U.S. patent application number 11/400219 was filed with the patent office on 2006-10-19 for interleukin-19.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Joseph J. Kenny, Craig A. Rosen.
Application Number | 20060233749 11/400219 |
Document ID | / |
Family ID | 26698980 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060233749 |
Kind Code |
A1 |
Rosen; Craig A. ; et
al. |
October 19, 2006 |
Interleukin-19
Abstract
The present invention concerns a novel human cytokine. In
particular, isolated nucleic acid molecules are provided encoding
interleukin-19 (IL-19). IL-19 polypeptides are also provided, as
are vectors, host cells and recombinant methods for producing the
same. The IL-19 polypeptides of the present invention can also be
used to raise polyclonal and monoclonal antibodies. The invention
further concerns therapeutic methods for modulating cytokine
production.
Inventors: |
Rosen; Craig A.;
(Laytonsville, MD) ; Kenny; Joseph J.; (Boston,
MA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
26698980 |
Appl. No.: |
11/400219 |
Filed: |
April 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10425961 |
Apr 30, 2003 |
7056681 |
|
|
11400219 |
Apr 10, 2006 |
|
|
|
09386380 |
Aug 31, 1999 |
6583270 |
|
|
10425961 |
Apr 30, 2003 |
|
|
|
08921382 |
Aug 29, 1997 |
5985614 |
|
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09386380 |
Aug 31, 1999 |
|
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60024882 |
Aug 30, 1996 |
|
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Current U.S.
Class: |
424/85.2 ;
435/320.1; 435/325; 435/69.52; 530/351; 536/23.5 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 38/00 20130101; Y02A 50/41 20180101; C07K 14/54 20130101 |
Class at
Publication: |
424/085.2 ;
530/351; 435/069.52; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07K 14/54 20060101 C07K014/54; C07H 21/04 20060101
C07H021/04; C12P 21/04 20060101 C12P021/04 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about -24 to
about 153 in SEQ ID NO:2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about -23 to about 153 in
SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide
comprising amino acids from about 1 to about 153 in SEQ ID NO:2;
(d) a nucleotide sequence encoding a polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97662; (e) a nucleotide sequence encoding the mature IL-19
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97662; and (f) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d) or (e).
2. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d) or (e) of claim 1 wherein
said polynucleotide which hybridizes does not hybridize under
stringent hybridization conditions to a polynucleotide having a
nucleotide sequence consisting of only A residues or of only T
residues.
3. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a IL-19 polypeptide having an amino acid sequence in (a), (b),
(c), (d), or (e) of claim 1.
4. The isolated nucleic acid molecule of claim 3, which encodes an
epitope-bearing portion of a IL-19 polypeptide comprising amino
acid residues from about -5 to about 4 in SEQ ID NO:2.
5. An isolated nucleic acid molecule, comprising a polynucleotide
having a sequence selected from the group consisting of the
nucleotide sequence of a fragment of the sequence shown in SEQ ID
NO: 1 or the complement thereof, wherein said fragment comprises at
least 50 contiguous nucleotides of SEQ ID NO: 1, provided that said
fragment does not have a sequence starting: (a) at nucleotide 9 and
ending at nucleotide 302 of SEQ ID NO:1 or any subfragment thereof
or the complement thereof; or (b) at nucleotide 438 and ending at
nucleotide 934 of SEQ ID NO:1 or any subfragment thereof or the
complement thereof.
6. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
7. A recombinant vector produced by the method of claim 6.
8. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 7 into a host cell.
9. A recombinant host cell produced by the method of claim 8.
10. A recombinant method for producing a IL-19 polypeptide,
comprising culturing the recombinant host cell of claim 9 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
11. An isolated IL-19 polypeptide having an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of: (a) amino acids from about -24 to about 153 in SEQ
ID NO:2; (b) amino acids from about -23 to about 153 in SEQ ID
NO:2; (c) amino acids from about 1 to about 153 in SEQ ID NO:2; (d)
the amino acid sequence of the IL-19 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97662; (e) the amino acid sequence of the mature IL-19
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97662; and (f) the amino acid
sequence of an epitope-bearing portion of any one of the
polypeptides of (a), (b), (c), (d) or (e).
12. An isolated polypeptide comprising an epitope-bearing portion
of the IL-19 protein, wherein said portion is selected from the
group consisting of: a polypeptide comprising amino acid residues
from about -5 to about 4 in SEQ ID NO:2; a polypeptide comprising
amino acid residues from about 64 to about 82 in SEQ ID NO:2; and a
polypeptide comprising amino acid residues from about 115 to about
125 in SEQ ID NO:2.
13. The isolated polypeptide of claim 11, which is produced or
contained in a recombinant host cell.
14. The isolated polypeptide of claim 11, wherein said recombinant
host cell is mammalian.
15. An isolated nucleic acid molecule comprising a polynucleotide
encoding an IL-19 polypeptide wherein, except for at least one to
fifty conservative amino acid substitution, said polypeptide has a
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide comprising amino acids from about
-24 to about 153 in SEQ ID NO:2; (b) a nucleotide sequence encoding
a polypeptide comprising amino acids from about -23 to about 153 in
SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide
comprising amino acids from about 1 to about 153 in SEQ ID NO:2;
(d) a nucleotide sequence encoding a polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97662; (e) a nucleotide sequence encoding the mature IL-19
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97662; and (f) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d) or (e).
16. An isolated IL-19 polypeptide wherein, except for at least one
to fifty conservative amino acid substitution, said polypeptide has
a sequence selected from the group consisting of: (a) amino acids
from about -24 to about 153 in SEQ ID NO:2; (b) amino acids from
about -23 to about 153 in SEQ ID NO:2; (c) amino acids from about 1
to about 153 in SEQ ID NO:2; (d) the amino acid sequence of the
IL-19 polypeptide having the amino acid sequence encoded by the
cDNA clone contained in ATCC Deposit No. 97662; (e) the amino acid
sequence of the mature IL-19 polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97662; and (f) the amino acid sequence of an epitope-bearing
portion of any one of the polypeptides of (a), (b), (c), (d) or
(e).
17. A method for treatment of an individual in need of a decreased
level of IFN-.gamma., TNF-.alpha., or IL-6 activity comprising
administering to said individual a composition comprising an
isolated polypeptide of claim 16.
18. A method for treatment of an individual in need of an increase
in IL-2 activity, comprising administering to said individual a
composition comprising an antagonist of interleukin-19 activity.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 10/425,961, filed Apr. 30, 2003, (now U.S. Pat. No. 7,056,681),
which is herein incorporated by reference; said 10/425,961 is a
divisional of U.S. application Ser. No. 09/386,380, filed Aug. 31,
1999, (now U.S. Pat. No. 6,583,270) which is herein incorporated by
reference; said 09/386,380 is a divisional of U.S. application Ser.
No. 08/921,382, filed Aug. 29, 1997, (now U.S. Pat. No. 5,985,614)
which is herein incorporated by reference; said 08/921,382 claims
the benefit of Provisional Application 60/024,882, filed on Aug.
30, 1996, which is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns a novel human cytokine. In
particular, isolated nucleic acid molecules are provided encoding
interleukin-19 (IL-19). IL-19 polypeptides are also provided, as
are vectors, host cells and recombinant methods for producing the
same. The invention further concerns therapeutic methods for
modulating cytokine production.
[0004] 2. Related Art
[0005] Interleukin-10 (IL-10) is a pleiotropic cytokine that has
been implicated as an important regulator of the functions of
lymphoid and myeloid cells. IL-10 blocks activation of cytokine
synthesis and several accessory functions of macrophages, thus
acting as a potent suppressor of the effector functions of
macrophages, T cells and NK cells. IL-10 has also been implicated
in the regulation of differentiation of B cells, mast cells and
thymocytes.
[0006] IL-10 was identified independently in two different lines of
experiments. One of these identified a B-cell-derived mediator
which co-stimulated active thymocytes (Suda et al., Cell Immunol.
129:228 (1990)). The other identification determined that IL-10 is
involved in the cross-regulation between two often mutually
exclusive effector arms of immunity carried out by T helper
(CD4.sup.+) subpopulations, Th1 (involved in cell-mediated immune
responses) and Th2 (involved in antibody-mediated immune
responses). In this role, IL-10 is expressed by Th2 cells and
functions to suppress cytokine production by Th1 cells, an activity
termed cytokine synthesis inhibitory factor (CSIF) activity.
[0007] cDNA clones encoding murine IL-10 (mIL-10) were isolated
based on the expression of CSIF activity (Moore et al., Science
248:1230-34 (1990)). cDNA clones encoding human IL-10 (hIL-10) were
subsequently identified by cross-hybridization with the mouse cDNA
(Vieira et al., Proc. Natl. Acad. Sci. USA 88:1172-1176 (1991)).
mIL-10 is expressed by mouse CD4.sup.+ Th2 cells, at least one
CD8.sup.+ clone, B lymphomas, T cells, activated mast cell lines,
activated macrophages, keratinocytes, and Ly-1 B (B-1) cells
(Fiorentino, D. F. et al., J. Exp. Med. 170:2081 (1989); Moore et
al., Science 248:1230-34 (1990); Hisatsune et al., Lymphokine
Cytokine Res. 11:87-93 (1992); Lin et al., Ann. N.Y. Acad. Sci.
651:581-583 (1992); O'Garra et al., Int. Immunol. 2: 821-832
(1990); MacNeil et al., J. Immunol. 145: 4167-4173 (1990);
Fiorentino et al., J. Immunol. 147:3815-3822 (1991)). hIL-10 is
expressed by human CD4.sup.+ T cells and Th0, Th1, and Th2 T cell
clones, by CD8.sup.+ T cells and clones (Yssel et al., J. Immunol.
149:2378-2384), monocytes/macrophages, keratinocytes, activated B
cells, B lymphomas, and Burkitt lymphoma lines infected with a
transforming EBV strain, but not with a non-transforming strain
(Vieira, P. et al., Proc. Natl. Acad. Sci. USA 88:1172-76 (1991);
de Waal-Malefyt, R. et al., J. Exp. Med. 174:1209-20 (1991); de
Waal-Malefyt, R. et al., J. Exp. Med. 174:915-24 (1991); Salgame,
P. et al., Science 254:279-82 (1991); Yamamura, M. et al., Science
254:277-79 (1991); Ralph, P. et al., J. Immunol. 148:808-14 (1992);
Benjamin, D. et al., Blood 80:1289-98 (1992)). Thus, IL-10 is not
strictly a Th2-specific cytokine, and its pattern of expression
resembles IL-6 more than IL-4 or IL-5 (Wang, S. C. et al.,
Transplant. Proc. 23:2920-22 (1991)). Like IL-6 but unlike most
other T cell derived cytokines, IL-10 expression is not inhibited
by cyclosporin or FK-506 (Wang, S. C. et al., Transplant Proc.
