U.S. patent application number 10/896297 was filed with the patent office on 2005-02-10 for interleukin-15 receptors.
Invention is credited to Anderson, Dirk M., Giri, Judith G..
Application Number | 20050032167 10/896297 |
Document ID | / |
Family ID | 27398929 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032167 |
Kind Code |
A1 |
Anderson, Dirk M. ; et
al. |
February 10, 2005 |
Interleukin-15 receptors
Abstract
There are disclosed Interleukin-15 Receptor (IL-15R) proteins,
DNAs and expression vectors encoding IL-15R, and processes for
producing IL-15R as products of recombinant cell cultures.
Inventors: |
Anderson, Dirk M.; (Seattle,
WA) ; Giri, Judith G.; (Chesterfield, MO) |
Correspondence
Address: |
IMMUNEX CORPORATION
LAW DEPARTMENT
1201 AMGEN COURT WEST
SEATTLE
WA
98119
US
|
Family ID: |
27398929 |
Appl. No.: |
10/896297 |
Filed: |
July 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10896297 |
Jul 20, 2004 |
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10385072 |
Mar 10, 2003 |
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6764836 |
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10385072 |
Mar 10, 2003 |
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08988197 |
Dec 10, 1997 |
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6548065 |
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08988197 |
Dec 10, 1997 |
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08435760 |
May 4, 1995 |
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08435760 |
May 4, 1995 |
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08300903 |
Sep 6, 1994 |
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5591630 |
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08300903 |
Sep 6, 1994 |
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08236919 |
May 6, 1994 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/7155
20130101 |
Class at
Publication: |
435/069.1 ;
530/350; 536/023.5; 435/320.1; 435/325 |
International
Class: |
C07K 014/715; C07H
021/04 |
Claims
We claim:
1. An isolated DNA encoding an IL-15 receptor (IL-15R), wherein the
DNA is selected from the group consisting of: (a) a DNA having a
nucleotide sequence as set forth in SEQ ID NO:1, nucleotides 91
through 789; (b) a DNA having a nucleotide sequence as set forth in
SEQ ID NO:6, nucleotides 34 through 753; (c) DNAs that hybridize to
the DNA sequences of (a) or (b) or their complementary strands
under conditions of high stringency, and which encode polypeptides
capable of binding IL-15; and (d) DNAs that, due to degeneracy of
the genetic code, encode polypeptides encoded by any of the
foregoing DNAs.
2. An isolated DNA according to claim 1 that encodes a human
IL-15R.
3. An isolated DNA according to claim 1 that encodes a soluble
IL-15R.
4. An isolated DNA according to claim 3 wherein the DNA is selected
from the group consisting of: (a) a DNA having a nucleotide
sequence as set forth in SEQ ID NO:1, nucleotides 91 through 612;
(b) a DNA having a nucleotide sequence as set forth in SEQ ID NO:6,
nucleotides 34 through 567; (c) a DNA that encodes a fragment of a
peptide encoded by the sequences of (a) or (b), which fragment is
capable of binding IL-15; (d) DNAs that hybridize to the DNA
sequences of (a), (b) or (c) or their complementary strands under
conditions of high stringency, and which encode a polypeptide
capable of binding IL-15; and (e) DNAs that, due to degeneracy of
the genetic code, encode a polypeptide encoded by any of the
foregoing DNAs.
5. An isolated DNA sequence according to claim 3 that encodes a
human IL-15R.
6. An isolated DNA according to claim 1, encoding a modified IL-15R
polypeptide having one or more changes in a primary amino acid
sequence, which changes are selected from the group consisting of:
inactivated N-linked glycosylation sites; modified KEX2 protease
cleavage sites; deleted cysteine residues; and conservative amino
acid substitutions, wherein the modified polypeptide binds
IL-15.
7. A recombinant expression vector comprising a DNA according to
claim 1.
8. A recombinant expression vector comprising a DNA according to
claim 4.
9. A recombinant expression vector comprising a DNA according to
claim 5.
10. A process for preparing an IL-15 receptor (IL-15R), comprising
culturing a host cell transformed or transfected with a recombinant
expression vector according to claim 7 under conditions promoting
expression, and recovering a polypeptide from the culture, wherein
the polypeptide is capable of binding IL-15.
11. A process for preparing an IL-15 receptor (IL-15R), comprising
culturing a host cell transformed or transfected with a recombinant
expression vector according to claim 8 under conditions promoting
expression, and recovering a polypeptide from the culture, wherein
the polypeptide is capable of binding IL-15.
12. A process for preparing an IL-15 receptor (IL-15R), comprising
culturing a host cell transformed or transfected with a recombinant
expression vector according to claim 9 under conditions promoting
expression, and recovering a polypeptide from the culture, wherein
the polypeptide is capable of binding IL-15.
13. An isolated IL-15 receptor (IL-15R) encoded by a DNA sequence
according to claim 1.
14. An IL-15 receptor (IL-15R) according to claim 13 which is a
soluble IL-15R.
15. An IL-15 receptor (IL-15R) according to claim 13, comprising a
polypeptide selected from the group consisting of: (a) a
polypeptide having an amino acid sequence as set forth in SEQ ID
NO:2, having an amino terminus selected from the group consisting
of amino acid 31, amino acid 34 and amino acid 35 of SEQ ID NO:2,
and a carboxy terminus selected from the group consisting of amino
acid 204 and amino acid 205 of SEQ ID NO:2; (b) a polypeptide
having an amino acid sequence as set forth in SEQ ID NO:6, having
an amino terminus selected from the group consisting of amino acid
12, amino acid 15 and amino acid 16 of SEQ ID NO:6, and a carboxy
terminus selected from the group consisting of an amino acid
between amino acid 78 and amino acid 189 of SEQ ID NO:6; and (c) a
fragment of a polypeptide having an amino acid sequence of the
polypeptide of (a) or (b), which fragment is capable of binding
IL-15.
16. Antibodies immunoreactive with mammalian IL-15 receptors.
17. An antibody according to claim 16 which is a monoclonal
antibody.
18. An antibody according to claim 17 which is a blocking
monoclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 08/988,197 filed Dec. 10, 1997, and issued as
U.S. Pat. No. 6,548,065 which is a continuation of U.S. patent
application Ser. No. 08/435,760, filed May 4, 1995, now abandoned,
which is a continuation-in-part application of U.S. patent
application Ser. No. 08/300,903, filed Sep. 6, 1994, and issued as
U.S. Pat. No. 5,591,630, which is a continuation-in-part
application of U.S. patent application Ser. No. 08/236,919, filed
May 6, 1994, now abandoned.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to cytokine
receptors, and more specifically, to Interleukin-15 receptors.
[0003] Interleukin-15 (IL-15) is a recently identified cytokine
with biological activities similar to IL-2 (Grabstein et al.,
Science 264:965, 1994). There is approximately 96% nucleotide
sequence identity and 96% amino acid sequence identity between
human and simian IL-15, and approximately 81% nucleotide sequence
identity and 73% amino acid sequence identity between human and
murine IL-15.
[0004] Northern analysis of a variety of human tissues indicated
that IL-15 mRNA is expressed by many human tissues and abundantly
by placenta and skeletal muscle. Significant levels of IL-15 mRNA
were also observed in other tissues including kidney, lung, liver,
and heart. The best sources of IL-15 mRNA so far observed have been
adherent mononuclear cells (monocyte enriched, PBM) and epithelial
and fibroblast cell lines such as CV-1/EBNA and IMTLH. Activated
peripheral blood T cells (PBT), a rich source of IL-2, express no
detectable IL-15 mRNA.
[0005] IL-15 shares many biological properties with Interleukin-2
("IL-2"). These properties include proliferation and activation of
human and murine T cells and the generation of lymphokine activated
killer cells (LAK), natural killer cells (NK) and cytotoxic T
lymphocytes (CTL). IL-15 also can co-stimulate with CD40 ligand
(CD40L) proliferation and immunoglobulin secretion by B
lymphocytes.
[0006] In view of the shared biological properties with IL-2, tests
were conducted to determine whether IL-15 uses any of the
components of the IL-2 receptor. IL-2 cell surface receptors
(IL-2R) contain at least three subunits, .alpha., .beta. and
.gamma. (Toshikazu et al., Science, 257: 379 (1992); see also
Minami et al., Annu. Rev. Immunol. 11,245, 1993, for a recent
review). The .beta. and .gamma. chains are required for high
affinity IL-2 binding and IL-2 signaling and are members of the
hematopoietin receptor superfamily. The .alpha. chain (or p55) is a
low affinity, non-signaling binding subunit, and the only cytokine
receptor member of a large family of binding proteins whose members
include complement receptor proteins (Perkins et al., Biochemistry
27:4004, 1988; Davie et al., Cold Spring Harb. Symp. Quant. Biol.
51:509, 1986). The .gamma. chain of the IL-2R has been shown
recently to be shared by receptors for several other cytokines
(IL-4, IL-7, IL-9; (Noguchi et al., Science 262:1877, 1993; Kondo,
et al., Science 262:1874, 1993; Kondo et al., Science 263:1453,
1994; Russell et al., Science 262:1880, 1993; Russell, et al.,
Science 266:1042, 1994) and designated the common .gamma. chain or
.gamma..sub.c.
[0007] Several lines of evidence suggest that there is an IL-15
specific binding protein. For example, an IL-3 dependent murine
cell line, 32D (J. S. Greenberger et al., Fed. Proc. 42: 2762
(1983)), expressed the complete IL-2R and proliferated in response
to IL-2, but cannot bind or respond to IL-15 (Grabstein et al.,
supra). Similarly, early murine pre-T cells derived from day 13
fetal liver that lack CD3, CD4 and CD8 expression (triple negative,
or TN, cells) expressed all three IL-2R subunits, proliferated in
response to IL-2, but did not bind or respond to IL-15 (Giri et
al., EMBO J. 13:2822, 1994). On the other hand, certain human cell
types and cell lines (e.g., umbilical vein endothelial cells,
fibroblasts and thymic and stromal cells) did not bind IL-2 but
bound IL-15 with high affinity (Giri et al., supra).
