 (SEQ ID NO: 11). Sequence
analysis of the full-length cDNA for human TSLPR polypeptide (as
contained in Clone 9604927) indicated that the human TSLPR gene
comprises an open reading frame of 1113 bp encoding a protein of
371 amino acids and possessing a potential signal peptide of 22
amino acids in length at its amino-terminus (FIGS. 3A-3B; predicted
signal peptide indicated by underline).
[0348] Clone 9508990 contains an open reading frame of 1137 bp
encoding a protein of 379 amino acids (FIGS. 4A-4B). This clone
essentially comprises the full-length human TSLPR polypeptide
sequence and an additional 8 amino acids at the carboxyl-terminus
corresponding to the FLAG epitope. FIG. 5 illustrates an amino acid
sequence alignment of murine TSLPR polypeptide (upper sequence) and
human TSLPR polypeptide (lower sequence). The availability of
murine and human TSLPR nucleic acid and amino acid sequences will
further aid in the elucidation of signal transduction pathways
utilized by TSLP.
EXAMPLE 2
TSLPR Polypeptide Expression
[0349] A cDNA construct encoding the entire open reading frame for
murine TSLPR was transcribed and translated in vitro in the
presence of .sup.35S-methionine and the product resolved by
SDS-PAGE. FIG. 6A illustrates an autoradiogram of the gel in which
a single species of approximately 40 kD was obtained.
[0350] FIG. 6B illustrates the immunoprecipitation of murine TSLPR
polypeptide in the growth factor-dependent pre-B-cell line NAG8/7
using a rabbit polyclonal antiserum raised against the
extracellular domain of murine TSLPR polypeptide. The rabbit
polyclonal antiserum was generated against murine TSLPR
polypeptide-glutathione S-transferase fusion protein which was
cloned into the pGEX4T2 expression vector (Pharmacia) and expressed
in bacteria. Prior to metabolic labeling, NAG8/7 cells were grown
in RPMI supplemented with 10% fetal bovine serum, antibiotics, and
TSLP.
[0351] NAG8/7 cells were metabolically labeled with
.sup.35S-methionine and cysteine, lysed in 50 mM Tris, pH 7.4, 150
mM NaCl, 1% Triton X-100, and protease inhibitors, and the lysates
incubated overnight with either rabbit polyclonal antiserum (lane
2) or pre-immune serum (lane 1). The immune complexes were captured
with Protein G sepharosel, washed in lysis buffer, and then
resolved by SDS-PAGE. The polyclonal antiserum specifically
immunoprecipitated a broad band of approximately 50 kD in a
pre-B-cell line NAG8/7 (FIG. 6B). The larger size of the
immunoprecipitated product as compared with the product generated
by in vitro translation is consistent with the addition of N-linked
carbohydrate moieties in the extracellular domain. Flow cytometric
analysis of transfected 293 cells and several hematopoietic cell
lines (i.e., 32D, BaF3, and WEHI-3) confirmed that murine TSLPR was
expressed at the cell surface.
EXAMPLE 3
TSLPR mRNA Expression
[0352] The tissue distribution of murine TSLPR was examined by
northern blot analysis.
[0353] A mouse multiple tissue northern blot (Clontech, Palo Alto,
Calif.) was screened with a .sup.32P-labeled TSLPR cDNA probe using
standard techniques. Murine TSLPR mRNA transcripts were detected in
nearly all of the tissues examined, with highest levels of
expression being detected in the lung, liver, and testis (FIG. 6C).
Lower levels of expression were detected in the heart, brain,
spleen, and skeletal muscle. Two transcripts of approximately 2 kb
and 2.2 kb were detected in some tissues, whereas only a single
transcript of approximately 2 kb was detected in other tissues. The
broad tissue distribution of murine TSLPR mRNA differs from the
relatively restricted lympho-hematopoietic pattern of expression
observed for .gamma..sub.c.
[0354] The expression of TSLPR mRNA can be localized by in situ
hybridization as follows. A panel of normal embryonic and adult
mouse tissues is fixed in 4% paraformaldehyde, embedded in
paraffin, and sectioned at 5 .mu.m. Sectioned tissues are
permeabilized in 0.2 M HCl, digested with Proteinase K, and
acetylated with triethanolamine and acetic anhydride. Sections are
prehybridized for 1 hour at 60.degree. C. in hybridization solution
(300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1.times.