23:2920 (1991)).
[0008] In an attempt to determine the in vivo role of IL-10, normal
mice were treated from birth to adulthood with IL-10-neutralizing
antibodies (Wang, S. C. et al., Transplant Proc. 23:2920 (1991);
Ishida, H. et al., J. Exp. Med. 175:1213-1220 (1992)). The
resulting phenotypic changes included an increased level of
circulating IFN-.gamma., TNF-.alpha. and IL-6, reduced serum IgM
and IgA, a marked depletion of peritoneal B cells, and an inability
to develop in vivo antibody responses to two bacterial antigens
known be combated with antibody produced by peritoneal B cells
(Hayakawa, K. et al., Annu. Rev. Immunol. 6:197-218 (1988)). The
reduction in peritoneal B cells was determined to be a consequence
of IFN-.gamma. elevation (Ishida, H. et al., J. Exp. Med. 175:1213
(1992)).
[0009] Other experiments have shown that IL-10 suppresses in vitro
production of inflammatory monokines such as TNF-.alpha. and IL-1.
This data corresponds to in vivo studies which show that IL-10
antagonists elevate the same inflammatory monokines. These results
predict a strong anti-inflammatory role for IL-10. In addition,
IL-10 antagonists may be useful to enhance Th1 immunity, which
could be beneficial in infectious diseases of viral origin, or
diseases involving intracellular pathogens.
[0010] The diverse biological activities of IL-10 have led to
predictions that both IL-10 and its antagonists will have a wide
range of clinical applications. It is clear that there is a
continuing need in the art for isolating novel cytokines capable of
mediating such diverse biological processes.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a human IL-19
polypeptide having the amino acid sequence in FIG. 1 [SEQ ID NO: 2]
or the amino acid sequence encoded by the cDNA clone deposited as
ATCC.RTM. Deposit Number 97662 on Jul. 17, 1996. The nucleotide
sequence determined by sequencing the deposited IL-19 cDNA clone,
which is shown in FIG. 1 [SEQ ID NO: 1], contains an open reading
frame encoding a polypeptide of about 177 amino acid residues
including an initiation codon at nucleotide positions 44-46, a
leader sequence of about 24 amino acid residues and a deduced
molecular weight of about 20.4 kDa. The 153 amino acid sequence of
the predicted mature IL-19 protein is shown in FIG. 1 (last 153
residues) and in SEQ ID NO:2 (from amino acid residue 1 to residue
153).
[0012] The present invention also relates to recombinant vectors
which include the isolated nucleic acid molecules of the present
invention and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of IL-19 polypeptides or peptides by
recombinant techniques.
[0013] The invention further provides an isolated IL-19 polypeptide
having an amino acid sequence encoded by a polynucleotide described
herein.
[0014] IL-19, which is secreted, and which has significant homology
to IL-10, is believed by the present inventors to be expressed
only, or at least primarily, in activated monocytes (FIG. 4). Thus,
detecting IL-19 gene expression in cells of the immune system is
useful for identifying activated monocytes. Further, for a number
of disorders, it is believed by the inventors that significantly
higher or lower levels of IL-19 gene expression can be detected in
bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal
fluid) taken from an individual having such a disorder, relative to
a "standard" IL-19 gene expression level, i.e., the IL-19
expression level in bodily fluids from an individual not having the
disorder. Thus, the invention provides a diagnostic method useful
during diagnosis of a disorder related to an abnormal level of
IL-19 gene expression, which involves (a) assaying IL-19 gene
expression level in cells or body fluid of that individual; (b)
comparing that IL-19 gene expression level with a standard IL-19
gene expression level, whereby an increase or decrease in the
assayed IL-19 gene expression level compared to the standard
expression level is indicative of a disorder. An additional aspect
of the invention is related to a method for treating an individual
in need of an increased level of IL-19 activity in the body, which
involves administering to such an individual a composition
comprising an IL-19 polypeptide of the invention. A still further
aspect of the invention is related to a method of treating an
individual in need of a decreased level of IL-19 activity in the
body, which involves administering to such an individual a
composition comprising an antagonist to IL-19 such as anti-IL-19
antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0015] FIG. 1 shows the nucleotide [SEQ ID NO: 1] and deduced amino
acid [SEQ ID NO:2] sequences of the complete IL-19 protein
determined by sequencing of the DNA clone contained in ATCC.RTM.
Deposit No. 97662. The protein has a leader sequence of about 24
amino acid residues (underlined) and a deduced molecular weight of
about 20.4 kDa. The amino acid sequence of the predicted mature
IL-19 protein is also shown in FIG. 1 (last 153 amino acids)
[residues 1-153 of SEQ ID NO:2].
[0016] FIG. 2 is a protein gel showing IL-19 protein expressed from
E. coli strain M15rep4 (see Example 1).
[0017] FIG. 3 is a protein gel showing full length and truncated
IL-19 proteins produced in an in vitro coupled
transcription/translation system (see Example 3).
[0018] FIG. 4 is a northern blot analysis of IL-19 expression in
human tissue (HL-60, THP-1, U937 and primary human monocytes) (see
Example 5).
[0019] FIG. 5 shows the regions of similarity between the amino
acid sequences of the IL-19 protein and human IL-10 (hIL-10) [SEQ
ID NO: 3].
[0020] FIG. 6 shows an analysis of the IL-19 amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, amino acid residues about 20 to about
28, about 88 to about 106, and about 139 to about 149 in FIG. 1
correspond to the shown highly antigenic regions of the IL-19
protein. These highly antigenic fragments in FIG. 1 correspond to
the following fragments, respectively, in SEQ ID NO:2 amino acid
residues about -5 to about 4, about 64 to about 82, and about 115
to about 125.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding the IL-19 protein
having the amino acid sequence shown in FIG. 1 [SEQ ID NO:2] which
was determined by sequencing a cloned cDNA. The IL-19 protein of
the present invention shares sequence homology with human IL-10
(FIG. 5) [SEQ ID NO:3]. The nucleotide sequence shown in FIG. 1
[SEQ ID NO:1] was obtained by sequencing the HMQBM23 cDNA clone
encoding an IL-19 polypeptide, which was deposited on Jul. 17, 1996
at the American Type Culture Collection, Patent Depository, 10801
University Boulevard, Manassas, Va. 20110-2209, and given accession
number 97662. The deposited clone is contained in the pBluescript
SK(-) plasmid (Stratagene, La Jolla, Calif.).
Nucleic Acid Molecules
[0022] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0023] Using the information provided herein, such as the
nucleotide sequence in FIG. 1, a nucleic acid molecule of the
present invention encoding an IL-19 polypeptide may be obtained
using standard cloning and screening procedures, such as those for
cloning cDNAs using mRNA as starting material. Illustrative of the
invention, the nucleic acid molecule described in FIG. 1 [SEQ ID
NO:1] was discovered in a cDNA library derived from human activated
monocytes. The determined nucleotide sequence of the IL-19 cDNA of
FIG. 1 contains an open reading frame encoding a protein of about
177 amino acid residues with an initiation codon at positions 44-46
of the nucleotide sequence shown in FIG. 1 [SEQ ID NO. 1], and a
predicted leader sequence of about 24 amino acid residues, and a
deduced molecular weight of about 20.4 kDa. The amino acid sequence
of the predicted mature IL-19 protein is from about amino acid
residue 25 to about residue 177 shown in FIG. 1 or amino acids
1-153 shown in SEQ ID NO:2. The IL-19 protein shown in FIG. 1 [SEQ
ID NO:2] is about 20% identical and about 46% similar to human
IL-10 (FIG. 5).
[0024] The present invention also provides the mature form(s) of
the IL-19 protein of the present invention. According to the signal
hypothesis, proteins secreted by mammalian cells have a signal or
secretory leader sequence which is cleaved from the mature protein
once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and
even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species on the protein. Further, it has long been known that the
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide. Therefore,
the present invention provides a nucleotide sequence encoding the
mature IL-19 polypeptides having the amino acid sequence encoded by
the cDNA clone contained in the host identified as ATCC.RTM.
Deposit No. 97662 and as shown in SEQ ID NO:2. By the mature IL-19
protein having the amino acid sequence encoded by the cDNA clone
contained in the host identified as ATCC.RTM. Deposit 97662 is
meant the mature form(s) of the IL-19 protein produced by
expression in a mammalian cell (e.g., COS cells, as described
below) of the complete open reading frame encoded by the human DNA
sequence of the clone contained in the vector in the deposited
host. As indicated below, the mature IL-19 having the amino acid
sequence encoded by the cDNA clone contained in ATCC.RTM. Deposit
No. 97662 may or may not differ from the predicted "mature" IL-19
protein shown in SEQ ID NO:2 (amino acids from about 1 to about
153) depending on the accuracy of the predicted cleavage site based
on computer analysis.
[0025] Methods for predicting whether a protein has a secretory
leader as well as the cleavage point for that leader sequence are
available. For instance, the methods of McGeoch (Virus Res.
3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690
(1986)) can be used. The accuracy of predicting the cleavage points
of known mammalian secretory proteins for each of these methods is
in the range of 75-80%. von Heinje, supra. However, the two methods
do not always produce the same predicted cleavage point(s) for a
given protein.
[0026] In the present case, the predicted amino acid sequence of
the complete IL-19 polypeptides of the present invention were
analyzed by a computer program ("PSORT") (K. Nakai and M. Kanehisa,
Genomics 14:897-911 (1992)), which is an expert system for
predicting the cellular location of a protein based on the amino
acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated. The analysis by the PSORT program predicted the
cleavage site between amino acids -1 and 1 in SEQ ID NO:2.
Thereafter, the complete amino acid sequences were further analyzed
by visual inspection, applying a simple form of the (-1,-3) rule of
von Heinje. von Heinje, supra. Thus, the leader sequence for the
IL-19 protein is predicted to consist of amino acid residues from
about -24 to about -1 in SEQ ID NO:2, while the mature IL-19
protein is predicted to consist of residues from about 1 to about
153.