[0008] Additionally, antibodies directed against the .alpha. chain
of the IL-2 receptor (anti-IL-2R.alpha.) have no effect on IL-15
(Grabstein et al., supra; Giri et al., supra). Antibodies directed
against the IL-2R.beta., however, are able to block the activity of
IL-15, suggesting that IL-15 uses the .beta. chain of IL-2R.
Similarly, some cells require the y chain of IL-2R for IL-15 signal
transduction (Giri et al., supra) IL-15 requires the p chain of the
IL-2R for all the biological activities tested, but the .alpha.
chain of the IL-2R is not required (Giri et al., supra; Grabstein
et al., supra). However, prior to the present invention, neither an
IL-15-specific binding protein, nor a DNA encoding such protein,
had been isolated.
SUMMARY OF THE INVENTION
[0009] The present invention provides isolated Interleukin-15
receptor (IL-15R) and isolated DNA sequences encoding IL-15R, in
particular, human and murine IL-15R, or analogs thereof.
Preferably, such isolated DNA sequences are selected from the group
consisting of (a) DNA sequences comprising a nucleotide sequence
derived from the coding region of a native IL-15R gene; (b) DNA
sequences capable of hybridization to a DNA of (a) under moderate
to high stringency conditions and that encode biologically active
IL-5R; and (c) DNA sequences that are degenerate as a result of the
genetic code to a DNA sequence defined in (a) or (b) and that
encode biologically active IL-15R. The present invention also
provides recombinant expression vectors or plasmids and transformed
host cells comprising the DNA sequences defined above, recombinant
IL-15R proteins produced using the recombinant expression vectors,
plasmids or transformed host cells, and processes for producing the
recombinant IL-15R proteins utilizing the expression vectors,
plasmids or transformed host cells.
[0010] The present invention also provides substantially
homogeneous preparations of IL-15R protein. The present invention
also provides compositions for use in assays for IL-15 or IL-15R,
purification of IL-15, or in raising antibodies to IL-15R,
comprising effective quantities of the IL-15R proteins of the
present invention.
[0011] These and other aspects of the present invention will become
evident upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates the inhibition of binding of radiolabeled
IL-15 to CTLL.2 cells by soluble murine IL-15 receptor
(HIS-IL15R).
[0013] FIG. 2 presents a sequence alignment between the murine
IL-15 receptor and the human IL15 receptor (clone W5). The top line
represents the amino acid sequence of human IL-15R; the bottom line
represents the amino acid sequence of murine IL-15R. The amino acid
sequence has been separated into several protein domains:
[0014] 1. signal sequence
[0015] 2. structural domain 1
[0016] 3. proline-rich, flexible hinge region
[0017] 4. structural domain 2
[0018] 5. transmembrane domain
[0019] 6. cytoplasmic domain
[0020] The primary amino acid sequence was also analyzed for
predicted structural characteristics, and found to share common
features with a group of complement factors, and the .alpha.
subunit of IL-2 receptor. Certain structural characteristics of the
IL-15R are also designated in FIG. 2:
[0021] .beta.: beta sheet
[0022] L: loop
[0023] bold: amino acids conserved among IL-15R and related
proteins (i.e., complement control proteins, IL-2 receptor a
chain)
[0024] shaded: putative IL-15 binding region
DETAILED DESCRIPTION OF THE INVENTION
[0025] "Interleukin-15 receptor," "IL-15R" and "IL-15R.alpha."
refer to proteins that are present on many cell types, including
cells of lymphoid origin, as well as non-lymphoid cells such as
fresh human endothelial cells, and stromal cells types from bone
marrow, fetal liver and thymic epithelium. As used herein, the
above terms include analogs or fragments of native and recombinant
IL-15R proteins with L-15-binding activity. Specifically included
are truncated, soluble or fusion forms of IL-15R protein as defined
below. In the absence of any species designation, IL-15R refers
generically to mammalian IL-15R, including but not limited to,
human, murine, and bovine IL-15R. Similarly, in the absence of any
specific designation for deletion mutants, the term IL-15R means
all forms of IL-15R, including mutants and analogs that possess
IL-15R biological activity.
[0026] "Soluble IL-15R" or "sIL-15R" as used in the context of the
present invention refer to proteins, or substantially equivalent
analogs, that are substantially similar to all or part of the
extracellular region of a native IL-15R and are secreted by the
host cell but retain the ability to bind IL-15. Soluble L-15R
proteins may also include part of the transmembrane region or part
of the cytoplasmic domain or other sequences, provided that the
soluble IL-15R proteins are capable of being secreted from the host
cell in which they are produced.
[0027] The term "isolated" or "purified", as used in the context of
this specification to define the purity of IL-15R protein or
protein compositions, means that the protein or protein composition
is substantially free of other proteins of natural or endogenous
origin and contains less than about 1% by mass of protein
contaminants residual of production processes. Such compositions,
however, can contain other proteins added as stabilizers, carriers,
excipients or co-therapeutics. IL-15R is purified to substantial
homogeneity if no other proteins of natural or endogenous origin,
or protein contaminants residual of production processes, are
detected in a polyacrylamide gel by silver staining.
[0028] The term "substantially similar," when used to define either
amino acid or nucleic acid sequences, means that a particular
subject sequence, for example, a mutant sequence, varies from a
reference sequence (e.g., a native sequence) by one or more
substitutions, deletions, or additions, the net effect of which is
to retain biological activity of the IL-5R protein as may be
determined, for example, in IL-15R binding assays, such as is
described in Example 1 below. Substantially similar analog protein
will be greater than about 30 percent similar to the corresponding
sequence of the native IL-15R. More preferably, the analog proteins
will be greater than about 80 percent identical to the
corresponding sequence of the native IL-15R. For fragments of
IL-15R proteins, (e.g., soluble IL-15R polypeptides), the term "80
percent identical" refers to that portion of the reference native
sequence that is found in the IL-15R fragment.
[0029] Computer programs are available for determining the percent
identity between two DNA or amino acid sequences (e.g., between a
mutant sequence and a native sequence). One example is the GAP
computer program, version 6.0, described by Devereux et al., Nucl.
Acids Res. 12:387 (1984) and available from the University of
Wisconsin Genetics Computer Group (UWGCG). The GAP program uses the
alignment method of Needleman and Wunsch, J. Mol. Biol. 48:443
(1970), as revised by Smith and Waterman, Adv. Appl. Math 2:482
(1981).
[0030] Alternatively, nucleic acid subunits and analogs are
"substantially similar" to the specific native DNA sequences
disclosed herein if (a) the DNA sequence is derived from the coding
region of a native mammalian IL-15R gene; (b) the DNA sequence is
capable of hybridization to a native IL-15R DNA sequence under
moderately stringent conditions (i.e., 50.degree. C., 2.times.SSC)
and encodes biologically active IL-15R protein; or (c) the DNA
sequence is degenerate as a result of the genetic code to one of
the foregoing native or hybridizing DNA sequences and encodes a
biologically active IL-15R protein. DNA sequences that hybridize to
a native IL-15R DNA sequence under conditions of high stringency,
and that encode biologically active IL-15R, are also encompassed by
the present invention. Moderate and high stringency hybridization
conditions are terms understood by the skilled artisan and have
been described in, for example, Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring
Harbor Laboratory Press (1989). IL-15R proteins encoded by the
foregoing DNA sequences are provided by the present invention.
[0031] "Recombinant DNA technology" or "recombinant", as used
herein, refers to techniques and processes for producing specific
polypeptides from microbial (e.g., bacterial, fungal or yeast) or
mammalian cells or organisms (e.g., transgenics) that have been
transformed or transfected with cloned or synthetic DNA sequences
to enable biosynthesis of heterologous peptides. Native
glycosylation patterns will only be achieved with mammalian cell
expression systems. Yeast provide a distinctive glycosylation
pattern. Prokaryotic cell expression (e.g., E. coli) will generally
produce polypeptides without glycosylation.
[0032] "Biologically active", as used throughout the specification
as a characteristic of IL-15R, means that a particular molecule
shares sufficient amino acid sequence similarity with a native
IL-15R protein to be capable of binding detectable quantities of
IL-15, preferably with affinity similar to native IL-15R.
[0033] A "DNA sequence" refers to a DNA polymer, in the form of a
separate fragment or as a component of a larger DNA construct, that
has been derived from DNA isolated at least once in substantially
pure form (i.e., free of contaminating endogenous materials) and in
a quantity or concentration enabling identification, manipulation,
and recovery of its component nucleotide sequences by standard
biochemical methods such as those outlined in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989). Such sequences are
preferably provided in the form of an open reading frame
uninterrupted by internal nontranslated sequences, or introns, that
are typically present in eukaryotic genes. Sequences of
non-translated DNA may be present 5' or 3' from an open reading
frame, where the same do not interfere with manipulation or
expression of the coding regions.
[0034] "Nucleotide sequence" refers to a heteropolymer of
deoxyribonucleotides. DNA sequences encoding the proteins provided
by this invention may be assembled from cDNA fragments and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene that is capable of being expressed in a
recombinant transcriptional unit.
[0035] "Recombinant expression vector" refers to a plasmid
comprising a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, promoters or enhancers, (2) a structural
or coding sequence that is transcribed into mRNA and translated
into protein, and (3) appropriate transcription and translation
initiation and termination sequences. Structural elements intended
for use in yeast expression systems preferably include a leader
sequence enabling extracellular secretion of translated protein by
a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it may include an
N-terminal methionine residue. This residue may optionally be
subsequently cleaved from the expressed recombinant protein to
provide a final product.