Denhardt's solution, 0.2% SDS, 10 mM DTT, 0.25 mg/ml tRNA, 25
.mu.g/ml polyA, 25 .mu.g/ml polyC and 50% formamide) and then
hybridized overnight at 60.degree. C. in the same solution
containing 10% dextran and 2.times.10.sup.4 cpm/.mu.l of a
.sup.33P-labeled antisense riboprobe complementary to the human
TSLPR gene. The riboprobe is obtained by in vitro transcription of
a clone containing human TSLPR cDNA sequences using standard
techniques.
[0355] Following hybridization, sections are rinsed in
hybridization solution, treated with RNaseA to digest unhybridized
probe, and then washed in 0.1.times.SSC at 55.degree. C. for 30
minutes. Sections are then immersed in NTB-2 emulsion (Kodak,
Rochester, N.Y.), exposed for 3 weeks at 4.degree. C., developed,
and counterstained with hematoxylin and eosin. Tissue morphology
and hybridization signal are simultaneously analyzed by darkfield
and standard illumination for brain (one sagittal and two coronal
sections), gastrointestinal tract (esophagus, stomach, duodenum,
jejunum, ileum, proximal colon, and distal colon), pituitary,
liver, lung, heart, spleen, thymus, lymph nodes, kidney, adrenal,
bladder, pancreas, salivary gland, male and female reproductive
organs (ovary, oviduct, and uterus in the female; and testis,
epididymus, prostate, seminal vesicle, and vas deferens in the
male), BAT and WAT (subcutaneous, peri-renal), bone (femur), skin,
breast, and skeletal muscle.
EXAMPLE 4
Biological Activity of Murine TSLPR Polypeptide
[0356] The similarity between murine TSLPR polypeptide and the
erythropoietin receptor suggested that murine TSLPR, like the
erythropoietin receptor, could be activated by homodimerization.
This was examined in a proliferation assay using a chimeric
construct derived from the extracellular and transmembrane domains
of the c-Kit receptor and the cytoplasmic domain of murine TSLPR
polypeptide. To generate this construct, the extracellular and
transmembrane domains of c-Kit and the cytoplasmic domain of TSLPR
were amplified by PCR and ligated into the retroviral vector
pMX-IRES-GFP using standard techniques.
[0357] IL-2-dependent CTLL2 cells were stably transfected with
expression constructs encoding c-Kit/TSLPR and c-Kit/.beta.,
c-Kit/.beta., and c-Kit/.gamma., or c-Kit/.gamma. alone. The
constructs for c-Kit/.beta. and c-Kit/.gamma. were as described by
Nelson et al., 1994, Nature 369: 333-36. Following transfection,
CTLL2 cells were deprived of IL-2, transferred into 48-well dishes
at 10,000 cells/well, and grown in the absence or presence of Stem
Cell Factor (SCF), the ligand for c-Kit. Cells were counted after 7
days of growth in culture.
[0358] FIG. 7 illustrates that when IL-2 was replaced by SCF, CTLL2
cells stably expressing chimeric c-Kit/TSLPR polypeptide were
unable to grow, suggesting that simple homodimerization of the
cytoplasmic domain of murine TSLPR polypeptide is insufficient to
induce a proliferative signal. Similar results have been obtained
in proliferation experiments using a chimeric c-Kit/.gamma..sub.c
polypeptide (Nelson et al., supra). Furthermore, when CTLL2 cells
were co-transfected with c-Kit/TSLPR and c-Kit/.beta., the cells
were still unable to proliferate. However, CTLL2 cells
co-transfected with c-Kit/.beta. and c-Kit/.gamma. were able to
proliferate following incubation with SCF. This suggested that the
cytoplasmic domain of the IL-2R.beta. chain could not cooperate
with the cytoplasmic domain of murine TSLPR polypeptide to initiate
proliferation, and that murine TSLPR polypeptide might oligomerize
with some other receptor to participate in signal transduction.