[0027] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, as well as the
variability of cleavage sites for leaders in different known
proteins, the actual IL-19 polypeptide encoded by the deposited
cDNA comprises about 177 amino acids, but may be anywhere in the
range of 170-183 amino acids; and the actual leader sequence of
this protein is about 24 amino acids, but may be anywhere in the
range of about 18 to about 29 amino acids.
[0028] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0029] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0030] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) with
an initiation codon at positions 44-46 of the nucleotide sequence
shown in FIG. 1 [SEQ ID NO:1]; DNA molecules comprising the coding
sequence for the mature IL-19 protein shown in FIG. 1 (last 153
amino acids) [SEQ ID NO:2]; and DNA molecules which comprise a
sequence substantially different from those described above but
which, due to the degeneracy of the genetic code, still encode the
IL-19 protein. Of course, the genetic code is well known in the
art. Thus, it would be routine for one skilled in the art to
generate the degenerate variants described above.
[0031] In addition, the invention provides nucleic acid molecules
having nucleotide sequences related to extensive portions of SEQ ID
NO: 1 which have been determined from the following related cDNA
clones: PO14 (SEQ ID NO:16).
[0032] Sequences of public ESTs that relate to a portion of SEQ ID
NO: 1 have the following GenBank Accession Numbers: AA151656 (SEQ
ID NO:12), AA151652 (SEQ ID NO:13), AA151733 (SEQ ID NO:14), and
AA151736 (SEQ ID NO:15).
[0033] In another aspect, the invention provides isolated nucleic
acid molecules encoding the IL-19 polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC.RTM. Deposit No. 97662 on Jul. 17, 1996. In a
further embodiment, nucleic acid molecules are provided encoding
the mature IL-19 polypeptide or the full-length IL-19 polypeptide
lacking the N-terminal methionine. The invention also provides an
isolated nucleic acid molecule having the nucleotide sequence shown
in SEQ ID NO:1 or the nucleotide sequence of the above-described
deposited cDNA clone. The invention further provides an isolated
nucleic acid molecule having the nucleotide sequence shown in FIG.
1 [SEQ ID NO:1] or the nucleotide sequence of the IL-19 cDNA
contained in the above-described deposited clone, or nucleic acid
molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping by in situ hybridization with
chromosomes and for detecting expression of the IL-19 gene in human
tissue, for instance, by Northern blot analysis.
[0034] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in SEQ ID
NO:1 is intended fragments at least about 15 nt, and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably, at least about 40 nt in
length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments 50, 75, 100, 125,
150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,
800, 825, 850, 875, 900, 925 or 950 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in SEQ ID NO:1. By a fragment at least 20 nt in
length, for example, is intended fragments which include 20 or more
contiguous bases from the nucleotide sequence of the deposited cDNA
or the nucleotide sequence as shown in SEQ ID NO:1.
[0035] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in
ATCC.RTM. Deposit No. 97662. By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0036] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0037] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:1). Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3' terminal poly(A) tract of the IL-19 cDNA
shown in SEQ ID NO:1), or to a complementary stretch of T (or U)
resides, would not be included in a polynucleotide of the invention
used to hybridize to a portion of a nucleic acid of the invention,
since such a polynucleotide would hybridize to any nucleic acid
molecule containing a poly (A) stretch or the complement thereof
(e.g., practically any double-stranded cDNA clone).
[0038] Since an IL-19 cDNA clone has been deposited and its
determined nucleotide sequence is provided in FIG. 1 [SEQ ID NO:1],
generating polynucleotides which hybridize to a portion of the
IL-19 cDNA molecule would be routine to the skilled artisan. For
example, restriction endonuclease cleavage or shearing by
sonication of the IL-19 cDNA clone could easily be used to generate
DNA portions of various sizes which are polynucleotides that
hybridize to a portion of the IL-19 cDNA molecule. Alternatively,
the hybridizing polynucleotides of the present invention could be
generated synthetically according to known techniques. Of course, a
polynucleotide which hybridizes only to a poly A sequence (such as
the 3' terminal poly(A) tract of the IL-19 cDNA shown in FIG. 1
[SEQ ID NO:1]), or to a complementary stretch of T (or U) resides,
would not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0039] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the IL-19 protein. In particular, such nucleic acid fragments of
the present invention include nucleic acid molecules encoding: a
polypeptide comprising amino acid residues from about -5 to about 4
in SEQ ID NO:2; a polypeptide comprising amino acid residues from
about 64 to about 82 in SEQ ID NO:2; and a polypeptide comprising
amino acid residues from about 115 to about 125 in SEQ ID NO:2. The
inventors have determined that the above polypeptide fragments are
antigenic regions of the IL-19 protein. Methods for determining
other such epitope-bearing portions of the IL-19 protein are
described in detail below.
[0040] As indicated, nucleic acid molecules of the present
invention which encode the IL-19 polypeptide may include, but are
not limited to those encoding the amino acid sequence of the mature
polypeptide, by itself; the coding sequence for the mature
polypeptide and additional sequences, such as those encoding the
about 24 amino acid leader or secretory sequence, such as a pre-,
or pro- or prepro-protein sequence; the coding sequence of the
mature polypeptide, with or without the aforementioned additional
coding sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing--including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (Qiagen, Inc.), among others, many of
which are commercially available. As described in Gentz et al.
Proc. Natl. Acad. Sci., USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson et al.,
Cell 37: 767-768 (1984). As discussed below, other such fusion
proteins include the IL-19 fused to Fc at the N- or C-terminus.
[0041] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the IL-19 protein. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, ed. Non-naturally occurring variants may be produced using
art-known mutagenesis techniques.
[0042] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding or non-coding regions or both. Alterations in
the coding regions may produce conservative or non-conservative
amino acid substitutions, deletions or additions. Especially
preferred among these are silent substitutions, additions and
deletions, which do not alter the properties and activities of the
IL-19 protein or portions thereof. Also especially preferred in
this regard are conservative substitutions.
[0043] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide
sequence encoding the polypeptide having the amino acid sequence in
SEQ ID NO:2; (b) a nucleotide sequence encoding the polypeptide
having the amino acid sequence in SEQ ID NO:2, but lacking the
N-terminal methionine; (c) a nucleotide sequence encoding the
polypeptide having the amino acid sequence at positions from about
1 to about 153 in SEQ ID NO:2; (d) a nucleotide sequence encoding
the polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.RTM. Deposit No. 97662; (e) a nucleotide
sequence encoding the mature IL-19 polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC.RTM.
Deposit No. 97662; or (f) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), (c), (d) or (e).
[0044] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding an IL-19 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the IL-19 polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0045] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in FIG. 1 or to the
nucleotide sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711. Bestfit uses the local homology
algorithm of Smith and Waterman (Advances in Applied Mathematics 2:
482-489 (1981)) to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0046] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 [SEQ ID NO: 1] or to the
nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having IL-19 activity. This is
because, even where a particular nucleic acid molecule does not
encode a polypeptide having IL-19 activity, one of skill in the art
would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having IL-19 activity
include, inter alia, (1) isolating the IL-19 gene or allelic
variants thereof in a cDNA library; (2) in situ hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the IL-19 gene as described in Verma et
al., Human Chromosomes: a Manual of Basic Techniques, Pergamon
Press, New York (1988); and (3) Northern Blot analysis for
detecting IL-19 mRNA expression in specific cell types (e.g.,
activated monocytes).
[0047] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 [SEQ ID NO:1] or to the
nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having IL-19 protein activity. By "a
polypeptide having IL-19 activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the IL-19 protein of the invention (either the
full-length protein, or preferably, the mature protein) as measured
in a particular biological assay.
[0048] IL-19 exhibits several biological activities which could
form the basis of such biological assays. In particular, it is
believed by the inventors that IL-19 has the property of modulating
the synthesis of at least one cytokine in the group consisting of
IFN-.gamma., lymphotoxin, IL-2, IL-3, and GM-CSF in a population of
T helper cells induced to synthesize one or more of these cytokines
by exposure to syngeneic antigen-presenting cells (APCs) and
antigen. In this activity, APCs are treated so that they become
incapable of replication, but their antigen-processing machinery
remains functional. This is conveniently accomplished by
irradiating the APCs, e.g. with about 1500-3000 R (gamma or
X-radiation) before mixing with the T-cells.
[0049] Alternatively, changes in levels of cytokine production may
be assayed in primary, or preferably, secondary mixed-lymphocyte
reactions (MLR), in which case syngeneic APCs need not be used.
MLRs are well-known in the art, e.g., Bradley, pp 162-166 in
Mishell et al., eds. Selected Methods in Cellular Immunology
(Freeman, San Francisco, 1980); and Battisto, et al., Meth, in
Enzymol. 150:83-91 (1987). Briefly, the cell populations are mixed,
one of the populations having been treated prior to mixing to
prevent proliferation, e.g., by irradiation. Preferably, the cell
populations are prepared at a concentration of about
2.times.10.sup.6 cells/ml in supplemented media, e.g. RPMI 1640
with 10% fetal calf serum. For both controls and test cultures, mix
0.05 ml of each population for the assay. For a secondary MLR, the
cells remaining after 7 days in the primary MLR are re-stimulated
by freshly prepared, irradiated stimulator cells. The sample
suspected of containing IL-19 may be added to the test cultures at
the time of mixing, and both controls and test cultures may be
assayed for cytokine production from 1-3 days after mixing.
[0050] Obtaining T cell populations and/or APC populations for
IL-19 assays employs techniques well known in the art which are
fully described in DiSabato et al., eds., Meth. in Enzymol. Vol.
108 (1984). APCs for the preferred IL-19 assay are peripheral blood
monocytes. These are obtained using standard techniques, e.g. as
described by the following articles in the aforementioned DiSabato
et al., eds., Meth. in Enzymol. Vol. 108 (1984): Boyum, pp. 88-102;
Mage, pp. 118-132; Litvin et al., pp. 298-302; Stevenson, pp.
242-249; and Romain, pp. 148-153, which references are herein
incorporated by reference. Preferably, helper T-cells are used in
the IL-19 assays, which are obtained by first separating
lymphocytes from the peripheral blood, and then selecting, e.g., by
panning or flow cytometry, helper cells using a commercially
available anti-CD4 antibody, e.g. OKT4, described in U.S. Pat. No.