[0036] "Recombinant microbial expression system" means a
substantially homogeneous monoculture of suitable host
microorganisms, for example, bacteria such as E. coli or yeast such
as S. cerevisiae, that has stably integrated a recombinant
transcriptional unit into chromosomal DNA or carries the
recombinant transcriptional unit as a component of a resident
plasmid. Generally, cells constituting the system are the progeny
of a single ancestral transformant. Recombinant expression systems
as defined herein will express heterologous protein upon induction
of the regulatory elements linked to the DNA sequence or synthetic
gene to be expressed.
[0037] Isolation of DNA Encoding IL-15R
[0038] As shown by Scatchard analysis of iodinated IL-15 binding,
activated PBT as well as antigen specific T cell clones express
only a few hundred receptors for IL-15. Cells from the murine Th2
CD4.sup.+ cell clone, D10 (Kaye et al., J. Immunol. 133:1339
(1984)), express up to 24,000 IL-15 receptors when cultured with
IL-2. A murine DNA sequence encoding murine IL-15R was isolated
from a cDNA library prepared using standard methods by reverse
transcription of polyadenylated RNA isolated from D10 cells.
Transfectants expressing biologically active IL-15R were initially
identified using a slide autoradiographic technique, substantially
as described by Gearing et al., EMBO J. 8:3667 (1989).
[0039] A D10 cDNA library in plasmid pDC304 was prepared as
described in Larsen et al., J. Exp. Med., 172:159 (1990). pDC304 is
derived from pDC302 previously described by Mosley et al., Cell,
59: 335-348 (1989) by deleting the adenovirus tripartite leader
(TPL) in pDC302.
[0040] Using this approach, approximately 20,000 cDNAs were
screened in pools of approximately 1000 cDNAs each using the slide
autoradiographic method until assay of one transfectant pool showed
multiple cells clearly positive for IL-15 binding. This pool was
then partitioned into pools of approximately 100 and again screened
by slide autoradiography and a positive pool was identified.
Individual colonies from this pool of approximately 100 were
screened until a single clone (clone D1-4-D5) was identified that
directed synthesis of a surface protein with detectable IL-15
binding activity. This clone was isolated and sequenced to
determine the sequence of the murine IL-15R cDNA clone, D1-4-D5.
The cloning vector pDC304 containing the murine IL-15R cDNA clone,
D1-4-D5, was deposited with the American Type Culture Collection,
12301 Parklawn Drive, Rockville, Md. 20852 USA ("ATCC") in
accordance with the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure on Apr. 22, 1994, under accession number ATCC
62659. The deposit was named "DI-4-D5 (pDC304:muIL-15R)" and
comprised an E. coli strain containing a murine IL-15R cDNA insert
that is made up of a 71-bp 5' noncoding region preceding an open
reading frame of 792 bp and a 995-bp 3' non coding region (the
3'-most approximately 200 bp of which is likely to be derived from
non-related sequence). The nucleotide sequence of the open reading
frame is disclosed in SEQ ID NO: 1. All restrictions on the
availability to the public of the material deposited will be
irrevocably removed upon the granting of a patent.
[0041] A probe may be constructed from the murine sequence and used
to screen various other mammalian cDNA libraries. cDNA clones that
hybridize to the murine probe are then isolated and sequenced.
[0042] Like most mammalian genes, mammalian IL-15R is encoded by a
multi-exon gene. IL-5R variants can be attributed to different mRNA
splicing events following transcription or from proteolytic
cleavage of the IL-15R protein, wherein the IL-15R binding property
is retained. Alternative splicing of mRNA may yield a truncated but
biologically active IL-15R protein, such as a soluble form of the
protein. Variations attributable to proteolysis include, for
example, differences in the N- or C-termini upon expression in
different types of host cells, due to proteolytic removal of one or
more terminal amino acids from the IL-5R protein (generally from
1-5 terminal amino acids). Signal peptides may be cleaved at
different positions in a given protein, resulting in variations of
the N-terminal amino acid of the mature protein. These IL-15R
variants share large regions of identity or similarity with the
cDNAs claimed herein and are considered to be within the scope of
the present invention.
[0043] Proteins and Analogs
[0044] The present invention provides recombinant mammalian IL-15R
polypeptides. Isolated IL-15R polypeptides of this invention are
substantially free of other contaminating materials of natural or
endogenous origin and contain less than about 1% by mass of protein
contaminants residual of production processes. The IL-5R
polypeptides of this invention are optionally without associated
native-pattern glycosylation.
[0045] Mammalian IL-15R of the present invention includes, by way
of example, primate, human, murine, canine, feline, bovine, ovine,
equine and porcine IL-15R. The amino acid sequence of a full length
murine IL-15R (i.e., including signal peptide, extracellular
domain, transmembrane region and cytoplasmic domain) is shown in
SEQ ID NOs:1 and 2. The amino acid sequence in SEQ ID NOs:1 and 2
predicts a type 1 membrane protein (i.e., a single transmembrane
region with a N-terminal extracellular domain and a C-terminal
cytoplasmic domain). The predicted signal peptide cleavage occurs
between amino acids 30 and 31 in SEQ ID NO:2. The predicted
transmembrane region includes amino acids 206 to 226 in SEQ ID
NO:2. Mammalian IL-15R cDNA can be obtained by cross species
hybridization, for example, by using a single stranded probe
derived from the murine IL-15R DNA sequence, SEQ ID NO:1, as a
hybridization probe to isolate IL-15R cDNAs from mammalian cDNA
libraries. The isolated IL-15R cDNAs then can be transfected into
expression vectors and host cells to express the IL-15R
proteins.
[0046] Derivatives of IL-15R within the scope of the invention also
include various structural forms of the primary protein that retain
biological activity. Due to the presence of ionizable amino and
carboxyl groups, for example, an IL-15R protein may be in the form
of acidic or basic salts, or may be in neutral form. Individual
amino acid residues may also be modified by oxidation or
reduction.
[0047] The primary amino acid structure may be modified by forming
covalent or aggregative conjugates with other chemical moieties,
such as glycosyl groups, lipids, phosphate, acetyl groups and the
like, or by creating amino acid sequence mutants. Covalent
derivatives are prepared by linking particular functional groups to
IL-15R amino acid side chains or at the N- or C-termini. Other
derivatives of IL-15R within the scope of this invention include
covalent or aggregative conjugates of IL-15R or its fragments with
other proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions.
[0048] When initially expressed in a recombinant system, IL-15R may
comprise a signal or leader polypeptide sequence (native or
heterologous) at the N-terminal region of the protein. The signal
or leader peptide co-translationally or post-translationally
directs transfer of the protein from its site of synthesis to its
site of function outside the cell membrane or wall, and is cleaved
from the mature protein during the secretion process. Further,
using conventional techniques, IL-15R polypeptides can be expressed
as polypeptide fusions comprising additional polypeptide sequences,
such as Fc or other immunoglobulin sequences, linker sequences, or
other sequences that facilitate purification and identification of
IL-15R polypeptides.
[0049] IL-15R derivatives may also be used as immunogens, reagents
in receptor-based immunoassays, or as binding agents for affinity
purification procedures of IL-15 or other binding ligands. IL-15R
derivatives may also be obtained by cross-linking agents, such as
m-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at
cysteine and lysine residues. IL-15R proteins may also be
covalently bound through reactive side groups to various insoluble
substrates, such as cyanogen bromide-activated,
bisoxirane-activated, carbonyldiimidazole-activated or
tosyl-activated agarose structures, or by adsorbing to polyolefin
surfaces (with or without glutaraldehyde cross-linking). Once bound
to a substrate, IL-15R may be used to selectively bind (for
purposes of assay or purification) anti-IL-15R antibodies or
IL-15.
[0050] The IL-15R proteins of the present invention encompass
proteins having amino acid sequences that vary from those of native
IL-15R proteins, but that retain the ability to bind IL-15. Such
variant proteins comprise one or more additions, deletions, or
substitutions of amino acids when compared to a native sequence,
but exhibit biological activity that is essentially equivalent to
that of native IL-15R protein. Likewise, the IL-15R-encoding DNA
sequences of the present invention encompass sequences that
comprise one or more additions, deletions, or substitutions of
nucleotides when compared to a native IL-15R DNA sequence, but that
encode an IL-15R protein that is essentially bioequivalent to a
native IL-15R protein. Examples of such variant amino acid and DNA
sequences (the "substantially similar" sequences discussed above)
include, but are not limited to, the following.
[0051] Bioequivalent analogs of IL-15R proteins may be constructed
by, for example, making various substitutions of residues or
sequences or deleting terminal or internal residues or sequences
not needed for biological activity. For example, cysteine residues
not essential for biological activity can be deleted or replaced
with other amino acids to prevent formation of unnecessary or
incorrect intramolecular disulfide bridges upon renaturation.
[0052] Another embodiment of the present invention involves
modification of adjacent dibasic amino acid residues to enhance
expression of IL-15R in yeast systems in which KEX2 protease
activity is present. Generally, substitutions should be made
conservatively; i.
[0053] e., the most preferred substitute amino acids are those
having physiochemical characteristics resembling those of the
residue to be replaced. Similarly, when a deletion or insertion
strategy is adopted, the potential effect of the deletion or
insertion on biological activity should be considered.