[0359] The similarity between murine TSLPR polypeptide and
.gamma..sub.c suggested that murine TSLPR may have the capacity to
bind to some of the members of the IL-2 cytokine subfamily. This
was examined in an affinity labeling assay using .sup.125I-labeled
IL-2, IL-4, IL-7, and IL-15. Prior to the addition of an
.sup.125I-labeled cytokine, 293 cells were reconstituted with the
cytokine specific subunits IL-2R.beta., IL-4R.alpha., or
IL-7R.alpha. in the presence of either .gamma..sub.c or murine
TSLPR polypeptide. None of the ligands examined exhibited binding
when murine TSLPR was co-expressed with a cytokine specific
subunit, even though the ligands efficiently bound when
.gamma..sub.c was co-expressed with a cytokine specific subunit.
This suggested that murine TSLPR polypeptide either bound a novel
cytokine or bound a known cytokine in conjunction with a novel or
untested subunit.
[0360] Thymic stromal lymphopoietin (TSLP) is a cytokine whose
biological activities overlap with those of IL-7. TSLP activity was
originally identified in the conditioned medium of a thymic stromal
cell line that supported the development of murine IgM.sup.+
B-cells from fetal liver hematopoietic progenitor cells (Friend et
al., 1994 Exp. Hematol. 22: 321-28). Moreover, TSLP can promote
B-cell lymphopoiesis in long-term bone marrow cultures and can
co-stimulate both thymocytes and mature T-cells (Friend et al.,
supra; Levin et al., 1999, J. Immunol. 162: 677-83).
[0361] Although IL-7 also possesses these activities (Suda et al.,
1989, Blood 74: 1936-41; Lee et al., 1989, J. Immunol. 142:
3875-83; Sudo et al., 1989, J. Exp. Med. 170: 333-38), TSLP is
unique in that it promotes B lymphopoiesis to the IgM.sup.+
immature B-cell stage, while IL-7 primarily facilitates production
of IgM.sup.- pre-B-cells (Levin et al., supra; Candeias et al.,
1997, Immunity 6: 501-08). One possible explanation for the
overlapping biological activities of IL-7 and TSLP is that TSLP
signals via a receptor containing the IL-7R.alpha. chain (Levin et
al., supra). However, antibody inhibition experiments have
indicated that TSLP does not require .gamma..sub.c to exert its
effects (Levin et al., supra). These results suggested that TSLP
would bind murine TSLPR polypeptide in the presence of
IL-7R.alpha..
[0362] The binding of TSLP to TSLPR polypeptide in the presence of
IL-7R.alpha. was examined in affinity labeling assays. Affinity
labeling assays were performed by adding 1-5 nM of
.sup.125I-labeled TSLP to 5.times.10.sup.6 293 cells transfected
with expression constructs for murine IL-7R.alpha., murine TSLPR
polypeptide, murine IL-7R.alpha. and murine TSLPR polypeptide, or
human IL-7R.alpha. and murine TSLPR polypeptide. Iodinated TSLP was
prepared by adding IODO-GEN (Pierce, Rockford, Ill.) and 2 mCi
.sup.125I to 1 .mu.g of TSLP. A specific activity of approximately
200-300 .mu.Ci/.mu.g was obtained by this method. Prior to affinity
labeling, 293 cells were transiently transfected using the calcium
phosphate method (Eppendorf-5 Prime, Boulder, Colo.). Following a 2
hour incubation with .sup.1251I-TSLP, cells were cross-linked with
0.1 mg/ml disuccinimidyl suberate (Pierce), lysed in lysis buffer,
and the lysates resolved by SDS-PAGE.
[0363] As shown in FIG. 8A, .sup.125I-TSLP bound to the heterodimer
of murine IL-7R.alpha. and murine TSLPR polypeptide (lane 4). The
upper band corresponds to cross-linked murine IL-7R.alpha. and the
lower band corresponds to cross-linked murine TSLPR polypeptide. In
addition, .sup.125I-TSLP also bound the heterodimer of human
IL-7R.alpha. and murine TSLPR polypeptide (lane 5). No TSLP binding
was observed with murine IL-7R.alpha. alone (lane 2).
[0364] Affinity labeling assays were also performed using a
FLAG-tagged version of murine TSLPR polypeptide. Murine TSLPR-FLAG
polypeptide was derived by PCR amplifying a fragment containing the
coding region of TSLPR polypeptide using a 3' primer containing
sequence corresponding to the FLAG epitope. This PCR product was
then subcloned into pCR3.1 (Invitrogen) and the resulting clone
analyzed by sequencing. Affinity labeling assays were performed as
described herein, with the exception that cell lysates were
immunoprecipitated with an anti-FLAG monoclonal M2 antibody. As
shown in FIG. 8B, following TSLPR immunoprecipitation, a
cross-linked TSLPR band was observed (lane 1), indicating that TSLP
exhibits weak binding to TSLPR alone.