4,381,295, and available form Ortho Pharmaceutical Corp. The
requisite techniques are fully disclosed by Boyum in Scand. JL.
Clin. Lab. Invest., 21(Suppl. 97):77 (1968) and in Meth. in
Enzymol. Vol. 108 (1984) (cited above) and by Bram et al., Meth. in
Enzymol. 121:737-748 (1986). Generally, PBLs are obtained from
fresh blood by Ficoll-Hypaque density gradient centrifugation.
[0051] A variety of antigens can be employed in the assay, e.g.
Keyhole limpet hemocyanin (KLH), fowl .gamma.-globulin, or the
like. More preferably, in place of antigen, helper T cells are
stimulated with anti-CD3 monoclonal antibody, e.g. OKT3 disclosed
in U.S. Pat. No. 4,361,549, in the assay.
[0052] Cytokine concentrations in control and test samples are
measured by standard biological and/or immunochemical assays.
Construction of immunochemical assays for specific cytokines is
well known in the art when the purified cytokine is available; e.g.
Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam,
1984); Tijssen, Practice and Theory of Enzyme Immunoassays
(Elsevier, Amsterdam, 1985); and U.S. Pat. No. 4,486,530, are
examples of the extensive literature on the subject. ELISA kits for
human IL-2, human IL-3, and human GM-CSF are commercially available
from Genzyme Corp. (Boston, Mass.); and an ELISA kit for human
IFN-.gamma. is commercially available from Endogen, Inc. (Boston,
Mass.). Polyclonal antibodies specific for human lymphotoxin are
available from genzyme, Corp., which can be used in a
radioimmunoassay for human lymphotoxin, e.g., Chard, An
Introduction to Radioimmunoassay and Related Techniques (Elsevier,
Amsterdam, 1982).
[0053] Biological assays of the cytokines listed above can also be
used to determine whether a sample has IL-19 activity, i.e.,
whether a sample modulates cytokine expression or activity in a
manner similar to IL-19. A biological assay for human lymphotoxins
disclosed by Aggarwal, Meth. in Enzymol. 116:441-447 (1985), and by
Matthews et al., in Lymphokines and Interferons: A Practical
Approach, Clemens et al., eds, IRL Press, Washington, D.C., 1987,
pp. 221-225. Human IL-2 and GM-CSF can be assayed with
factor-dependent cell lines CTLL-2 and KG-1, available from the
ATCC.RTM. under accession numbers TIB 214 and CCL246, respectively.
Human IL-3 can be assayed by its ability to simulate the formation
of a wide range of hematopoietic cell colonies in soft agar
cultures, e.g. as described by Metcalf, The Hematopoietic Colony
Stimulating Factors (Elsevier, Amsterdam, 1984). IFN-.gamma. can be
quantified with anti-viral assays, e.g. Meager, pp. 129-147, in
Clemens et al., eds (cited above).
[0054] Cytokine production can also be determined by mRNA analysis.
Cytokine mRNAs can be measured by cytoplasmic dot hybridization, as
described by White et al. (J. Biol. Chem. 257:8569-8572 (1982)) and
Gillespie et al., U.S. Pat. No. 4,483,920, both of which are hereby
incorporated by reference. Other approaches include dot blotting
using purified RNA, e.g. Chapter 6 in Hames et al., eds, Nucleic
Acid Hybridization: A Practical Approach, IRL PRess, Washington
D.C., 1985.
[0055] Some samples to be tested for IL-19 activity must be
pretreated to remove cytokines that might interfere with the assay.
For example, IL-2 increases the production of IFN-.gamma. in some
cells. Thus, depending on the T helper cells used in the assay,
IL-2 may have to be removed from the sample being tested. Such
removals are conveniently accommodated by passing a sample over a
standard anti-cytokine affinity column.
[0056] Thus, by using an assay such as those described above, the
effect of the substance suspected of having IL-19 activity on the
activity of any one of a number of cytokines may be compared to the
IL-19 protein of the invention, in order to determine if the sample
indeed has IL-19 activity.
[0057] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIG. 1 [SEQ ID NO:1] will encode a polypeptide "having IL-19
protein activity." In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing one of the
above-described comparison assays. It will be further recognized in
the art that, for such nucleic acid molecules that are not
degenerate variants, a reasonable number will also encode a
polypeptide having IL-19 protein activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly affect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid).
[0058] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U., et
al., "Deciphering the Message in Protein Sequences: Tolerance to
Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein
the authors indicate that were surprisingly tolerant of amino acid
substitutions.
Vectors and Host Cells
[0059] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of IL-19 polypeptides or portions thereof by
recombinant techniques.
[0060] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0061] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0062] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0063] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0064] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0065] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination, and in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will include a translation initiating
AUG at the beginning and a termination codon appropriately
positioned at the end of the polypeptide to be translated.
[0066] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal
cells such as CHO, COS and Bowes melanoma cells; and plant cells.
Appropriate culture media and conditions for the above-described
host cells are known in the art.
[0067] Among vectors preferred for use in bacteria include pA2,
pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic
vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from
Pharmacia. Other suitable vectors will be readily apparent to the
skilled artisan.
[0068] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0069] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986).
[0070] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0071] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0072] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant regions of immunoglobin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL5-receptor has been fused with Fc portions for the purpose
of high-throughput screening assays to identify antagonists of
hIL-5. See, D. Bennett et al., Journal of Molecular Recognition
8:52-58 (1995) and K. Johanson et al., The Journal of Biological
Chemistry 270(16):9459-9471 (1995).
[0073] The IL-19 protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification.
[0074] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
IL-19 Polypeptides and Peptides
[0075] The invention further provides an isolated IL-19 polypeptide
having the amino acid sequence encoded by the deposited cDNA, or
the amino acid sequence in FIG. 1 [SEQ ID NO:2], or a peptide or
polypeptide comprising a portion of the above polypeptides. The
terms "peptide" and "oligopeptide" are considered synonymous (as is
commonly recognized) and each term can be used interchangeably as
the context requires to indicate a chain of at least two amino
acids coupled by peptidyl linkages. The word "polypeptide" is used
herein for chains containing more than ten amino acid residues. All
oligopeptide and polypeptide formulas or sequences herein are
written from left to right and in the direction from amino terminus
to carboxy terminus.
[0076] It will be recognized in the art that some amino acid
sequence of the IL-19 polypeptide can be varied without significant
effect of the structure or function of the protein. If such
differences in sequence are contemplated, it should be remembered
that there will be critical areas on the protein which determine
activity. In general, it is possible to replace residues which form
the tertiary structure, provided that residues performing a similar
function are used. In other instances, the type of residue may be
completely unimportant if the alteration occurs at a non-critical
region of the protein.
[0077] Thus, the invention further includes variations of the IL-19
polypeptide which show substantial IL-19 polypeptide activity or
which include regions of IL-19 protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions. As indicated above,
guidance concerning which amino acid changes are likely to be
phenotypically silent can be found in Bowie, J. U., et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990).
[0078] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0079] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the IL-19
protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0080] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993) describes certain mutations resulting in
selective binding of TNF-.alpha. to only one of the two known types
of TNF receptors. Thus, the IL-19 of the present invention may
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
[0081] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0082] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of substitutions
for any given IL-19 polypeptide will not be more than 50, 40, 30,
25, 20, 15, 10, 5 or 3.
[0083] Amino acids in the IL-19 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in
vitro, or in vitro proliferative activity. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al., Science 255:306-312 (1992)).
[0084] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell. For example, a recombinantly produced version of the
IL-19 polypeptide can be substantially purified by the one-step
method described in Smith and Johnson, Gene 67:31-40 (1988).
[0085] The polypeptides of the present invention include the
polypeptide encoded by the deposited cDNA including the leader; the
mature polypeptide encoded by the deposited cDNA minus the leader
(i.e., the mature protein); a polypeptide comprising amino acids
about -24 to about 153 in SEQ ID NO:2; a polypeptide comprising
amino acids about -23 to about 153 in SEQ ID NO:2; a polypeptide
comprising amino acids about 1 to about 153 in SEQ ID NO:2; as well
as polypeptides which are at least 80% identical, more preferably
at least 90% or 95% identical, still more preferably at least 96%,
97%, 98% or 99% identical to those described above and also include
portions of such polypeptides with at least 30 amino acids and more
preferably at least 50 amino acids.
[0086] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of an
IL-19 polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the IL-19
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0087] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIG. 1 [SEQ ID NO:2] or to the
amino acid sequence encoded by deposited cDNA clone can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0088] The polypeptide of the present invention is useful as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0089] As described in detail below, the polypeptides of the
present invention can be used to raise polyclonal and monoclonal
antibodies, which are useful in diagnostic assays for detecting
IL-19 protein expression as described below or as agonists and
antagonists capable of enhancing or inhibiting IL-19 protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" IL-19 protein binding proteins which
are also candidate agonist and antagonist according to the present
invention. The yeast two hybrid system is described in Fields and
Song, Nature 340:245-246 (1989).
[0090] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. These immunogenic epitopes are believed to be confined
to a few loci on the molecule. On the other hand, a region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen, H. M., Meloen, R. H. and Barteling, S.
J. "Use of peptide synthesis to probe viral antigens for epitopes
to a resolution of a single amino acid." Proc. Natl. Acad. Sci. USA
81:3998-4002 (1984).
[0091] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., et al., "Antibodies that react with predetermined
sites on proteins." Science 219:660-666 (1983). Peptides capable of
eliciting protein-reactive sera are frequently represented in the
primary sequence of a protein, can be characterized by a set of
simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals.
[0092] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. See, for instance, Wilson, et al., Cell 37:767-778
at 777.
[0093] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 15 to about 30 amino acids
contained within the amino acid sequence of a polypeptide of the
invention.
[0094] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate IL-19-specific antibodies include:a
polypeptide comprising amino acid residues from about -5 to about 4
in SEQ ID NO:2; a polypeptide comprising amino acid residues from
about 64 to about 82 in SEQ ID NO:2; and a polypeptide comprising
amino acid residues from about 115 to about 125 in SEQ ID NO:2. As
indicated above, the inventors have determined that the above
polypeptide fragments are antigenic regions of the IL-19
protein.
[0095] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. Houghten, R.
A. "General method for the rapid solid-phase synthesis of large
numbers of peptides: specificity of antigen-antibody interaction at
the level of individual amino acids." Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Pat. No. 4,631,211 to
Houghten et al. (1986).