[0054] Substantially similar polypeptide sequences, as defined
above, generally comprise a like number of amino acid sequences,
although C-terminal truncations for the purpose of constructing
soluble IL-15Rs will contain fewer amino acid sequences. In order
to preserve the biological activity of IL-15Rs, deletions and
substitutions will preferably result in homologous or
conservatively substituted sequences, meaning that a given residue
is replaced by a biologically similar residue. Examples of
conservative substitutions include substitution of one aliphatic
residue for another, such as Ile, Val, Leu, or Ala for one another,
or substitutions of one polar residue for another, such as between
Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative
substitutions, for example, substitutions of entire regions having
similar hydrophobicity characteristics, are well known. Moreover,
particular amino acid differences between human, murine and other
mammalian IL-15Rs are suggestive of additional conservative
substitutions that may be made without altering the essential
biological characteristics of IL-15R.
[0055] The present invention includes IL-15R with or without
associated native-pattern glycosylation. IL-15R expressed in yeast
or mammalian expression systems, e.g., COS-7 cells, may be similar
or slightly different in molecular weight and glycosylation pattern
than the native molecules, depending upon the expression system.
Expression of IL-15R DNAs in bacteria such as E. coli provides
non-glycosylated molecules. Functional mutant analogs of mammalian
IL-15R having inactivated N-glycosylation sites can be produced by
oligonucleotide synthesis and ligation or by site-specific
mutagenesis techniques. These analog proteins can be produced in a
homogeneous, reduced-carbohydrate form in good yield using yeast
expression systems. N-glycosylation sites in eukaryotic proteins
are characterized by the amino acid triplet Asn-A.sub.1-Z, where
A.sub.1 is any amino acid except Pro, and Z is Ser or Thr. In this
sequence, asparagine provides a side chain amino group for covalent
attachment of carbohydrate. Such sites can be eliminated by
substituting another amino acid for Asn or for residue Z, deleting
Asn or Z, or inserting a non-Z amino acid between A.sub.1 and Z, or
an amino acid other than Asn between Asn and A.sub.1.
[0056] Subunits of IL-15R may be constructed by deleting terminal
or internal residues or sequences. Particularly preferred sequences
include those in which the transmembrane region and intracellular
domain of IL-15R are deleted or substituted with hydrophilic
residues to facilitate secretion of the receptor into the cell
culture medium. Soluble IL-15R proteins may also include part of
the transmembrane region, provided that the soluble IL-15R protein
is capable of being secreted from the cell. The resulting protein
is referred to as a soluble IL-15R molecule that retains its
ability to bind IL-15. The present invention contemplates such
soluble IL-15R constructs corresponding to all or part of the
extracellular region of IL-15R. The resulting soluble IL-15R
constructs are then inserted and expressed in appropriate
expression vectors and assayed for the ability to bind IL-15, as
described in Example 1. Biologically active soluble IL-15Rs (i.e.,
those which bind IL-15) resulting from such constructions are also
contemplated to be within the scope of the present invention.
Soluble IL-15Rs can be used to inhibit IL-15, for example, in
ameliorating undesired effects of IL-15, in vitro or in vivo. For
example, significant levels of IL-15 mRNA occur in kidney, lung,
liver, and heart, organs that may be transplanted. Soluble IL-15Rs
are thus likely to be useful as IL-15 antagonists in preventing or
treating graft rejection. Soluble IL-15Rs can also be used as
components of quantitative or qualitative assays for IL-15, or for
affinity purification of IL-15.
[0057] Mutations in nucleotide sequences constructed for expression
of the above-described variant or analog IL-15R proteins should, of
course, preserve the reading frame phase of the coding sequences
and preferably will not create complementary regions that could
hybridize to produce secondary mRNA structures such as loops or
hairpins that would adversely affect translation of the receptor
mRNA. Although a mutation site may be predetermined, it is not
necessary that the nature of the mutation per se be predetermined.
For example, in order to select for optimum characteristics of
mutants at a given site, random mutagenesis may be conducted at the
target codon and the expressed IL-15R mutants screened for the
desired activity.
[0058] Not all mutations in the nucleotide sequence that encodes
IL-15R will be expressed in the final product. For example,
nucleotide substitutions may be made to enhance expression,
primarily to avoid secondary structure loops in the transcribed
mRNA (see EPA 75,444A, incorporated herein by reference), or to
provide codons that are more readily translated by the selected
host, e.g., the well-known E. coli preference codons for E. coli
expression (see U.S. Pat. No. 4,425,437, column 6). The known
degeneracy of the genetic code permits variation of a DNA sequence
without altering the amino acid sequence, since a given amino acid
may be encoded by more than one codon.
[0059] Mutations can be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked
by restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
[0060] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
having particular codons altered according to the substitution,
deletion, or insertion required. Examples of methods of making the
alterations set forth above are disclosed by Walder et al., Gene
42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik,
BioTechniques, 12-19 (1985); Smith et al., Genetic Engineering:
Principles and Methods, Plenum Press (1981); and U.S. Pat. Nos.
4,518,584 and 4,737,462.
[0061] The IL-15R proteins of the present invention encompass
proteins encoded by (a) a DNA sequence derived from the coding
region of a native IL-15R gene or (b) a DNA sequence capable of
hybridization to a native IL-15R DNA of (a) under moderate to high
stringency conditions and that encodes biologically active L-15R.
IL-15R proteins encoded by a DNA molecule that varies from the DNA
sequences of SEQ ID NO:1, wherein one strand of the DNA molecule
will hybridize to the DNA sequence presented in SEQ ID NO:1,
include, but are not limited to, IL-15R fragments (soluble or
membrane-bound) and IL-15R proteins comprising inactivated
N-glycosylation site(s), inactivated KEX2 protease processing
site(s), and/or conservative amino acid substitution(s), as
described above. IL-15R proteins encoded by DNA derived from other
mammalian species, wherein the DNA will hybridize to the murine DNA
of SEQ ID NO:1, are also encompassed.
[0062] Both monovalent forms and polyvalent forms of IL-15R are
useful in the compositions and methods of this invention.
Polyvalent forms possess multiple IL-15R binding sites for IL-15
ligand. For example, a bivalent soluble IL-15R may consist of two
tandem repeats of the extracellular region of IL-15R, separated by
a linker region. Two IL-15R polypeptides (each capable of binding
IL-15) may be joined by any suitable means, e.g., using one of the
commercially available cross-linking reagents used to attach one
polypeptide to another (Pierce Chemical Co., Rockford, Ill.).
Alternatively, a fusion protein comprising multiple IL-15R
polypeptides joined by peptide linkers may be produced using
recombinant DNA technology. Suitable peptide linkers comprise a
chain of amino acids, preferably from 20 to 100 amino acids in
length. The linker advantageously comprises amino acids selected
from the group consisting of glycine, asparagine, serine,
threonine, and alanine. Examples of suitable peptide linkers and
the use of such peptide linkers are found in U.S. Pat. No.
5,073,627.
[0063] Alternate polyvalent forms may also be constructed, for
example, by chemically coupling IL-15R to any clinically acceptable
carrier molecule, a polymer selected from the group consisting of
Ficoll, polyethylene glycol or dextran using conventional coupling
techniques. Alternatively, IL-15R may be chemically coupled to
biotin, and the biotin-IL-15R conjugate then allowed to bind to
avidin, resulting in tetravalent avidin/biotin/IL-15R molecules.
IL-15R may also be covalently coupled to dinitrophenol (DNP) or
trinitrophenol (TNP) and the resulting conjugate precipitated with
anti-DNP or anti-TNP-IgM, to form decameric conjugates with a
valency of 10 for IL-15R binding sites.
[0064] A recombinant chimeric antibody molecule may also be
produced having IL-5R sequences substituted for the variable
domains of either or both of the immunoglobulin molecule heavy and
light chains and having unmodified constant region domains. For
example, chimeric IL-15R/IgG.sub.1 may be produced from two
chimeric genes--an IL-15R/human K light chain chimera
(IL-15R/C.sub..kappa.) and an IL-15R/human .gamma.1 heavy chain
chimera (IL-15R/C.sub..gamma.-1). Following transcription and
translation of the two chimeric genes, the gene products assemble
into a single chimeric antibody molecule having IL-15R displayed
bivalently. Such polyvalent forms of IL-15R may have enhanced
binding affinity for IL-15 ligand. Additional details relating to
the construction of such chimeric antibody molecules are disclosed
in WO 89/09622 and EP 315062.
[0065] Expression of Recombinant IL-15R
[0066] The present invention provides recombinant expression
vectors to amplify or express DNA encoding IL-15R. Recombinant
expression vectors are replicable DNA constructs that have
synthetic or cDNA-derived DNA fragments encoding mammalian IL-15R
or bioequivalent analogs operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, microbial, viral or insect genes. A transcriptional unit
generally comprises an assembly of (1) a genetic element or
elements having a regulatory role in gene expression, for example,
transcriptional promoters or enhancers, (2) a structural or coding
sequence that is transcribed into mRNA and translated into protein,
and (3) appropriate transcription and translation initiation and
termination sequences, as described in detail below. Such
regulatory elements may include an operator sequence to control
transcription, a sequence encoding suitable mRNA ribosomal binding
sites. The ability to replicate in a host, usually conferred by an
origin of replication, and a selection gene to facilitate
recognition of transformants may additionally be incorporated. DNA
regions are operably linked when they are functionally related to
each other. For example, DNA for a signal peptide (secretory
leader) is operably linked to DNA for a polypeptide if it is
expressed as a precursor that participates in the secretion of the
polypeptide; a promoter is operably linked to a coding sequence if
it controls the transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is
positioned so as to permit translation. Generally, operably linked
means contiguous and, in the case of secretory leaders, contiguous
and in reading frame. Structural elements intended for use in yeast
expression systems preferably include a leader sequence enabling
extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a
leader or transport sequence, it may include an N-terminal
methionine residue. This residue may optionally be subsequently
cleaved from the expressed recombinant protein to provide a final
product.
[0067] DNA sequences encoding mammalian IL-15Rs that are to be
expressed in a microorganism will preferably contain no introns
that could prematurely terminate transcription of DNA into mRNA.