[0365] To examine whether murine IL-7 could compete for TSLP
binding in cells expressing TSLPR polypeptide and IL-7R.alpha.,
competition assays were performed. Cellular lysates were analyzed
as described herein, with the exception that increasing amounts of
unlabeled murine IL-7 were added with .sup.125I-TSLP. As shown in
FIG. 8C, an excess of murine IL-7 inhibited the binding of TSLP to
the IL-7R.alpha./TSLPR polypeptide heterodimer. The affinity
labeling assays illustrated the cooperativity of IL-7R.alpha. and
murine TSLPR polypeptide for binding TSLP. These assays also
established that IL-7 can compete for the binding of TSLP, which
has implications for potential competition between these two
cytokines in vivo.
[0366] The binding of TSLP to 293 cells transfected with murine
IL-7R.alpha. and murine TSLPR polypeptide, or murine IL-7R.alpha.
alone, was analyzed in a displacement binding assay. Following two
washes, 1.times.10.sup.6 transfected 293 cells were incubated in a
constant amount of .sup.125I-labeled TSLP (approximately 20,000
cpm) and varying amounts of unlabeled TSLP. Following a 3 hour
incubation, treated cells were separated from the medium by
centrifugation in olive oil and N-butylphthalate. Cell-bound
radioactivity was measured using a gamma counter.
[0367] As shown in FIG. 9A, non-specific binding of .sup.125I-TSLP
was observed with cells transfected with murine IL-7R.alpha. alone
(or vector alone), while specific binding of .sup.125I-TSLP was
observed with cells transfected with both IL-7R.alpha. and TSLPR
polypeptide, with excess unlabeled TSLP competing for binding of
.sup.125I-TSLP. Cells transfected with TSLPR polypeptide alone
exhibited very low binding. Analysis of binding data by Scatchard
transformation was performed using the LIGAND computer program
(Munson and Rodbard, 1980, Anal. Biochem. 107: 220-39). The K.sub.d
for the binding of TSLP to cells expressing TSLPR polypeptide and
IL-7R.alpha. was determined to be approximately 13 nM (FIG. 9B). In
seven independent experiments, the K.sub.d was found to range from
1.2 to 40 nM. Due to the very low binding activity of TSLP for
cells expressing TSLPR polypeptide alone, it was not possible to
determine the K.sub.d for these cells. Displacement binding assays
were also performed using NAG8/7 cells, which constitutively
express TSLP receptors and proliferate in response to TSLP (Friend
et al., supra; Levin et al., supra). In these displacement binding
assays, 5.times.10.sup.6 NAG8/7 cells were incubated in a constant
amount of .sup.125I-labeled TSLP (approximately 180,000 cpm) and
varying amounts of unlabeled TSLP. The remainder of the assay was
performed as described herein. As shown in FIG. 9C, the Scatchard
transformation of binding data obtained using NAG8/7 cells
suggested the cells expressed a single class of receptors having a
K.sub.d of approximately 2.2 nM--results that are similar to those
obtained using the transfected 293 cells.
[0368] Displacement binding assays were also performed to compare
the displacement of .sup.125I-labeled TSLP by IL-7 or unlabeled
TSLP in 293 cells transfected with TSLPR polypeptide and
IL-7R.alpha.. FIG. 9D illustrates that murine IL-7 competes for
binding to TSLPR polypeptide.
[0369] It has been previously shown that treatment of NAG8/7 cells
with either IL-7 or TSLP activates STAT5 (Friend et al., supra;
Levin et al., supra). The possible role of TSLPR polypeptide in
STAT5 activation was analyzed in CAT assays using HepG2 cells.
Expression constructs for IL-7R.alpha. and TSLPR, or IL-7R.alpha.
and .gamma..sub.c, were introduced into HepG2 cells with the
pHRRE-CAT vector by calcium phosphate transfection. The pHRRE-CAT
vector contains eight tandem copies of the 27 bp cytokine-inducible
hematopoietin receptor response element and STAT5b (Ziegler et al.,
1995, Eur. J. Immunol. 25: 399-404). Transfected cells were allowed
to recover overnight, after which the cells were trypsinized and
plated in 6-well culture dishes. The cells were allowed to adhere
to the plates during a 24 hour incubation, and the cells were then
incubated in serum free medium containing 100 ng/ml of either IL-7
or TSLP, for an additional 24 hours.