[0096] As one of skill in the art will appreciate, IL-19
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric IL-19 protein or protein fragment alone
(Fountoulakis et al., J Biochem 270:3958-3964 (1995)).
[0097] The entire disclosure of each document cited in this section
on "Polypeptides and Peptides" is hereby incorporated herein by
reference
Chromosome Assays
[0098] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0099] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of an interleukin-19
protein gene. This can be accomplished using a variety of well
known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose. Typically, in
accordance with routine procedures for chromosome mapping, some
trial and error may be necessary to identify a genomic probe that
gives a good in situ hybridization signal.
[0100] In some cases, in addition, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified portion.
[0101] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of portions from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0102] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
probes from the cDNA as short as 50 or 60 bp. For a review of this
technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC
TECHNIQUES, Pergamon Press, New York (1988).
[0103] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, MENDELIAN INHERITANCE IN MAN, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0104] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0105] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
Treatment of Pathological Conditions by IL-19 Inhibition of
Cytokine Production
[0106] As noted above, IL-19 is secreted by activated monocytes,
and shares significant homology with human IL-10. Thus, it is
believed by the inventors that IL-19 is active in inhibiting
cytokine production during the mammalian immune response. The
cytokines whose production may be affected by IL-19 include
IFN-.gamma., TNF-.alpha., and IL-6.
[0107] One such activity of IL-19 is the ability to limit excessive
production of gamma-interferon (IFN-.gamma.), and hence the
consequent effects of such production, including major
histocompatibility (MHC) associated auto-immune diseases. As
increased IFN-.gamma. expression has been implicated in an increase
of MHC genes, which may then increase the chance of an autoimmune
response against the MHC-overexpressing cells, the ability to limit
IFN-.gamma. expression may be therapeutically valuable in the
treatment of clinical manifestations of such MHC disorders. These
include rheumatoid arthritis, systemic lupus erythematosus (SLE),
myasthenia gravis, insulin-dependent diabetes mellilitus, and
thyroiditis.
[0108] The down regulation of IFN-.gamma. by IL-19 may also be
therapeutically valuable in treating parasitic infections such as
leishmaniasis. Levels of IFN-.gamma., IL-2 and IL-4 are all
involved in the regulation of the life cycle of this parasite.
Thus, the ability to regulate the production of these cytokines by
IL-19 will be therapeutically valuable.
[0109] Given the activities modulated by IL-19, it is readily
apparent that a substantially altered (increased or decreased)
level of expression of IL-19 in an individual compared to the
standard or "normal" level produces pathological conditions such as
those described above. It will also be appreciated by one of
ordinary skill that, since the IL-19 protein of the invention is
translated with a leader peptide suitable for secretion of the
mature protein from the cells which express IL-19, when IL-19
protein (particularly the mature form) is added from an exogenous
source to cells, tissues or the body of an individual, the protein
will exert its modulating activities on any of its target cells.
Therefore, it will be appreciated that conditions caused by a
decrease in the standard or normal level of IL-19 activity in an
individual, can be treated be administration of IL-19 protein.
Thus, the invention further provides a method of treating an
individual in need of an increased level of IL-19 activity
comprising administering to such an individual a pharmaceutical
composition comprising an amount of an isolated IL-19 polypeptide
of the invention, particularly a mature form of the IL-19 protein
of the invention, effective to increase the IL-19 activity level in
such an individual.
[0110] One of ordinary skill will appreciate that effective amounts
of the IL-19 polypeptides for treating an individual in need of an
increased level of IL-19 activity can be determined empirically for
each condition where administration of IL-19 is indicated. The
polypeptide having IL-19 activity may be administered in
pharmaceutical compositions in combination with one or more
pharmaceutically acceptable excipients. It will be understood that,
when administered to a human patient, the total daily usage of the
pharmaceutical compositions of the present invention will be
decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular patient will depend upon a variety of factors
including the type and degree of the response to be achieved; the
specific composition an other agent, if any, employed; the age,
body weight, general health, sex and diet of the patient; the time
of administration, route of administration, and rate of excretion
of the composition; the duration of the treatment; drugs (such as a
chemotherapeutic agent) used in combination or coincidental with
the specific composition; and like factors well known in the
medical arts.
[0111] For example, it is predicted that satisfactory results are
obtained by oral administration of a polypeptide having IL-19
activity in dosages on the order of from 0.05 to 10 mg/kg/day,
preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2
mg/kg/day, administered once or, in divided doses, 2 to 4 times per
day. On administration parenterally, for example by i.v. drip or
infusion, dosages on the order of from 0.01 to 5 mg/kg/day,
preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0
mg/kg/day can be used. Suitable daily dosages for patients are thus
on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg
p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5
to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably
2.5 to 50 mg i.v.
[0112] Dosaging may also be arranged in a patient specific manner
to provide a predetermined concentration of an IL-19 activity in
the blood, as determined by an RIA technique, for instance. Thus
patient dosaging may be adjusted to achieve regular on-going trough
blood levels, as measured by RLA, on the order of from 50 to 1000
ng/ml, preferably 150 to 500 ng/ml.
[0113] Pharmaceutical compositions of the invention may be
administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, drops or transdermal patch), bucally, or as an oral or
nasal spray. By "pharmaceutically acceptable carrier" is meant a
non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or formulation auxiliary of any type. The term
"parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion.
[0114] Pharmaceutical compositions of the present invention for
parenteral injection can comprise pharmaceutically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions as well as sterile powders for reconstitution into
sterile injectable solutions or dispersions just prior to use.
Examples of suitable aqueous and nonaqueous carriers, diluents,
solvents or vehicles include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like),
carboxymethylcellulose and suitable mixtures thereof, vegetable
oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper fluidity can be maintained, for example, by
the use of coating materials such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants.
[0115] The compositions of the present invention may also contain
adjuvants such as preservatives, wetting agents, emulsifying
agents, and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents such as sugars, sodium
chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents
which delay absorption such as aluminum monostearate and
gelatin.
[0116] In some cases, in order to prolong the effect of the
pharmaceutical composition, it is desirable to slow the absorption
of the drug from subcutaneous or intramuscular injection. This may
be accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle.
[0117] Injectable depot forms are made by forming microencapsulated
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are also prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body
tissues.
[0118] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0119] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compounds are mixed with at least one item
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0120] Solid compositions of a similar type may also be employed as
fillers in soft and hard filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0121] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They may optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of embedding compositions which can be used
include polymeric substances and waxes.
[0122] The active compounds can also be in micro-encapsulated form,
if appropriate, with one or more of the above-mentioned
excipients.
[0123] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compounds, the liquid
dosage forms may contain inert diluents commonly used in the art
such as, for example, water or other solvents, solubilizing agents
and emulsifiers such as ethyl alcohol, isoptopyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures
thereof.
[0124] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.
[0125] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar, and tragacanth, and mixtures thereof.
[0126] The active polypeptide can also be administered in the form
of liposomes. As is known in the art, liposomes are generally
derived from phospholipids or other lipid substances. Liposomes are
formed by mono- or multi-lamellar hydrated liquid crystals that are
dispersed in an aqueous medium. Any non-toxic, physiologically
acceptable and metabolizable lipid capable of forming liposomes can
be used. The present compositions in liposome form can contain, in
addition to the agent or inhibitor, stabilizers, preservatives,
excipients, and the like. The preferred lipids are the
phospholipids and the phosphatidyl cholates (lecithins), both
natural and synthetic. Methods to form liposomes are known in the
art. See, for example, Prescott, Ed., Methods in Cell Biology,
Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et
seq.
Use of IL-19 in Adoptive Immunotherapy of Cancer
[0127] Another therapeutic application of IL-19 is the
administration of IL-19 in adoptive immunotherapy to prevent or
reduce the production of cytokines believed to be responsible for
many of the deleterious side effects currently encountered in
adoptive immunotherapy. As used herein, "Adoptive immunotherapy"
means therapy wherein functional cancer-fighting immune cells are
transferred to a patient. The cancer-fighting immune cells
preferably comprise tumor infiltrating lymphocytes (TILS)
originating from the patient himself. As is known in the art, while
IL-2 is useful in adoptive immunotherapy due to its ability to
activate the killer cells transferred to the patent (Rosenberg et
al., Ann. Rev. Immunol. 4: 681-709 (1988)), the severe side effects
caused directly or indirectly by IL-2 have been an obstacle to the
development of routine treatment protocols based on this approach
(Hsu, D-H et al., WO 92/12726). As IL-19 is believed to be capable
of preventing or reducing the production of cytokines responsible
for these side effects, TILs cultured in the presence of both IL-2
and IL-19 prior to administration, wherein the administration of
IL-2 and IL-19 is continued after the administration of those TILs
to the patient, may reduce the deleterious side effects typical of
adoptive immunotherapy.
Use of IL-19 Antagonists in the Restoration of Immunocompetency to
T Helper Cells in HIV-Infected Patients
[0128] Another therapeutic application of the IL-19 polypeptide of
the invention is the use of the polypeptide to identify IL-19
antagonists, such as an antibody specific for binding to IL-19,
which can then be used to increase the production of IL-2 in T
helper cells. For example, patients infected with human
immunodeficiency virus (HIV) have a decreased level of IL-2
production in non-virally infected T helper cells. IL-19 is
believed to be capable of preventing or reducing the production of
IL-2. Therefore, the administration of IL-19 antagonists may result
in an increase in IL-2 production in HIV-infected patients. As IL-2
is responsible for T cell proliferation, the maintenance of IL-2
production is beneficial to HIV-infected patients.
[0129] Antagonists specific for IL-19 can be made by mutating the
amino acid sequence of IL-19 using standard mutagenesis methods
well known to those of ordinary skill in the art. Such methods
include the use of Ml 3 vectors to introduce single-site mutations,
to delete random amino acids from IL-19, or to add amino acids. The
resulting muteins are then tested in standard assays for the
ability to compete with non-mutated IL-19, including assays which
test the ability of the mutein, as compared with the IL-19 protein
of the invention, to enhance IL-2 dependent proliferation of T
cells in vitro.
[0130] Other suitable IL-19 antagonists include an antibody
specific for binding to IL-19 (aIL-19) which interferes with its
binding to the T helper receptor. Production of such antibodies has
been described in full above.