However, premature termination of transcription may be desirable,
for example, where it would result in mutants having advantageous
C-terminal truncations, for example, deletion of a transmembrane
region to yield a soluble receptor not bound to the cell membrane.
Due to code degeneracy, there can be considerable variation in
nucleotide sequences encoding the same amino acid sequence. Other
embodiments include sequences capable of hybridizing to SEQ ID NO:1
under at least moderately stringent conditions (50.degree. C.,
2.times.SSC) and other sequences hybridizing or degenerate to those
that encode biologically active IL-15R polypeptides.
[0068] Recombinant IL-15R DNA is expressed or amplified in a
recombinant expression system comprising a substantially
homogeneous monoculture of suitable host microorganisms, for
example, bacteria such as E. coli or yeast such as S. cerevisiae,
that have stably integrated (by transformation or transfection) a
recombinant transcriptional unit into chromosomal DNA or carry the
recombinant transcriptional unit as a component of a resident
plasmid. Mammalian host cells are preferred for expressing
recombinant IL-15R. Generally, cells constituting the system are
the progeny of a single ancestral transformant. Recombinant
expression systems as defined herein will express heterologous
protein upon induction of the regulatory elements linked to the DNA
sequence or synthetic gene to be expressed.
[0069] Transformed host cells are cells that have been transformed
or transfected with IL-15R vectors constructed using recombinant
DNA techniques. Transformed host cells ordinarily express IL-15R,
but host cells transformed for purposes of cloning or amplifying
IL-15R DNA do not need to express IL-15R. Expressed IL-15R will be
deposited in the cell membrane or secreted into the culture
supernatant, depending on the IL-15R DNA selected. Suitable host
cells for expression of mammalian IL-15R include prokaryotes, yeast
or higher eukaryotic cells under the control of appropriate
promoters. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include established cell lines of mammalian origin as described
below. Cell-free translation systems could also be employed to
produce mammalian IL-15R using RNAs derived from the DNA constructs
of the present invention. Appropriate cloning and expression
vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts are described by Pouwels et al., Cloning Vectors: A
Laboratory Manual, Elsevier, New York (1985).
[0070] Prokaryotic expression hosts may be used for expression of
IL-15R that do not require extensive proteolytic and disulfide
processing. Prokaryotic expression vectors generally comprise one
or more phenotypic selectable markers, for example a gene encoding
proteins conferring antibiotic resistance or supplying an
autotrophic requirement, and an origin of replication recognized by
the host to ensure amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium, and various species within the
genera Pseudomonas, Streptomyces, and Staphyolococcus, although
others may also be employed as a matter of choice.
[0071] Useful expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well-known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 and pGEX (Pharmacia Fine
Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison,
Wis., USA). These pBR322 "backbone" sections are combined with an
appropriate promoter and the structural sequence to be expressed.
E. coli is typically transformed using derivatives of pBR322, a
plasmid derived from an E. coli species (Bolivar et al., Gene 2:95
(1977)). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells.
[0072] Promoters commonly used in recombinant microbial expression
vectors include the 13-lactamase (penicillinase) and lactose
promoter system (Chang et al., Nature 275:615 (1978); and Goeddel
et al., Nature 281:544 (1979)), the tryptophan (trp) promoter
system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); and EPA
36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, p. 412 (1982)). A
particularly useful bacterial expression system employs the phage
.lambda.P.sub.L promoter and cI857ts thermolabile repressor.
Plasmid vectors available from the American Type Culture Collection
that incorporate derivatives of the .lambda.P.sub.L promoter
include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092)
and pPLc28, resident in E. coli RR1 (ATCC 53082).
[0073] Recombinant IL-15R proteins may also be expressed in yeast
hosts, preferably from the Saccharomyces species, such as S.
cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces
may also be employed. Yeast vectors will generally contain an
origin of replication from the 2.mu. yeast plasmid or an
autonomously replicating sequence (ARS), promoter, DNA encoding
IL-15R, sequences for polyadenylation and transcription termination
and a selection gene. Preferably, yeast vectors will include an
origin of replication and selectable marker permitting
transformation of both yeast and E. coli, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 or URA3 gene,
that provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, and a promoter derived
from a highly expressed yeast gene to induce transcription of a
structural sequence downstream. The presence of the TRP1 or URA3
lesion in the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan or uracil.
[0074] Suitable promoter sequences in yeast vectors include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes
(Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); and Holland et al.,
Biochem. 17:4900 (1978)). Suitable vectors and promoters for use in
yeast expression are further described in R. Hitzeman et al., EPA
73,657.
[0075] Preferred yeast vectors can be assembled using DNA sequences
from pUC18 for selection and replication in E. coli (Amp.sup.r gene
and origin of replication) and yeast DNA sequences including a
glucose-repressible ADH2 promoter and .alpha.-factor secretion
leader. The ADH2 promoter has been described by Russell et al., J.
Biol. Chem. 258:2674 (1982) and Beier et al., Nature 300:724
(1982). The yeast .alpha.-factor leader, that directs secretion of
heterologous proteins, can be inserted between the promoter and the
structural gene to be expressed (see, e.g., Kurjan et al., Cell
30:933 (1982); and Bitter et al., Proc. Natl. Acad. Sci. USA
81:5330 (1984)). The leader sequence may be modified to contain,
near its 3' end, one or more useful restriction sites to facilitate
fusion of the leader sequence to foreign genes. Suitable yeast
transformation protocols are known to those of skill in the art
(see Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978);
Sherman et al., Laboratory Course Manual for Methods in Yeast
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1986)).
[0076] Host strains transformed by vectors comprising the ADH2
promoter may be grown for expression in a rich medium consisting of
1% yeast extract, 2% peptone, and 1% or 4% glucose supplemented
with 80 .mu.g/ml adenine and 80 .mu.g/ml uracil. Derepression of
the ADH2 promoter occurs upon exhaustion of medium glucose. Crude
yeast supernatants are harvested by filtration and held at
4.degree. C. prior to further purification.
[0077] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells is particularly
preferred because such proteins are generally correctly folded,
appropriately modified and completely functional. Examples of
suitable mammalian host cell lines include the COS-7 lines of
monkey kidney cells, described by Gluzman, Cell 23:175 (1981), and
other cell lines capable of expressing a heterologous gene in an
appropriate vector including, for example, L cells, C127, 3T3,
Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian
expression vectors may comprise nontranscribed elements such as an
origin of replication, a suitable promoter and enhancer linked to
the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a polyadenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
Bio/Technology 6:47 (1988).
[0078] The transcriptional and translational control sequences in
expression vectors to be used in transforming vertebrate cells may
be provided by viral sources. For example, commonly used promoters
and enhancers are derived from Polyoma, Adenovirus 2, Simian Virus
40 (SV40), and human cytomegalovirus. DNA sequences derived from
the SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the other genetic elements required for expression of a
heterologous DNA sequence. The early and late promoters are
particularly useful because both are obtained easily from the virus
as a fragment that also contains the SV40 viral origin of
replication (Fiers et al., Nature 273:113 (1978)). Smaller or
larger SV40 fragments may also be used, provided the approximately
250 bp sequence extending from the Hind III site toward the BglI
site located in the viral origin of replication is included.
Further, mammalian genomic IL-15R promoter, control and/or signal
sequences may be used, provided such control sequences are
compatible with the host cell chosen. Exemplary vectors can be
constructed as disclosed by Okayama and Berg, Mol. Cell. Biol.
3:280 (1983).
[0079] A useful system for stable high level expression of
mammalian receptor cDNAs in C127 murine mammary epithelial cells
can be constructed substantially as described by Cosman et al.,
Mol. Immunol. 23:935 (1986).
[0080] In preferred aspects of the present invention, recombinant
expression vectors comprising IL-15R cDNAs are stably integrated
into a host cell's DNA. Elevated levels of expression product are
achieved by selecting for cell lines having amplified numbers of
vector DNA. Cell lines having amplified numbers of vector DNA are
selected, for example, by transforming a host cell with a vector
comprising a DNA sequence that encodes an enzyme that is inhibited
by a known drug. The vector may also comprise a DNA sequence that
encodes a desired protein. Alternatively, the host cell may be
co-transformed with a second vector that comprises the DNA sequence
that encodes the desired protein. The transformed or co-transformed
host cells are then cultured in increasing concentrations of the
known drug, thereby selecting for drug-resistant cells. Such
drug-resistant cells survive in increased concentrations of the
toxic drug by over-production of the enzyme that is inhibited by
the drug, frequently as a result of amplification of the gene
encoding the enzyme. Where drug resistance is caused by an increase
in the copy number of the vector DNA encoding the inhibiting
enzyme, there is a concomitant co-amplification of the vector DNA
encoding the desired protein (e.g., IL-15R) in the host cell's
DNA.
[0081] A preferred system for such co-amplification uses the gene
for dihydrofolate reductase (DHFR), that can be inhibited by the
drug methotrexate (MTX). To achieve co-amplification, a host cell
that lacks an active gene encoding DHFR is either transformed with
a vector that comprises DNA sequence encoding DHFR and a desired
protein, or is co-transformed with a vector comprising a DNA
sequence encoding DHFR and a vector comprising a DNA sequence
encoding the desired protein. The transformed or co-transformed
host cells are cultured in media containing increasing levels of
MTX, and those cell lines that survive are selected.