[0370] The CAT activity and fold stimulation after normalizing for
transfection efficiencies is shown in FIG. 10. No increase in CAT
activity was seen after TSLP stimulation in the presence of
IL-7R.alpha. alone (lane 2) or with IL-7R.alpha. and .gamma..sub.c
(lane 7).
[0371] However, if TSLPR polypeptide was co-transfected, a dramatic
increase in CAT activity was observed following TSLP stimulation
(lane 5). This demonstrates that the presence of TSLPR polypeptide
is required for TSLP signaling. While co-transfection of
.gamma..sub.c and IL-7R.alpha. had no effect on TSLP-dependent
reporter activity, this combination effectively mediated
IL-7-dependent reporter activation (lane 9).
[0372] A number of cytokine receptor chains are shared by more than
one cytokine. The best known examples are gp130, which is shared by
IL-6, IL-11, ciliary neurotropic factor, leukemia inhibitory
factor, oncostatin M, and cardiotrophin-1 (Hirano et al., 1997,
Cytokine Growth Factor Rev. 8: 241-52; Taga and Kishimoto, 1997,
Annu. Rev. Immunol. 15: 797-819), .gamma..sub.c, which is shared by
IL-3, IL-5, and GM-CSF (Miyajima et al., 1997, Leukemia 11: 418-22;
Guthridge et al., 1998, Stem Cells 16: 301-13; Burdach et al.,
1998, Curr. Opin. Hematol. 5: 177-80), and .gamma..sub.c, which is
shared by IL-2, IL-4, IL-7, IL-9, and IL-15 (Noguchi et al., 1993,
Science 262: 1877-80; Kondo et al., 1994, supra; Kondo et al.,
1993, supra; Russell et al., 1994, supra; Takeshita et al., supra;
Russell et al., 1993, supra; Giri et al., supra; Kimura et al.,
supra). The list of cytokine receptor chains that serve as
components of more than one cytokine receptor includes IL-2R.beta.,
which is a component of both the IL-2 and IL-15 receptors, and
IL-4R.alpha., which is a component of both the IL-4 and IL-13
receptors. The cytokine receptor subunit IL-7R.alpha. can now be
added to this list as the data presented herein demonstrates that
this subunit is a component of both the IL-7 and TSLP
receptors.
[0373] The observation of defects in T-cell and B-cell development
in Il7.sup.-/- mice (von Freeden-Jeffrey et al., 1995, J. Exp. Med.
181: 1519-26) suggests that TSLP cannot fully compensate for the
loss of IL-7. An examination of the functional cooperation of
IL-7R.alpha. in TSLP signaling may help to explain the differences
in B-cell development in Il7r.sup.-/- and Il7.sup.-/- mice
(Candeias et al., 1997, Immunity 6: 501-08; von Freeden-Jeffrey et
al., supra; Peschon et al., 1994, J. Exp. Med. 180: 1955-60; He et
al., 1997, J. Immurzol. 158: 2592-99). The further characterization
of TSLPR polypeptide will aid this investigation.
EXAMPLE 5
Production of TSLPR Polypeptides
[0374] A. Expression of TSLPR Polypeptides in Bacteria
[0375] PCR is used to amplify template DNA sequences encoding a
TSLPR polypeptide using primers corresponding to the 5' and 3' ends
of the sequence. The amplified DNA products may be modified to
contain restriction enzyme sites to allow for insertion into
expression vectors. PCR products are gel purified and inserted into
expression vectors using standard recombinant DNA methodology. An
exemplary vector, such as pAMG21 (ATCC no. 98113) containing the
lux promoter and a gene encoding kanamycin resistance is digested
with Bam HI and Nde I for directional cloning of inserted DNA. The
ligated mixture is transformed into an E. coli host strain by
electroporation and transformants are selected for kanamycin
resistance. Plasmid DNA from selected colonies is isolated and
subjected to DNA sequencing to confirm the presence of the
insert.