[0131] The antibodies used in the method of the invention
preferably are autologous for the patient, thereby minimizing
further immunological problems. However, as immunodeficient
individuals in need of this treatment will tend to be less reactive
to non-self antibodies, non-self antibodies derived from cells of
the same species will also be useful.
[0132] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Expression and Purification of IL-19 in E. coli
[0133] The DNA sequence encoding the mature IL-19 protein in the
deposited cDNA clone was amplified using PCR oligonucleotide
primers specific to the amino terminal sequences of the IL-19
protein and to vector sequences 3' to the gene. Additional
nucleotides containing restriction sites to facilitate cloning were
added to the 5' and 3' sequences respectively.
[0134] The 5' oligonucleotide primer had the sequence 5' GGC ATG
CCA TGG AGT TAC AGT GTG TTT CCC 3' [SEQ ID NO:4] (sequences
specific to the IL-19 nucleotide sequence are underlined), and
included an NcoI restriction site.
[0135] The 3' primer had the sequence 5' GGA AGA TCT AGC TGA GGA
CAT TAC 3' [SEQ ID NO:5] containing the underlined 15 nucleotides
complementary to the last 15 nucleotides immediately after the
IL-19 protein coding sequence in FIG. 1. The 3' primer included a
BglII restriction site.
[0136] The restriction sites were convenient to restriction enzyme
sites in the bacterial expression vector pQE60, which were used for
bacterial expression in these examples. (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, Calif., 91311). pQE60 encodes ampicillin
antibiotic resistance ("Ampr") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, a ribosome binding
site ("RBS"), a 6-His tag and restriction enzyme sites. The
amplified IL-19 protein DNA and the vector pQE60 both were digested
with NcoI and BglII and the digested DNAs were then ligated
together. Insertion of the IL-19 protein DNA into the restricted
pQE60 vector placed the IL-19 protein coding region downstream of
and operably linked to the vector's IPTG-inducible promoter and
in-frame with an initiating ATG appropriately positioned for
translation of IL-19 protein.
[0137] The ligation mixture was transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kan.sup.r"), was used in carrying out the illustrative
example described here. This strain, which is only one of many that
are suitable for expressing IL-19 protein, is available
commercially from Qiagen.
[0138] Transformants were identified by their ability to grow on LB
plates in the presence of ampicillin and kanamycin. Plasmid DNA was
isolated from resistant colonies and the identity of the cloned DNA
was confirmed by restriction analysis.
[0139] Clones containing the desired constructs were grown
overnight ("O/N") in liquid culture in LB media supplemented with
both ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml).
[0140] The O/N culture was used to inoculate a large culture, at a
dilution of approximately 1:100 to 1:250. The cells were grown to
an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalactopyranoside ("IPTG") was then added to a
final concentration of 0.1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently were incubated further for 4 hours (FIG. 2 shows
IL-19 protein induction; samples removed 0, 3, 5, 6, and 24 hours
after the addition of IPTG were run on a 12.5% polyacrylamide gel
and stained with brilliant blue). The mobility of the IL-19 protein
is indicated by an arrow. Cells were then harvested by
centrifugation and disrupted by gentle shaking overnight in 6M
guanidine HCl in 50 mM NaPO.sub.4 buffer at pH 8.0 at 4.degree. C.
The lysate was then centrifuged and passed over a Sepharose CL-4B
(Pharmacia) column. The flowthrough was then passed over a column
containing activated Ni.sup.2+-NTA-agarose (Qiagen). IL-19 protein
was collected from the column in a fraction consisting of 6M
guanidine HCl pH 5.0. Guanidine HCl was removed from the
IL-19-containing fraction by dialysis against successively reduced
concentrations of guanidine in phosphate-buffered saline (PBS) at
pH 5.5.
Example 2
Cloning and Expression of IL-19 in a Baculovirus Expression
System
[0141] The cDNA sequence encoding the full length IL-19 protein in
the deposited clone was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
[0142] The 5' primer had the sequence 5' GGC GGG ATC CCG CCA TCA
TGA AGT TAC AGT GTG TTT CCC 3' [SEQ ID NO:6] containing the
underlined BamHI restriction enzyme site followed by 29 bases of
the sequence of IL-19 protein in FIG. 1. Inserted into an
expression vector, as described below, the 5' end of the amplified
fragment encoding IL-19 provided an efficient signal peptide. An
efficient signal for initiation of translation in eukaryotic cells,
as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) is
appropriately located in the vector portion of the construct.
[0143] The 3' primer had the sequence 5'CCC AAG CTT GGT ACC TCA TCA
AGC TGA GGA CAT TAC 3' [SEQ ID NO:7] containing the underlined
Asp718 restriction site followed by nucleotides complementary to
the last 21 nucleotides of the IL-19 coding sequence set out in
FIG. 1.
[0144] The amplified fragment was isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then was digested with BamHI and
Asp718 and again was purified on a 1% agarose gel. This fragment is
designated herein F2.
[0145] The vector and pA2-GP were used to express the IL-19 protein
in the baculovirus expression system, using standard methods, as
described in Summers et al., A MANUAL OF METHODS FOR BACULOVIRUS
VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural
Experimental Station Bulletin No. 1555 (1987). This expression
vector contains the strong polyhedrin promoter of the Autographa
californica nuclear polyhedrosis virus (AcMNPV) followed by
convenient restriction sites. The signal peptide of AcMNPV gp67,
including the N-terminal methionine, is located just upstream of a
BamHI site. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For an easy
selection of recombinant virus the beta-galactosidase gene from E.
coli is inserted in the same orientation as the polyhedrin promoter
and is followed by the polyadenylation signal of the polyhedrin
gene. The polyhedrin sequences are flanked at both sides by viral
sequences for cell-mediated homologous recombination with wild-type
viral DNA to generate viable virus that express the cloned
polynucleotide. The IL-19 protein was also expressed in baculovirus
using the pA2 vector.
[0146] Many other baculovirus vectors could be used in place of
pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of
skill readily will appreciate, that construction provides
appropriately located signals for transcription, translation,
trafficking and the like, such as an in-frame AUG and a signal
peptide, as required. Such vectors are described in Luckow et al.,
Virology 170: 31-39 (1989), among others.
[0147] The plasmid was digested with the restriction enzymes BamHI
and Asp718 and then was dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA was
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein "V2."
[0148] Fragment F2 and the dephosphorylated plasmid V2 were ligated
together with T4 DNA ligase. E. coli HB101 cells were transformed
with ligation mix and spread on culture plates. Bacteria were
identified that contained the plasmid with the human IL-19 gene by
digesting DNA from individual colonies using BamHI and Asp718 and
then analyzing the digestion product by gel electrophoresis. The
sequence of the cloned fragment was confirmed by DNA sequencing.
This plasmid is designated herein pBacIL-19.
[0149] 5 .mu.g of the plasmid pBacIL-19 was co-transfected with 1.0
.mu.g of a commercially available linearized baculovirus DNA
("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofection method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 .mu.g of
BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid pBacCK.beta.-15
were mixed in a sterile well of a microtiter plate containing 50
.mu.l of serum-free Grace's medium (Life Technologies Inc.,
Gaithersburg, Md.). Afterwards 10 .mu.l Lipofectin plus 90 .mu.l
Grace's medium are added, mixed and incubated for 15 minutes at
room temperature. Then the transfection mixture was added drop-wise
to Sf9 insect cells (ATCC.RTM. CRL 1711) seeded in a 35 mm tissue
culture plate with 1 ml Grace's medium without serum. The plate was
rocked back and forth to mix the newly added solution. The plate
was then incubated for 5 hours at 27.degree. C. After 5 hours the
transfection solution was removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation is
continued at 27.degree. C. for four days.
[0150] After four days the supernatant was collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with "Blue Gal" (Life Technologies Inc.,
Gaithersburg) was used to allow easy identification and isolation
of gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0151] Four days after serial dilution, the virus was added to the
cells. After appropriate incubation, blue stained plaques were
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses was then resuspended in an Eppendorf tube
containing 200 .mu.l of Grace's medium. The agar was removed by a
brief centrifugation and the supernatant containing the recombinant
baculovirus was used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes were
harvested and then they were stored at 4.degree. C. A clone
containing properly inserted HESSB I, II and III were identified by
DNA analysis including restriction mapping and sequencing. This is
designated herein as V-IL-19.
[0152] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-IL-19 at a multiplicity of infection ("MOI") of about
2 (about 1 to about 3). Six hours later the medium was removed and
was replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Gaithersburg). 42 hours
later, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) were added. The cells
were further incubated for 16 hours and then they were harvested by
centrifugation, lysed and the labeled proteins were visualized by
SDS-PAGE and autoradiography.
Example 3
In vitro Transcription/translation of IL-19 Protein
[0153] Recombinant IL-19 proteins were prepared using the TNT
Coupled Wheat Germ Extract System (Promega, Madison, Wis.).
[0154] 25 .mu.l of TNT wheat germ extract, 10 U T3 RNA polymerase,
1 mM amino acid mixture (methionine-free), 4 .mu.Ci
.sup.35S-methionine (1000 Ci/mmol), 40 U RNasin (Promega, Madison,
Wis.), and 1 .mu.g template DNA were combined in a final volume of
50 .mu.l and incubated at 30.degree. C. for 2 h. Coupled
transcription/translation reactions included the following template
DNAs: (1) No DNA, (2) pBluescript, (3) nucleotides 44-577
(corresponding to amino acids 1-177) of the IL-19 sequence shown in
FIG. 1 cloned into pBluescript, (4) nucleotides 116-577
(corresponding to amino acids 25-177 of FIG. 1 or residues 1-153 of
SEQ ID NO:2) of the IL-19 coding sequence cloned into pBluescript,
(5) a gel-purified PCR product derived from the template described
in (3) and amplified using M13 forward and reverse primers in a
standard PCR reaction. Samples were heated to 95.degree. C. for 10
minutes and a 5 .mu.l aliquot of each sample was then loaded onto a
15% polyacrylamide gel. The gel was run at 100 volts for
approximately 2 hours. The gel was then dried and exposed to X-ray
film for 3 days at room temperature. The mobility of full-length
and truncated (no signal sequence) IL-19 proteins are indicated in
FIG. 3. An apparent molecular mass marker (M) shows the relative
mobilities of 14.3, 21.5, 30, 46, 66, 97.4, and 220 kD
proteins.