[0082] A particularly preferred co-amplification system uses the
gene for glutamine synthetase (GS), that is responsible for the
synthesis of glutamine from glutamate and ammonia using the
hydrolysis of ATP to ADP and phosphate to drive the reaction. GS is
subject to inhibition by a variety of inhibitors, for example
methionine sulphoximine (MSX). Thus, IL-15R can be expressed in
high concentrations by co-amplifying cells transformed with a
vector comprising the DNA sequence for GS and a desired protein, or
co-transformed with a vector comprising a DNA sequence encoding GS
and a vector comprising a DNA sequence encoding the desired
protein, culturing the host cells in media containing increasing
levels of MSX and selecting for surviving cells. The GS
co-amplification system, appropriate recombinant expression vectors
and cells lines, are described in the following PCT applications:
WO 87/04462, WO 89/01036, WO 89/10404 and WO 86/05807.
[0083] Recombinant proteins are preferably expressed by
co-amplification of DHFR or GS in a mammalian host cell, such as
Chinese Hamster Ovary (CHO) cells, or alternatively in a murine
myeloma cell line, such as SP2/0-Ag14 or NSO or a rat myeloma cell
line, such as YB2/3.0-Ag2O, disclosed in PCT applications WO
89/10404 and WO 86/05807.
[0084] Vectors derived from retroviruses may be employed in
mammalian host cells. A preferred retroviral expression vector is
tgLS(+) HyTK, described in PCT application WO 92/08796.
[0085] A preferred eukaryotic vector for expression of IL-15R DNA
is disclosed below in Example 1. This vector, referred to as
pDC304, was derived from pDC302 previously described by Mosley et
al., Cell, 59: 335-348 (1989) by deleting the adenovirus tripartite
leader in pDC302.
[0086] Sense and Antisense Sequences
[0087] The present invention provides both double-stranded and
single-stranded IL-15R DNA, and IL-15R mRNA as well. The
single-stranded IL-15R nucleic acids have use as probes to detect
the presence of hybridizing IL-15R nucleic acids (e.g., in in vitro
assays) and as sense and antisense molecules to block expression of
IL-15R.
[0088] In one embodiment, the present invention provides antisense
or sense molecules comprising a single-stranded nucleic acid
sequence (either RNA or DNA) capable of binding to target IL-15R
mRNA (sense) or IL-15R DNA (antisense) sequences. These antisense
or sense molecules may comprise a fragment of the coding region of
IL-15R cDNA, and, in one embodiment, are oligonucleotides
comprising at least about 14 nucleotides, preferably from about 14
to about 30 nucleotides, of an IL-15R cDNA sequence. The ability to
create an antisense or sense oligonucleotide based upon a cDNA
sequence for a given protein is described in, for example, Stein
and Cohen, Cancer Res. 48:2659 (1988) and van der Krol et al.,
BioTechniques 6:958 (1988).
[0089] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means. The
oligonucleotides thus may be used to block expression of IL-15R
proteins. Uses of the antisense and sense nucleic acid sequences
include, but are not limited to, use as research reagents. The
biological effects of blocking IL-5R expression in cultured cells
may be studied, for example. The oligonucleotides also may be
employed in developing therapeutic procedures that involve blocking
IL-5R expression in vivo.
[0090] Antisense or sense oligonucleotides further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or
other sugar linkages, such as those described in WO 91/06629) and
wherein such sugar linkages are resistant to endogenous nucleases.
Such oligonucleotides with resistant sugar linkages are relatively
stable in vivo (i.e., capable of resisting enzymatic degradation)
but retain sequence specificity for binding to target nucleotide
sequences. Other examples of sense or antisense oligonucleotides
include those oligonucleotides that are covalently linked to
organic moieties such as those described in WO 90/10448, or to
other moieties that increase affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0091] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any suitable
method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. A preferred method involves insertion
of the antisense or sense oligonucleotide into a suitable
retroviral vector, then contacting the target cell with the
retrovirus vector containing the inserted sequence, either in vivo
or ex vivo. Suitable retroviral vectors include, but are not
limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived
from M-MuLV), or the double copy vectors designated DCT5A, DCT5B
and DCT5C (see PCT Application US 90/02656).
[0092] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by attaching
the oligonucleotide to a molecule that binds to the target cell, as
described in WO 91/04753. The oligonucleotide may be attached to
molecules that include, but are not limited to, antibodies, growth
factors, other cytokines, or other ligands that bind to cell
surface receptors.
[0093] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0094] The following examples are offered by way of illustration,
and not by way of limitation. Those skilled in the art will
recognize that variations of the invention embodied in the examples
can be made, especially in light of the teachings of the various
references cited herein, the disclosures of which are incorporated
by reference.
EXAMPLES
Example 1
Isolation and Expression of cDNAs Encoding Murine IL-15R
[0095] A. Radiolabeling of IL-15. Recombinant flag simian IL-15
expressed in yeast (SEQ ID NO:3) was purified by passage over a
Phenyl Sepharose CL-4B column (Pharmacia, Piscataway, N.J.)
followed by two passages over reverse phase HPLC C4 columns
(Vydac), the first using a pyridine acetate/propanol buffer system,
the second in trifluoro-acetic acid acetonitrile system. Fractions
containing pure IL-15 were dried under nitrogen and radiolabeled
using the enzymobead iodination reagent (BioRad, Richmond VA) as
described by in Park et al., J. Exp. Med., 165:1201-1206 (1987).
The biological activity of radiolabeled IL-15 was assessed using
the mitochondrial stain MTT (3-4,5-Dimethylthiazol-2-yl)-2-
,5-diphenyl tetrazolium bromide; thiazol blue (Sigma, St. Dextran,
as described by Cosman et al., Nature, 312:768-771 (1984)).
[0096] B. Binding To Intact Cells. A source for IL-15R was selected
by screening various murine and human cells lines and tissues for
expression of IL-15R based on their ability to bind .sup.125I-IL-15
that was prepared as described above in Example 1A. For the binding
assays, a phthalate oil separation method (Dower et al., J.
Immunol. 132:751 (1984)) was performed as described by Park et al.,
J. Biol. Chem 261:4177 (1986) and Park et al., Proc. Natl. Acad.
Sci. USA 84:5267 (1987) on candidate cells grown in suspension
culture. Nonspecific binding of .sup.125I-IL-15 was measured in the
presence of a 200-fold or greater molar excess of unlabeled IL-15.
Sodium azide (0.2%) was included in all binding assays to inhibit
internalization of .sup.125I-IL-15 at 37.degree. C. Activated PBT
and well as antigen specific T cell clones expressed only a few
hundred receptors for IL-15. Cells from the murine Th2 CD4.sup.+
cell clone, D10 (Kaye et al., J. Immunol., 133:1339 (1984)),
expressed up to 24,000 IL-15 receptors when cultured with IL-2.
[0097] C. Construction and Screening of cDNA Library.
Polyadenylated mRNA was prepared from a DIO cell line and cDNAs
were prepared using standard techniques. The DIO line is a producer
of murine IL-15R. cDNA ends were adapted with Bgl II adaptors:
1 5'-GATCTTGGAACGAGACGACCTGCT-3' (SEQ ID NO:4)
3'-AACCTTGCTCTGCTGGACGA-5' (SEQ ID NO:5)
[0098] and cloned into vector pDC304.
[0099] COS-7 cells were transfected with miniprep DNA from pools of
cDNA clones directly on glass slides and cultured for 2-3 days to
permit transient expression of IL-15R. The slides containing the
transfected cells were then incubated with medium containing
125I-labeled IL-15, washed to remove unbound labeled IL-15, fixed
with glutaraldehyde, and dipped in liquid autoradiographic emulsion
and exposed in the dark. After developing the slides, they were
individually examined with a microscope and positive cells
expressing IL-15R were identified by the presence of
autoradiographic silver grains against a light background.
Approximately 20,000 cDNAs were screened in pools of approximately
1000 cDNAs each using the slide autoradiographic method until assay
of one transfectant pool showed multiple cells clearly positive for
IL-15 binding. This pool was then partitioned into pools of
approximately 100 and again screened by slide autoradiography and a
positive pool was identified. Individual colonies from this pool of
approximately 100 were screened until a single clone (clone
D1-4-D5) was identified that directed synthesis of a surface
protein with detectable IL-15 binding activity. This clone was
isolated and sequenced to determine the sequence of the murine
IL-15R cDNA clone, D1-4-D5. The cloning vector pDC304 containing
the murine IL-15R cDNA clone, D1-4-D5, was deposited with the
American Type Culture Collection ("ATCC") under accession number
ATCC 62659. The murine IL-15R cDNA insert is made up of a 71-bp 5'
noncoding region before an open reading frame of 792 bp and a
995-bp 3' non coding region. The nucleotide sequence of the open
reading frame is disclosed in SEQ ID NO:1. The amino acid sequence
of a full length murine IL-15R (i.e., including signal peptide,
extracellular domain, transmembrane region and cytoplasmic domain)
is shown in SEQ ID NOs:1 and 2. The amino acid sequence in SEQ ID
NOs:1 and 2 predicts a type 1 membrane protein (i.e., a single
transmembrane region with a N-terminal extracellular domain and a
C-terminal cytoplasmic domain). A predicted signal peptide cleavage
occurs between amino acids 30 and 31 in SEQ ID NO:2; amino acids 32
and 33 are predicted to form another, preferred, cleavage site. The
predicted transmembrane region includes amino acids 206 to 226 in
SEQ ID NO:2.
[0100] D. Recombinant IL-15R Binding. Plasmid DNA from IL-15
receptor expression plasmid was used to transfect a sub-confluent
layer of monkey COS-7 cells using DEAE-dextran followed by
chloroquine treatment, as described by Luthman et al., Nucl Acids
Res. 11: 1295 (1983) and McCutchan et al., J. Natl. Cancer Inst.
41:351 (1968). The cells were then grown in culture for three days
to permit transient expression of the inserted sequences. After
three days, the cell monolayers were assayed for .sup.125I-L-15
binding essentially as described by Park, et al., J. Exp. Med.