[0376] Transformed host cells are incubated in 2xYT medium
containing 30 .mu.g/mL kanamycin at 30.degree. C. prior to
induction. Gene expression is induced by the addition of
N-(3-oxohexanoyl)-dl-homoserine lactone to a final concentration of
30 ng/mL followed by incubation at either 30.degree. C. or
37.degree. C. for six hours. The expression of TSLPR polypeptide is
evaluated by centrifugation of the culture, resuspension and lysis
of the bacterial pellets, and analysis of host cell proteins by
SDS-polyacrylamide gel electrophoresis. Inclusion bodies containing
TSLPR polypeptide are purified as follows. Bacterial cells are
pelleted by centrifugation and resuspended in water. The cell
suspension is lysed by sonication and pelleted by centrifugation at
195,000.times.g for 5 to 10 minutes. The supernatant is discarded,
and the pellet is washed and transferred to a homogenizer. The
pellet is homogenized in 5 mL of a Percoll solution (75% liquid
Percoll and 0.15 M NaCl) until uniformly suspended and then diluted
and centrifuged at 21,600.times.g for 30 minutes. Gradient
fractions containing the inclusion bodies are recovered and pooled.
The isolated inclusion bodies are analyzed by SDS-PAGE.
[0377] A single band on an SDS polyacrylamide gel corresponding to
E. coli-produced TSLPR polypeptide is excised from the gel, and the
N-terminal amino acid sequence is determined essentially as
described by Matsudaira et al., 1987, J. Biol. Chem. 262:
10-35.
[0378] B. Expression of TSLPR Polypeptide in Mammalian Cells
[0379] PCR is used to amplify template DNA sequences encoding a
TSLPR polypeptide using primers corresponding to the 5' and 3' ends
of the sequence. The amplified DNA products may be modified to
contain restriction enzyme sites to allow for insertion into
expression vectors. PCR products are gel purified and inserted into
expression vectors using standard recombinant DNA methodology. An
exemplary expression vector, pCEP4 (Invitrogen, Carlsbad, Calif.),
that contains an Epstein-Barr virus origin of replication, may be
used for the expression of TSLPR polypeptides in 293-EBNA-1
cells.
[0380] Amplified and gel purified PCR products are ligated into
pCEP4 vector and introduced into 293-EBNA cells by lipofection. The
transfected cells are selected in 100 .mu.g/mL hygromycin and the
resulting drug-resistant cultures are grown to confluence. The
cells are then cultured in serum-free media for 72 hours. The
conditioned media is removed and TSLPR polypeptide expression is
analyzed by SDS-PAGE.
[0381] TSLPR polypeptide expression may be detected by silver
staining. Alternatively, TSLPR polypeptide is produced as a fusion
protein with an epitope tag, such as an IgG constant domain or a
FLAG epitope, which may be detected by Western blot analysis using
antibodies to the peptide tag.
[0382] TSLPR polypeptides may be excised from an SDS-polyacrylamide
gel, or TSLPR fusion proteins are purified by affinity
chromatography to the epitope tag, and subjected to N-terminal
amino acid sequence analysis as described herein.
[0383] C. Expression and Purification of TSLPR Polypeptide in
Mammalian Cells
[0384] TSLPR polypeptide expression constructs are introduced into
293 EBNA or CHO cells using either a lipofection or calcium
phosphate protocol.
[0385] To conduct functional studies on the TSLPR polypeptides that
are produced, large quantities of conditioned media are generated
from a pool of hygromycin selected 293 EBNA clones. The cells are
cultured in 500 cm Nunc Triple Flasks to 80% confluence before
switching to serum free media a week prior to harvesting the media.
Conditioned media is harvested and frozen at -20.degree. C. until
purification.
[0386] Conditioned media is purified by affinity chromatography as
described below.
[0387] The media is thawed and then passed through a 0.2 .mu.m
filter. A Protein G column is equilibrated with PBS at pH 7.0, and
then loaded with the filtered media. The column is washed with PBS
until the absorbance at A.sub.280 reaches a baseline. TSLPR
polypeptide is eluted from the column with 0.1 M Glycine-HCl at pH
2.7 and immediately neutralized with 1 M Tris-HCl at pH 8.5.
Fractions containing TSLPR polypeptide are pooled, dialyzed in PBS,
and stored at -70.degree. C.