Example 4
Cloning and Expression in Mammalian Cells
[0155] Most of the vectors used for the transient expression of the
IL-19 protein gene sequence in mammalian cells should carry the
SV40 origin of replication. This allows the replication of the
vector to high copy numbers in cells (e.g. COS cells) which express
the T antigen required for the initiation of viral DNA synthesis.
Any other mammalian cell line can also be utilized for this
purpose.
[0156] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular signals
can also be used (e.g. human actin promoter). Suitable expression
vectors for use in practicing the present invention include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,
Sweden), pRSVcat (ATCC.RTM. 37152), pSV2dhfr (ATCC.RTM. 37146) and
pBC12MI (ATCC.RTM. 67109). Mammalian host cells that could be used
include, human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail
QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
[0157] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0158] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
is a useful marker to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) cells are often used for the production of
proteins.
[0159] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology 5(3):438-447 (1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g. with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 4(a)
Cloning and Expression in COS Cells
[0160] The expression plasmid, pIL-19 HA, is made by cloning a cDNA
encoding IL-19 into the expression vector pcDNA4 (which can be
obtained from Invitrogen, Inc.).
[0161] The expression vector pcDNA4 contains: (1) an E. coli origin
of replication effective for propagation in E. coli and other
prokaryotic cells; (2) an ampicillin resistance gene for selection
of plasmid-containing prokaryotic cells; (3) an SV40 origin of
replication for propagation in eukaryotic cells; (4) a CMV
promoter, a polylinker, an SV40 intron, and a polyadenylation
signal arranged so that a cDNA conveniently can be placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
[0162] A DNA fragment encoding the IL-19 protein and an HA tag
fused in frame to its 3' end is cloned into the polylinker region
of the vector so that recombinant protein expression is directed by
the CMV promoter. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein described by Wilson et al.,
Cell 37: 767 (1984). The fusion of the HA tag to the target protein
allows easy detection of the recombinant protein with an antibody
that recognizes the HA epitope.
[0163] The plasmid construction strategy is as follows. The IL-19
cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above regarding the
construction of expression vectors for expression of IL-19 in E.
coli. To facilitate detection, purification and characterization of
the expressed IL-19, one of the primers contains a hemagglutinin
tag ("HA tag") as described above.
[0164] Suitable primers include the following, which are used in
this example. The 5' primer, containing the underlined BamHI site,
an AUG start codon and 22 bp of the 5' coding region has the
following sequence: TABLE-US-00002 (SEQ ID NO: 8) 5' GGC GGG ATC
CCG CCA TGA AGT TAC AGT GTG TTT CCC 3'.
[0165] The 3' primer, containing the underlined Asp 718 site, a
stop codon, 10 codons thereafter forming the hemagglutinin HA tag,
and 25 bp of 3' coding sequence (at the 3' end) has the following
sequence: TABLE-US-00003 (SEQ ID NO: 9) 5' CCC AAG CTT GGT ACC TCA
TCA GAA AGC GTA GTC TGG GAC GTC GTA TGG GTA AGC TGA GGA CAT TAC TTC
ATG ATT C 3'.
[0166] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with HindIII and XhoI and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037), and the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis and gel
sizing for the presence of the IL-19-encoding fragment.
[0167] For expression of recombinant IL-19, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of IL-19 by the vector.
[0168] Expression of the IL-19/HA fusion protein is detected by
radiolabelling and immunoprecipitation, using methods described in,
for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing .sup.35S-cysteine for 8
hours. The cells and the media are collected, and the cells are
washed and then lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson et al. cited above. Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE gels and autoradiography. An expression
product of the expected size is seen in the cell lysate, which is
not seen in negative controls.
Example 4(b)
Cloning and Expression in CHO Cells
[0169] The vector pC4 is used for the expression of IL-19 protein.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr [ATCC.RTM.
Accession No. 37146]. Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary- or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., et al., J. Biol. Chem.
253:1357-1370 (1978); Hamlin, J. L. and Ma, C. Biochem. et Biophys.
Acta 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.
Biotechnology 9:64-68 (1991). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene it is
usually co-amplified and over-expressed. It is state of the art to
develop cell lines carrying more than 1,000 copies of the genes.
Subsequently, when the methotrexate is withdrawn, cell lines
contain the amplified gene integrated into the chromosome(s).
[0170] Plasmid pC4 contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology
5:438-447 (1985) plus a fragment isolated from the enhancer of the
immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, PvuII, and Nrul. Behind these
cloning sites the plasmid contains translational stop codons in all
three reading frames followed by the 3' intron and the
polyadenylation site of the rat preproinsulin gene. Other high
efficient promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. For the polyadenylation of the mRNA other signals, e.g.,
from the human growth hormone or globin genes can be used as
well.
[0171] Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0172] The plasmid pC4 is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0173] The DNA sequence encoding IL-19 protein is amplified using
PCR oligonucleotide primers specific to the amino terminal sequence
of the IL-19 protein and to carboxy terminal sequence 3' to the
gene. Additional nucleotides containing restriction sites to
facilitate cloning are added to the 5' and 3' sequences
respectively.
[0174] The 5' primer has the sequence 5' GGC GGG ATC CCG CCA TCA
TGA AGT TAC AGT GTG TTT CCC 3' (SEQ ID NO: 10), containing the
underlined BamHI restriction enzyme site followed by Kozak sequence
and 23 bases of the sequence of IL-19 in FIG. 1. The 3' primer has
the sequence 5'CCC AAG CTT GGT ACC TCA TCA AGC TGA GGA CAT TAC 3'
(SEQ ID NO: 11), containing the underlined Asp 718 restriction site
followed by nucleotides complementary to 15 bp of the nucleotide
sequence preceding the stop codon in FIG. 1. The restrictions sites
are convenient to restriction enzyme sites in the CHO expression
vector pC4.
[0175] The amplified fragments are isolated from a 1% agarose gel
as described above and then digested with the endonucleases BamHI
and Asp718 and then purified again on a 1% agarose gel.
[0176] The isolated fragment and the dephosphorylated vector are
then ligated with T4 DNA ligase. E. coli HB101 cells are then
transformed and bacteria identified that contained the plasmid pC4
inserted in the correct orientation using the restriction enzyme
BamHI. The sequence of the inserted gene is confirmed by DNA
sequencing.
Transfection of CHO-DHFR-Cells
[0177] Chinese hamster ovary cells lacking an active DHFR enzyme
are used for transfection. 5 .mu.g of the expression plasmid pC4
are cotransfected with 0.5 .mu.g of the plasmid pSVneo using the
lipofecting method (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the gene neo from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) and cultivated from 10-14 days. After this period, single
clones are trypsinized and then seeded in 6-well petri dishes using
different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200
nM, 400 nM, 800 nM). Clones growing at the highest concentrations
of methotrexate are then transferred to new 6-well plates
containing even higher concentrations of methotrexate (1 .mu.M, 2
.mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is repeated until
clones grow at a concentration of 100 .mu.M-200 .mu.M.
[0178] The expression of the desired gene product is analyzed by
Western blot analysis and SDS-PAGE or by reverse phase HPLC
analysis.
Example 5
Tissue Distribution of IL-19 Protein Expression
[0179] Northern blot analysis was carried out to examine the levels
of expression of IL-19 protein in human tissues, using methods
described by, among others, Sambrook et al., cited above.
[0180] HL-60, THP-1 and U937 cell lines and primary human monocytes
isolated by adherence from a mixed leukocyte population were grown
for 12 hours either in the presence (+) or absence (-) of bacterial
lipopolysaccharide (LPS). Total RNA was prepared from the cultures
with TRIzol Reagent (Life Technologies, Gaithersburg, Md.)
essentially as described by the manufacturer. Total RNA (10 .mu.g)
was dried completely, resuspended in a formamide/formaldehyde
loading buffer, and resolved by electrophoresis through a 1%
agarose gel containing 2.2 M formaldehyde. The gel was transferred
overnight in 20.times.SSC to a nylon membrane (Boehringer Mannheim,
Indianapolis, Ind.). Probe DNA was prepared by PCR amplifying the
entire IL-19 insert shown in FIG. 1 using M13 Forward and Reverse
primers. Probe DNA (25 ng) was labeled with .sup.32p using the
RediPrime Random Primer Labeling Kit (Amersham Life Science) to a
specific activity of greater than 10.sup.6 CPM/ng. The blot was
hybridized with denatured probe in a 10 ml Hybrizol hybridization
solution overnight at 42.degree. C. The blot was then washed in
approximately 100 ml of 0.2.times.SSC/0.1% SDS at 25.degree. C. for
20 minutes, and then twice in approximately 100 ml of
0.2.times.SSC/0.1% SDS at 65.degree. C. for 15 min. The blot was
then exposed to x-ray film for 5 days, and is shown in FIG. 4. The
arrow shows the migration of the IL-19-specific RNA. An RNA marker
shows the relative mobilities of 0.24, 1.35, 2.37, 4.40, 7.46, and
9.49 kb RNAs.
[0181] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0182] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0183] The disclosures of all patents, patent applications, and
publications referred to herein are hereby incorporated by
reference.