166:476 (1987). Non-specific binding of .sup.125I-IL-15 was
measured in the presence of 200-fold or greater excess of unlabeled
IL-15. Initial binding studies of .sup.125I-IL-15 to COS cells
transfected with IL-15R cDNA clone D1-4-D5 showed very high levels
of expression (.about.500,000 sites/cell), with an estimated
affinity of 1.0-2.2 nM, which is much lower that the affinity of
the native receptor on DIO cells.
[0101] E. Soluble IL-15R. A soluble murine IL-15 receptor was
prepared by deleting the transmembrane and cytoplasmic domains,
with the C-terminal end corresponding to Thr at amino acid 204 of
SEQ ID NO:1, and adding 5 C-terminal Histidines. The soluble IL-15
receptor was biologically active, as demonstrated by the fact that
it inhibited binding of radiolabeled IL-15 to cells expressing
membrane bound IL-15 receptor (FIG. 1).
Example 2
Preparation of Monoclonal Antibodies to IL-15R
[0102] Preparations of purified recombinant IL-15R, for example,
human IL-15R, or transfected COS cells expressing high levels of
IL-15R are employed to generate monoclonal antibodies against
IL-15R using conventional techniques, for example, those disclosed
in U.S. Pat. No. 4,411,993. Such antibodies are likely to be useful
in interfering with IL-15 binding to IL-15R, for example, in
ameliorating undesired effects of IL-15, or as components of
diagnostic or research assays for IL-15 or soluble IL-15R.
[0103] To immunize mice or rats, IL-15R immunogen is emulsified in
complete Freund's adjuvant and injected in amounts ranging from
10-100 .mu.g, subcutaneously. Ten to twenty-one days later, the
immunized animals are boosted with additional immunogen emulsified
in incomplete Freund's adjuvant and periodically boosted thereafter
on a weekly to biweekly immunization schedule. Serum samples are
periodically taken by retro-orbital bleeding or tail-tip excision
for testing by dot-blot assay (antibody sandwich) or ELISA
(enzyme-linked immunosorbent assay). Other assay procedures are
also suitable. Following detection of an appropriate antibody
titer, positive animals are given an intravenous injection of
antigen in saline. Three to four days later, the animals are
sacrificed, splenocytes harvested, and fused with an appropriate
murine myeloma cell line. Hybridoma cell lines generated by this
procedure are plated in multiple microtiter plates in a HAT
selective medium (hypoxanthine, aminopterin, and thymidine) to
inhibit proliferation of non-fused cells, myeloma hybrids, and
spleen cell hybrids.
[0104] Hybridoma clones thus generated can be screened by ELISA for
reactivity with IL-15R, for example, by adaptations of the
techniques disclosed by Engvall et al., Immunochem. 8:871 (1971)
and in U.S. Pat. No. 4,703,004. Clones that produce antibodies that
bind IL-15R and inhibit binding of IL-15 to IL-15R (blocking or
neutralizing antibodies) can also be isolated. Positive clones are
then injected into the peritoneal cavities of syngeneic animals to
produce ascites containing high concentrations (>1 mg/ml) of
anti-IL-15R monoclonal antibody. The resulting monoclonal antibody
can be purified by ammonium sulfate precipitation followed by gel
exclusion chromatography, and/or affinity chromatography based on
binding of antibody to Protein A of Staphylococcus aureus.
Example 3
Isolation and Expression of cDNAs Encoding Human IL-15R
[0105] A. Binding of IL-15 to Human Cells
[0106] Various human cell lines and tissues were screened for the
ability to bind radiolabeled IL-15 substantially as described in
Example 1. High affinity binding was observed on activated
peripheral blood mononuclear cells, activated monocytes and some
EBV-transformed cell lines. High affinity binding was also measured
on human fibroblast lines such as W126-VA4 (ATCC CCL 95.1; Kd: 27
pM; 1,400 sites per cell), a glioblastoma cell line, A-172 (ATCC
CRL 1620; Kd 50-138 pM; 1,560-4,350 sites pre cell), and vascular
endothelial cells (both venous and arterial origin, Kd 33 pM, 990
sites per cell; Kd 163 pM, 1,920 sites per cell, respectively).
Cross-linking of radiolabeled IL-15 to receptors present on the
surface of A172 cells showed a major IL-15 binding protein with an
estimated molecular weight of 55-65 Kd, a size similar to that seen
on the D10 murine cell line by cross-linking.
[0107] B. Clone WS
[0108] A cDNA encoding human IL-15R was isolated by cross-species
hybridization of the murine IL-15R cDNA to a human cDNA library
prepared from the human cell line W126-VA4-VA4 in the bacteriophage
kgt10 vector. Preparation of the library is described in U.S. Pat.
No. 5,194,375, issued Mar. 16, 1993 (DNAs Encoding IL-7 Receptors).
The W126-VA4 library was screened with a random prime labeled
murine IL-15R cDNA probe in 50% formamide buffer (50% formamide, Sx
SSC, 20 mM EDTA, 2.times. Denhardt's, 1% SDS, 0.1% sarcosyl, SO mM
KHPO.sub.4 pH 6.5, 300 .mu.g/ml salmon sperm DNA) using
1.times.10.sup.6 cpm of probe/ml of hybridization solution, at
42.degree. C. for 16-20 hours. The filters were washed once with
6.times.SSC/0.1% SDS at room temperature, followed by several
moderate stringency washes in 2.times.SSC/0.1% SDS at 55.degree.
C.
[0109] Approximately 500,000 plaques of the amplified
.lambda.gt10/WI26-VA4 library were screened by standard methods,
using the random-prime labeled murine IL-15R probe, which contained
the entire coding region as well as 5' and 3' flanking non-coding
regions. A single hybridizing plaque was identified,
plaque-purified, and its cDNA insert amplified by PCR, purified,
and sequenced. This clone, designated `W5,` shared about 65%
identity at the nucleotide level and 56% identity at the amino acid
level with the murine cDNA, `D1-4-D5` (SEQ ID NO:1). The nucleotide
and predicted amino acid sequence of W5 are shown in SEQ ID NOs:6
and 7.
[0110] As compared to the full-length murine clone D1-4-D5, W5
appeared to be missing a small portion of the expected 5'
sequences, i.e., about 125 bp compared to the murine clone,
indicating that W5 did not contain the coding region for the first
part of a putative IL-15R signal peptide (missing 19 amino acids
compared to the murine clone). The amino acid sequence in SEQ ID
NOs:6 and 7 predicts a type 1 membrane protein (i.e., a single
transmembrane region with an N-terminal extracellular domain and a
C-terminal cytoplasmic domain). The predicted transmembrane region
includes amino acids 190 to 210 of SEQ ID NOs:6 and 7. Binding of
IL-15R to IL-15 is mediated through the extracellular domain of
IL-15R as shown in FIG. 2, all or portions of which are involved in
binding.
[0111] A signal peptide cleavage is predicted to occur between
amino acids 14 and 15 in SEQ ID NO:6. For murine IL-15 receptor, a
signal peptide cleavage is predicted to occur between amino acids
32 and 33 in SEQ ID NO:2, or alternatively, between amino acids 30
and 31 in SEQ ID NO:2. Because of the similarity between murine and
human IL-15 receptor in this region, and because the murine IL-7
leader sequence (see below) utilizes a Thr residue as the mature
N-terminal amino acid following the leader, the Thr at residue 12
of SEQ ID NOs:6 and 7 was chosen as the mature N-terminus of a
human IL-15 receptor construct.
[0112] The mature peptide coding domain of W5 (nucleotides 34
through 753 of SEQ ID NO:6), and the remaining 3' non-coding
sequence, was fused to the coding domain for the signal peptide of
murine IL-7 in the expression vector pDC206 (similar to pDC201,
described in Sims et al., Science 241: 585, 1988, with the addition
of the murine IL-7 leader sequence, which is described in U.S. Pat.
No. 4,965,195, issued Oct. 23, 1990). Transfection of this
recombinant plasmid into COS-7 cells followed by cell-surface
binding of radiolabeled human IL-15 substantially as described in
Example 1 showed that this plasmid encoded a biologically active
polypeptide, i.e., one which bound IL-15. The clone W5 construct
containing the murine IL-7 leader sequence in pDC206 was deposited
with the American Type Culture Collection ("ATCC", 12301 Parklawn
Dr., Rockville, Md. 20852, USA), under the conditions of the
Budapest Treaty on Sep. 1, 1994, and given accession number ATCC
69690.
[0113] C. Clone P1
[0114] A .lambda.gt10 library from human peripheral blood
lymphocytes, prepared as described in U.S. Ser. No. 08/094,669,
filed Jul. 20, 1993, and in Idzerda et al., J. Exp. Med. 171:861
(1990), was screened for the presence of a full-length clone
encoding human IL-15R using a random prime labeled human IL15R cDNA
probe consisting of the entire W5 cDNA without the poly-A tail
(which had been removed by digestion of the cDNA with Ssp I
followed by gel purification of the remaining fragment, resulting
in a fragment of approximately 1465 bp), using substantially the
same conditions as described for screening of the A1172 library
(described below). The resulting sequence of the cDNA insert from
this clone (SEQ ID NOs:8 and 9) exhibited an in-frame insertion of
153 basepairs at the mature N-terminus (amino acids 24 through 74
of SEQ ID NOs:8 and 9), an in-frame deletion of 99 basepairs
downstream of the mature N-terminus that deleted nucleotides 236
through 334 of SEQ ID NO:6 (the sequence encoding amino acids 79
though 112, with the substitution of a Lys residue encoded by the
codon AAG, the equivalent of nucleotides 235, 335 and 336 of SEQ ID
NO:6), and also contained additional 5' sequence as compared to
clone W5 (amino acids 1 though 10 of SEQ ID NOs:8 and 9), but still
did not contain an initiator Met.