[0388] For Factor Xa cleavage of the human TSLPR polypeptide-Fc
fusion polypeptide, affinity chromatography-purified protein is
dialyzed in 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl.sub.2 at pH 8.0.
The restriction protease Factor Xa is added to the dialyzed protein
at 1/100 (w/w) and the sample digested overnight at room
temperature.
EXAMPLE 6
Production of Anti-TSLPR Polypeptide Antibodies
[0389] Antibodies to TSLPR polypeptides may be obtained by
immunization with purified protein or with TSLPR peptides produced
by biological or chemical synthesis.
[0390] Suitable procedures for generating antibodies include those
described in Hudson and Bay, Practical Immunology (2nd ed.,
Blackwell Scientific Publications).
[0391] In one procedure for the production of antibodies, animals
(typically mice or rabbits) are injected with a TSLPR antigen (such
as a TSLPR polypeptide), and those with sufficient serum titer
levels as determined by ELISA are selected for hybridoma
production. Spleens of immunized animals are collected and prepared
as single cell suspensions from which splenocytes are recovered.
The splenocytes are fused to mouse myeloma cells (such as
Sp2/0-Ag14 cells), are first incubated in DMEM with 200 U/mL
penicillin, 200 .mu.g/mL streptomycin sulfate, and 4 mM glutamine,
and are then incubated in HAT selection medium (hypoxanthine,
aminopterin, and thymidine). After selection, the tissue culture
supernatants are taken from each fusion well and tested for
anti-TSLPR antibody production by ELISA.
[0392] Alternative procedures for obtaining anti-TSLPR antibodies
may also be employed, such as the immunization of transgenic mice
harboring human Ig loci for production of human antibodies, and the
screening of synthetic antibody libraries, such as those generated
by mutagenesis of an antibody variable domain.
EXAMPLE 7
Expression of TSLPR Polypeptide in Transgenic Mice
[0393] To assess the biological activity of TSLPR polypeptide, a
construct encoding a TSLPR polypeptide/Fc fusion protein under the
control of a liver specific ApoE promoter is prepared. The delivery
of this construct is expected to cause pathological changes that
are informative as to the function of TSLPR polypeptide. Similarly,
a construct containing the full-length TSLPR polypeptide under the
control of the beta actin promoter is prepared. The delivery of
this construct is expected to result in ubiquitous expression.
[0394] To generate these constructs, PCR is used to amplify
template DNA sequences encoding a TSLPR polypeptide using primers
that correspond to the 5' and 3' ends of the desired sequence and
which incorporate restriction enzyme sites to permit insertion of
the amplified product into an expression vector. Following
amplification, PCR products are gel purified, digested with the
appropriate restriction enzymes, and ligated into an expression
vector using standard recombinant DNA techniques. For example,
amplified TSLPR polypeptide sequences can be cloned into an
expression vector under the control of the human .beta.-actin
promoter as described by Graham et al., 1997, Nature Genetics, 17:
272-74 and Ray et al., 1991, Genes Dev. 5: 2265-73.
[0395] Following ligation, reaction mixtures are used to transform
an E. coli host strain by electroporation and transformants are
selected for drug resistance. Plasmid DNA from selected colonies is
isolated and subjected to DNA sequencing to confirm the presence of
an appropriate insert and absence of mutation. The TSLPR
polypeptide expression vector is purified through two rounds of
CsCl density gradient centrifugation, cleaved with a suitable
restriction enzyme, and the linearized fragment containing the
TSLPR polypeptide transgene is purified by gel electrophoresis. The
purified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2 mM
EDTA at a concentration of 2 mg/mL.
[0396] Single-cell embryos from BDF1.times.BDF1 bred mice are
injected as described (PCT Pub. No. WO 97/23614). Embryos are
cultured overnight in a CO.sub.2 incubator and 15-20 two-cell
embryos are transferred to the oviducts of a pseudopregnant CD1
female mice. Offspring obtained from the implantation of
microinjected embryos are screened by PCR amplification of the
integrated transgene in genomic DNA samples as follows.
[0397] Ear pieces are digested in 20 mL ear buffer (20 mM Tris, pH
8.0, 10 mM EDTA, 0.5% SDS, and 500 mg/mL proteinase K) at
55.degree. C. overnight. The sample is then diluted with 200 mL of
TE, and 2 mL of the ear sample is used in a PCR reaction using
appropriate primers.