Sequence CWU 1
1
17 1 966 DNA Homo sapiens CDS (44)..(574) sig_peptide (44)..(115)
mat_peptide (116)..(574) 1 ggcacgagca caaggagcag cccgcaagca
ccaagtgaga ggc atg aag tta cag 55 Met Lys Leu Gln tgt gtt tcc ctt
tgg ctc ctg ggt aca ata ctg ata ttg tgc tca gta 103 Cys Val Ser Leu
Trp Leu Leu Gly Thr Ile Leu Ile Leu Cys Ser Val -20 -15 -10 -5 gac
aac cac ggt ctc agg aga tgt ctg att tcc aca gac atg cac cat 151 Asp
Asn His Gly Leu Arg Arg Cys Leu Ile Ser Thr Asp Met His His -1 1 5
10 ata gaa gag agt ttc caa gaa atc aaa aga gcc atc caa gct aag gac
199 Ile Glu Glu Ser Phe Gln Glu Ile Lys Arg Ala Ile Gln Ala Lys Asp
15 20 25 acc ttc cca aat gtc act atc ctg tcc aca ttg gag act ctg
cag atc 247 Thr Phe Pro Asn Val Thr Ile Leu Ser Thr Leu Glu Thr Leu
Gln Ile 30 35 40 att aag ccc tta gat gtg tgc tgc gtg acc aag aac
ctc ctg gcg ttc 295 Ile Lys Pro Leu Asp Val Cys Cys Val Thr Lys Asn
Leu Leu Ala Phe 45 50 55 60 tac gtg gac agg gtg ttc aag gat cat cag
gag cca aac ccc aaa atc 343 Tyr Val Asp Arg Val Phe Lys Asp His Gln
Glu Pro Asn Pro Lys Ile 65 70 75 ttg aga aaa atc agc agc att gcc
aac tct ttc ctc tac atg cag aaa 391 Leu Arg Lys Ile Ser Ser Ile Ala
Asn Ser Phe Leu Tyr Met Gln Lys 80 85 90 act ctg cgg caa tgt cag
gaa cag agg cag tgt cac tgc agg cag gaa 439 Thr Leu Arg Gln Cys Gln
Glu Gln Arg Gln Cys His Cys Arg Gln Glu 95 100 105 gcc acc aat gcc
acc aga gtc atc cat gac aac tat gat cag ctg gag 487 Ala Thr Asn Ala
Thr Arg Val Ile His Asp Asn Tyr Asp Gln Leu Glu 110 115 120 gtc cac
gct gct gcc att aaa tcc ctg gga gag ctc gac gtc ttt cta 535 Val His
Ala Ala Ala Ile Lys Ser Leu Gly Glu Leu Asp Val Phe Leu 125 130 135
140 gcc tgg att aat aag aat cat gaa gta atg tcc tca gct tgatgacaag
584 Ala Trp Ile Asn Lys Asn His Glu Val Met Ser Ser Ala 145 150
gaacctgtat agtgatccag ggatgaacac cccctgtgcg gtttactgtg ggagacagcc
644 caccttgaag gggaaggaga tggggaaggc cccttgcagc tgaaagtccc
actggctggc 704 ctcaggctgt cttattccgc ttgaaaatag ccaaaaagtc
tactgtggta tttgtaataa 764 actctatctg ctgaaagggc ctgcaggcca
tcctgggagt aaagggctgc cttcccatct 824 aatttattgt gaagtcatat
agtccatgtc tgtgatgtga gccaagtgat atcctgtagt 884 acacattgta
ctgagtggtt tttctgaata aattccatat tttacctatg gaaaaaaaaa 944
aaaaaaaaaa aaaaaaaaaa aa 966 2 177 PRT Homo sapiens 2 Met Lys Leu
Gln Cys Val Ser Leu Trp Leu Leu Gly Thr Ile Leu Ile -20 -15 -10 Leu
Cys Ser Val Asp Asn His Gly Leu Arg Arg Cys Leu Ile Ser Thr -5 -1 1
5 Asp Met His His Ile Glu Glu Ser Phe Gln Glu Ile Lys Arg Ala Ile
10 15 20 Gln Ala Lys Asp Thr Phe Pro Asn Val Thr Ile Leu Ser Thr
Leu Glu 25 30 35 40 Thr Leu Gln Ile Ile Lys Pro Leu Asp Val Cys Cys
Val Thr Lys Asn 45 50 55 Leu Leu Ala Phe Tyr Val Asp Arg Val Phe
Lys Asp His Gln Glu Pro 60 65 70 Asn Pro Lys Ile Leu Arg Lys Ile
Ser Ser Ile Ala Asn Ser Phe Leu 75 80 85 Tyr Met Gln Lys Thr Leu
Arg Gln Cys Gln Glu Gln Arg Gln Cys His 90 95 100 Cys Arg Gln Glu
Ala Thr Asn Ala Thr Arg Val Ile His Asp Asn Tyr 105 110 115 120 Asp
Gln Leu Glu Val His Ala Ala Ala Ile Lys Ser Leu Gly Glu Leu 125 130
135 Asp Val Phe Leu Ala Trp Ile Asn Lys Asn His Glu Val Met Ser Ser
140 145 150 Ala 3 178 PRT Homo sapiens 3 Met His Ser Ser Ala Leu
Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Ser Pro
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45
Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50
55 60 Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly
Cys 65 70 75 80 Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu
Val Met Pro 85 90 95 Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala
His Val Asn Ser Leu 100 105 110 Gly Glu Asn Leu Lys Thr Leu Arg Leu
Arg Leu Arg Arg Cys His Arg 115 120 125 Phe Leu Pro Cys Glu Asn Lys
Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 Ala Phe Asn Lys Leu
Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150 155 160 Phe Asp
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 165 170 175
Arg Asn 4 30 DNA Homo sapiens 4 ggcatgccat ggagttacag tgtgtttccc 30
5 24 DNA Homo sapiens 5 ggaagatcta gctgaggaca ttac 24 6 39 DNA Homo
sapiens 6 ggcgggatcc cgccatcatg aagttacagt gtgtttccc 39 7 36 DNA
Homo sapiens 7 cccaagcttg gtacctcatc aagctgagga cattac 36 8 36 DNA
Homo sapiens 8 ggcgggatcc cgccatgaag ttacagtgtg tttccc 36 9 75 DNA
Homo sapiens 9 cccaagcttg gtacctcatc agaaagcgta gtctgggacg
tcgtatgggt aagctgagga 60 cattacttca tgatt 75 10 39 DNA Homo sapiens
10 ggcgggatcc cgccatcatg aagttacagt gtgtttccc 39 11 36 DNA Homo
sapiens 11 cccaagcttg gtacctcatc aagctgagga cattac 36 12 588 DNA
Artificial cDNA 12 ataggtaaaa tatggaattt attcagaaaa accactcagt
acaatgtgta ctacaggata 60 tcacttggct cacatcacag acatggacta
tatgacttca caataaatta gacgggaagg 120 cagcccttta ctcccaggat
ggcctgcagg cctttcagca gatagagttt attacaaata 180 ccacagtaga
ctttttggct attttcaagc ggaataagac agcctgaggc cagccagtgg 240
gactttcagc tgccnggggc cttccccatc tccttcccct tcaaggtggg ctgtctccca
300 cagtaaaccg cacanggggt gttcatccct ggatcactat acaggttcct
tgtcatcaag 360 ctgaggacat tacttcatga ttcttattaa tccaggctag
aaagacgtcg agctctccca 420 gggatttaat ggcagcagcg tggacctcca
gctgatcata gttgtcatgg atgactctgg 480 tggcattggt ggcttctgnc
tgcagtgaca atgcctctgt tctgacattg cgcagagttt 540 tctgcatgta
gangaagagt tggcaatgng ctgattttct caagattt 588 13 514 DNA Artificial
cDNA 13 anaggtaaaa tatggaattt attcagaaaa accactcagt acaatgtgta
ctacaggata 60 tcacttggct cacatcacag acatggacta tatgacttca
caataaatta gangggaagg 120 cagcccttta ctcccaggat ggcctgcagg
cctttcagca gatagagttt attacaaata 180 ccacagtaga ctttttggct
accttcaagc ggaataagac agcctgaggc cagccagtgg 240 gactttcagc
tgccnggggc cttccccatc tccttcccct tcaaggtggg ctgtctccca 300
cagtaaaccg cacncggggt gttcatccct ggatcactat acaggttcct tgtcatcaag
360 ctgaggacat tacttcatga ttcttattaa tccaggctag aaagacgtcg
agctctccca 420 gggatttaat ggcagcagcg tggaactcca gctgatcata
gttgtcatgg atgacnctgg 480 tggnttggtg gcttccggct gcagtgacat gcct 514
14 574 DNA Artificial cDNA 14 accccaaaat cttgagaaaa atcagcagca
ttgccgncgn gtnngggggt gngggggagg 60 ngnngagnng nncnctntaa
gagnccncnn aaangngttg ggaccaatgc caccagagtc 120 atccatgaca
actatgatca gctggaggtc cacgctgctg ccattaaatc cctgggagag 180
ctcgacgtct ttctagcctg gattaatang aagnatggag gnnggtgngn ngcggggttg
240 tgaggnngct gggangnggn gctgggtnng gagtngnngt tgccgcgnnt
nggggagnna 300 gtgcancctg aaggggaagg agatggggaa ggccccttgc
agctgaaagt cccactggct 360 ggcctcaggc tgtcttattc cgcttgaaaa
tagccaaaaa gtctactgtg gtatttgtaa 420 taaactctat ctgctgaaag
ggcctgcagc aatcctggga gtaagggctg ccttcccanc 480 taatttattg
tgaagtcata tagtccatgt ctgtgatgtg agccaagtga tatctgtagt 540
acacattgta ctgagtggtt ttctgaataa ttca 574 15 594 DNA Artificial
cDNA 15 caccccaaaa tcttgagaaa aatcagcagc attgccagnn cgnggcnggn
ccgtgngcnn 60 gnngnngnnn ncnggnngcn cncnttaaaa agccnnnnnn
angggttcng ggnacccaat 120 gccaccagag tcatccatga caactatgat
cagctggagg tccacgctgc tgccattaaa 180 tccctgggag agctcgacgt
ctttctagcc tggattaata agaagcaggg ngggnggcgg 240 ngngggcgtn
ccgtgnncgg gnnaggggng ggncggtgng nnngcgcgnc gnggtggann 300
nggtngcggn ggnngcgctg gggangtgnt nggaagggcc ctgcagctga aagtcccact
360 ggctggcctc aggtgtctta ttccgcttga aaatagccaa aaagtctact
gtggtatttg 420 taataaactc tatctgctga aagggcctgc agcattcctg
ggagtaaagg gctgccttcc 480 catctaattt attgtgaagt catatagtcc
atgtctgtga tgtgagccaa gtgatatcct 540 gtagtacaca ttgtactgag
tggtttttct gaataaattc atatttacct taaa 594 16 270 DNA Artificial
cDNA 16 gcactacttc cagaacacac aaggcctgat cttcgtggtg gacagcaatg
acagagagcg 60 tgtgaacgag gcccgtnagg agctcatgag gatgctggcc
gaggacgagn tccgggatgc 120 tgtcctcctg gtgttcgcca acaagcagga
cctccccaac gncatgaatg cggccgagat 180 cacagacaag ctggggctgc
actcactacg ccacaggaac tggtacattc aggccacctn 240 cgncaccagc
ggcgacgggc tctatgaagg 270 17 30 DNA Homo sapiens 17 acctgaggtc
tgatggcaaa gtccaagaat 30
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