[0115] D. Clone A212
[0116] A library prepared from A172 cells as described in U.S. Ser.
No. 08/265,086, filed Jun. 17, 1994, was screened for the presence
of a full-length clone encoding human IL-15R. DNA (1-5 .mu.g) from
library pools (approximately 1000 cDNA clones/pool) was digested
with Sal I to release the inserted DNA, electrophoresed (1%
agarose, Tris-borate gel), and blotted to a nylon membrane. The
blot was probed with a random prime labeled human IL15R cDNA probe
consisting of the entire W5 cDNA minus the poly A tail, under
conditions of high stringency (50% formamide, 42.degree. C.
hybridization for 16-20 hr, followed by washing at 2.times.SSC at
room temperature for 5 minutes followed by 0.1.times.SSC/0.1% SDS
at 68.degree. C.). The blot was autoradiographed, and a pool with a
positive signal (i.e. hybridizing band) was chosen for isolation of
individual clones by colony hybridization.
[0117] A portion of the frozen glycerol stock of the pool of cDNA
clones corresponding to the Southern blot signal was diluted and
plated onto appropriate plates (LB+ampicillin). Colonies were
lifted onto nylon membranes and the membranes processed using
standard techniques. The membranes were hybridized and washed under
stringent conditions as described above, and a colony corresponding
to a positive hybridizing signal was grown, its plasmid DNA
purified and sequenced. The resulting sequence of the cDNA insert
from this clone (SEQ ID NOs:10 and 11) exhibited the same in-frame
deletion of 99 basepairs downstream of the mature N-terminus as
clone P1 (a deletion of nucleotides 236 through 34 of SEQ ID NO:6,
the sequence encoding amino acids 79 though 112, with the
substitution of a Lys residue encoded by the codon AAG, the
equivalent of nucleotides 235, 335 and 336 of SEQ ID NO:6). The
plasmid was transfected into COS cells, and the ability of the
encoded protein to bind IL-15 determined using slide
autoradiography with .sup.125I-labeled human IL-15 substantially as
described in Example 1. Clone A212 also encoded a biologically
active polypeptide, i.e., one which bound IL-15. Additionally,
clone A212 exhibited a complete signal peptide as compared to clone
W5, as indicated by the presence of additional 5' sequence and an
initiator Met.
[0118] E. Clone A133
[0119] A second clone was isolated from the A172 library described
above, under substantially the same conditions. The nucleotide and
amino acid sequence of the A133 clone are shown in SEQ ID NOs:12
and 13. This clone exhibited an incomplete 5' region which began at
the equivalent of nucleotide 355 of clone W5 (SEQ ID NO:6), and an
in-frame insertion downstream of the transmembrane region that
results in a different cytoplasmic tail coding domain (amino acids
97 through 117 of SEQ ID NOs:12 and 13). A hybrid construct
encoding the 5' half of W5 fused to A133 to give the alternate
cytoplasmic tail (SEQ ID NO:14) was prepared, and expressed
substantially as described above for clone W5. Cell-surface binding
experiments using radiolabeled human IL-15 substantially as
described in Example 1 showed that this hybrid construct encoded a
polypeptide that bound IL-15.
[0120] SEQ ID NO:15 presents the predicted amino acid sequence of a
composite human IL-15R containing the signal peptide of clone A212
and the coding region of clone W5. SEQ ID NO:15 also contains an
Xaa at amino acid 182, wherein Xaa is Asn or Thr. Clones W5 and P1
contain a Thr at the equivalent position (W5: amino acid 166 of SEQ
ID NOs:6 and 7; P1: amino acid 194 of SEQ ID NOs:8 and 9), whereas
clones A212 and A133 contain an Asn at the equivalent position
(A212: amino acid 149 of SEQ ID NOs:10 and 11; A133: amino acid 48
of SEQ ID NOs:13 and 14). The Asn/Thr substitution does not affect
binding of IL-15, as evidenced by the fact that both clones W5 and
A212 encoded a peptide that bound IL-15, and may be due to allelic
variation.
Example 4
Characterization of the Role of the .alpha. Subunit of the
IL-15R
[0121] A. Functional Role for the .alpha. Subunit of the IL-15R on
Murine Cells
[0122] In initial binding experiments with COS-7 cells transfected
with the murine IL-15Ra cDNA clone D5, in excess of
5.times.10.sup.5 receptors/cell were detected, a level too high to
obtain accurate measurements of IL-15 binding to these cells. More
accurate measurements of the affinity of IL-15Ra for IL-15 were
obtained using the murine IL-3 dependent 32D cell line, which
constitutively expresses the IL-2R a and Yc chains, but failed to
respond to IL-15 (Grabstein, et al., 1994, supra) as a model
system. 32D cells stably expressing various components of the IL-2
and IL-15 receptors were derived and tested for their ability to
proliferate in response to IL-15.
[0123] The original 32D cell line responded to IL-2, but a subline,
32D-01, which had lost the ability to respond to IL-2 (presumably
because it no longer expressed sufficient levels of IL-2R.beta.)
was used in these experiments. The murine IL-2R.beta. chain was
introduced into 32D-01, resulting in a line designated
32Dm.beta.-5, which had the ability to proliferate in response to
IL-2 but not IL-15. No detectable IL-15 binding to 32D-01 or
32Dm.beta.-5 was seen by cytofluorometric analysis, suggesting that
the level of IL-15Ra was very low on these cells. Direct binding
with .sup.125I-IL-15 confirmed this result (see below).
[0124] To test the role of IL-15R.alpha., 32D-01 cells were
transfected with the IL-15R.alpha. cDNA, which resulted in a line
expressing the .alpha. chain, 32Dm15R.alpha.-102. Although these
cells bound high levels of IL-15 as evidenced by both
cytofluorometric analysis and radiolabeled IL-15 binding, they were
unable to proliferate in response to IL-15. The 32Dm15R.alpha.-102
cells, like the parental 32D-01, did not express detectable levels
of IL-2R.beta.. A cell line termed 32Dm.beta.m15R.alpha.-3,
co-expressing both IL-15R.alpha. and IL-2R.beta. (.gamma..sub.c is
constitutively expressed) was derived, which was able to
proliferate in response to IL-15 and IL-2, with a pattern similar
to proliferation of the D10 cell line (from which D1-4-D5 ["D5"]
was cloned). This result demonstrates that the ability of murine
cells to respond to simian IL-15 is dependent on the level of
IL-15Ra expression and confirms the requirement for IL-2Rp.
[0125] B. IL-15R.alpha. Binds IL-15 with High Affinity
[0126] Preliminary equilibrium binding experiments with
.sup.125I-simian IL-15 indicated that the IL-15R.alpha. chain alone
was binding IL-15 with very high affinity; therefore, the optimal
binding conditions necessary to accurately measure this affinity
under equilibrium conditions, as well as to measure whether a
receptor complex containing the .beta. and .gamma..sub.c chains
along with the IL-15R.alpha. chain exhibited an enhanced affinity
for IL-15, were assessed. The parental 32D-01 cell line expressed
an average of 100.+-.33 IL-15 binding sites per cell, with an
affinity (K.sub.a) of 1.4.+-.0.4.times.10.sup.11 M.sup.-1, which is
similar to the affinity of IL-2 binding to the
IL-2R.alpha./.beta./.gamma- ..sub.c complex. The 32Dm15R.alpha.-102
cells, transfected with the IL-15R.alpha. chain, exhibited a much
higher level of IL-15 binding with the same very high affinity
(average of 15300.+-.3700 sites per cell with a K.sub.a of
1.5.+-.0.9.times.10.sup.11 M.sup.-1). Given the low expression of
IL-2R.beta. on these cells, the majority of these sites must
reflect binding to the IL-15R.alpha. chain alone. This suggests
that the low amount of IL-15 binding on the 32D-01 cells is due to
endogenous IL-15R.alpha..
[0127] The affinity of the receptors on both of these 32D lines is
very similar to the affinity of the native IL-15R on the D10 cells
from which the IL-15R.alpha. t subunit was cloned (average K.sub.a
of 1.3.+-.0.5.times.10.sup.11 M-1). Although D10 cells express
several hundred copies of IL-2R.beta., inferred from the number of
high affinity IL-2 binding sites (.about.500 sites/cell), a second
component of binding in these cells which might correspond to a
higher affinity .alpha./.beta. or .alpha./.beta./.gamma..sub.c
complex was not detected. This result was substantiated by analysis
of the 32Dm.beta.m15R.alpha.-3 cells, co-expressing both
recombinant IL-15R.alpha. and IL-2R.beta. subunits. These cells
showed binding characteristics very similar to those exhibited by
the 32Dm15R.alpha.-102 cells, with an average K.sub.a of
2.2.+-.0.3.times.10.sup.11 M.sup.-1, and 12800.+-.2700
receptors/cell.
[0128] In both D10 and 32Dm.beta.m15R.alpha.-3 cells,
overexpression of the IL-15R.alpha. relative to the .beta. subunit
might serve to obscure a small higher affinity component. This
possibility was addressed by analyzing binding to the 32Dm.beta.-5
cell line, which had been transfected with the .beta. subunit
alone. These cells showed a single high affinity binding site that
was essentially identical to the parental 32D-01 line, with an
average K.sub.a of 1.9.+-.0.5.times.10.sup.11 M.sup.-1 and 40.+-.15
sites per cell, presumably due to low level expression of
endogenous IL-15R.alpha.. The observation that the 32Dm.beta.-5
cell line did not display any additional IL-15 binding sites
relative to the 32D-01 parent line indicated that simian IL-15 is
unable to bind with any detectable affinity to complexes of murine
.beta. and .gamma..sub.c, in the absence of the IL-15R.alpha.
chain.
Sequence CWU 1
1
* * * * *