[0398] At 8 weeks of age, transgenic founder animals and control
animals are sacrificed for necropsy and pathological analysis.
Portions of spleen are removed and total cellular RNA isolated from
the spleens using the Total RNA Extraction Kit (Qiagen) and
transgene expression determined by RT-PCR. RNA recovered from
spleens is converted to cDNA using the SuperScript.TM.
Preamplification System (Gibco-BRL) as follows. A suitable primer,
located in the expression vector sequence and 3' to the TSLPR
polypeptide transgene, is used to prime cDNA synthesis from the
transgene transcripts.
[0399] Ten mg of total spleen RNA from transgenic founders and
controls is incubated with 1 mM of primer for 10 minutes at
70.degree. C. and placed on ice. The reaction is then supplemented
with 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl.sub.2, 10 mM of
each dNTP, 0.1 mM DTT, and 200 U of SuperScript II reverse
transcriptase. Following incubation for 50 minutes at 42.degree.
C., the reaction is stopped by heating for 15 minutes at 72.degree.
C. and digested with 2U of RNase H for 20 minutes at 37.degree. C.
Samples are then amplified by PCR using primers specific for TSLPR
polypeptide.
[0400] Determining the phenotypes of Tslp.sup.-/- or Tslpr.sup.-/-
mice will also assist in defining the exact role of TSLP.
EXAMPLE 8
Biological Activity of TSLPR Polypeptide in Transgenic Mice
[0401] Prior to euthanasia, transgenic animals are weighed,
anesthetized by isofluorane and blood drawn by cardiac puncture.
The samples are subjected to hematology and serum chemistry
analysis. Radiography is performed after terminal exsanguination.
Upon gross dissection, major visceral organs are subject to weight
analysis.
[0402] Following gross dissection, tissues (i.e., liver, spleen,
pancreas, stomach, the entire gastrointestinal tract, kidney,
reproductive organs, skin and mammary glands, bone, brain, heart,
lung, thymus, trachea, esophagus, thyroid, adrenals, urinary
bladder, lymph nodes and skeletal muscle) are removed and fixed in
10% buffered Zn-Formalin for histological examination. After
fixation, the tissues are processed into paraffin blocks, and 3 mm
sections are obtained. All sections are stained with hematoxylin
and exosin, and are then subjected to histological analysis.
[0403] The spleen, lymph node, and Peyer's patches of both the
transgenic and the control mice are subjected to immunohistology
analysis with B cell and T cell specific antibodies as follows. The
formalin fixed paraffin embedded sections are deparaffinized and
hydrated in deionized water. The sections are quenched with 3%
hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh,
Pa.), and incubated in rat monoclonal anti-mouse B220 and CD3
(Harlan, Indianapolis, Ind.). Antibody binding is detected by
biotinylated rabbit anti-rat immunoglobulins and peroxidase
conjugated streptavidin (BioGenex, San Ramon, Calif.) with DAB as a
chromagen (BioTek, Santa Barbara, Calif.). Sections are
counterstained with hematoxylin.
[0404] After necropsy, MLN and sections of spleen and thymus from
transgenic animals and control littermates are removed. Single cell
suspensions are prepared by gently grinding the tissues with the
flat end of a syringe against the bottom of a 100 mm nylon cell
strainer (Becton Dickinson, Franklin Lakes, N.J.). Cells are washed
twice, counted, and approximately 1.times.10.sup.6 cells from each
tissue are then incubated for 10 minutes with 0.5 .mu.g
CD16/32(Fc.gamma.III/II) Fc block in a 20 .mu.L volume. Samples are
then stained for 30 minutes at 2-8.degree. C. in a 100 .mu.L volume
of PBS (lacking Ca.sup.+ and Mg.sup.+), 0.1% bovine serum albumin,
and 0.01% sodium azide with 0.5 .mu.g antibody of FITC or
PE-conjugated monoclonal antibodies against CD90.2 (Thy-1.2), CD45R
(B220), CD11b (Mac-1), Gr-1, CD4, or CD8 (PharMingen, San Diego,
Calif.). Following antibody binding, the cells are washed and then
analyzed by flow cytometry on a FACScan (Becton Dickinson).
[0405] While the present invention has been described in terms of
the preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations that come within the scope of the invention as
claimed.
* * * * *