U.S. patent application number 10/313135 was filed with the patent office on 2003-06-12 for receptor for oncostatin m.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Cosman, David J., Mosley, Bruce.
Application Number | 20030109003 10/313135 |
Document ID | / |
Family ID | 26940161 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109003 |
Kind Code |
A1 |
Mosley, Bruce ; et
al. |
June 12, 2003 |
Receptor for oncostatin M
Abstract
A novel polypeptide functions as the .beta. chain of an
oncostatin M receptor and is thus designated OSM-R.beta..
Heterodimeric receptor proteins comprising OSM-R.beta. and gp130
bind oncostatin M and find use in inhibiting biological activities
mediated by oncostatin M.
Inventors: |
Mosley, Bruce; (Seattle,
WA) ; Cosman, David J.; (Bainbridge Island,
WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
LAW DEPARTMENT
51 UNIVERSITY STREET
SEATTLE
WA
98101
|
Assignee: |
Immunex Corporation
|
Family ID: |
26940161 |
Appl. No.: |
10/313135 |
Filed: |
December 6, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10313135 |
Dec 6, 2002 |
|
|
|
09455962 |
Dec 6, 1999 |
|
|
|
6524817 |
|
|
|
|
09455962 |
Dec 6, 1999 |
|
|
|
09058264 |
Apr 10, 1998 |
|
|
|
6010886 |
|
|
|
|
09058264 |
Apr 10, 1998 |
|
|
|
08308881 |
Sep 12, 1994 |
|
|
|
5783672 |
|
|
|
|
08308881 |
Sep 12, 1994 |
|
|
|
08249553 |
May 26, 1994 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 14/70503 20130101;
C07K 2319/00 20130101; C07K 16/248 20130101; G01N 33/6863 20130101;
C07K 2319/30 20130101; C07K 16/2866 20130101; C07K 14/7155
20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5; 530/388.22 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/72; C07K 016/28; C07H 021/04 |
Claims
What is claimed is:
1. A receptor capable of binding oncostatin M, comprising a gp130
polypeptide covalently linked to an oncostatin M receptor B-chain
(OSM-RB) polypeptide, wherein said gp130 polypeptide is a
biologically active polypeptide encoded by a DNA selected from the
group consisting of: a) DNA comprising the coding region of the
nucleotide sequence presented in SEQ ID NO:1; b) DNA capable of
hybridizing under highly stringent conditions to the DNA of (a);
and c) DNA that encodes the amino acid sequence presented in SEQ ID
NO:2; and wherein said OSM-R.beta. polypeptide is a biologically
active polypeptide encoded by a DNA selected from the group
consisting of: a') DNA comprising the coding region of the
nucleotide sequence presented in SEQ ID NO:5; b') DNA capable of
hybridizing under highly stringent conditions to the DNA of (a');
and c') DNA that encodes the amino acid sequence presented in SEQ
ID NO:6.
2. A receptor according to claim 1, wherein said receptor comprises
a soluble gp130 polypeptide covalently linked to a soluble
OSM-R.beta. polypeptide.
3. A receptor according to claim 1 wherein said receptor comprises
gp130 covalently linked to OSM-R.beta. via a peptide linker.
4. A receptor according to claim 3, wherein said receptor is a
recombinant fusion protein of the formula: R.sub.1-L-R.sub.2 or
R.sub.2-L-R.sub.1 wherein R.sub.1 represents a soluble gp130;
R.sub.2 represents a soluble OSM-R.beta., and L represents a
peptide linker.
5. A receptor according to claim 4, wherein said soluble gp130
comprises amino acids -22 to y or 1 to y of SEQ ID NO:2, wherein y
represents an integer between 308 and 597, inclusive; and said
soluble OSM-R.beta. comprises amino acids -27 to x or 1 to x of SEQ
ID NO:6, wherein x is an integer between 432 and 714,
inclusive.
6. An isolated DNA sequence encoding the receptor of claim 4.
7. A recombinant expression vector comprising the DNA sequence of
claim 6.
8. A process for preparing a receptor according to claim 4,
comprising culturing a host cell transformed with an expression
vector comprising a DNA sequence that encodes said fusion protein
under conditions that promote expression of said fusion protein,
and recovering said fusion protein.
9. A receptor according to claim 2 comprising a first fusion
polypeptide that comprises an antibody Fc region polypeptide
attached to the C-terminus of a soluble gp130, and a second fusion
polypeptide that comprises an antibody Fc region polypeptide
attached to the C-terminus of a soluble OSM-R.beta., wherein said
first fusion polypeptide is linked to said second fusion
polypeptide via disulfide bonds between the Fc region
polypeptides.
10. A process for preparing a receptor according to claim 9,
comprising culturing a host cell co-transfected with a first
expression vector encoding said first fusion polypeptide and with a
second expression vector encoding said second fusion polypeptide
under conditions that promote expression of said first and second
fusion polypeptides, and recovering said receptor.
11. A receptor according to claim 9, wherein said soluble gp130
comprises amino acids -22 to y or 1 to y of SEQ ID NO:2, wherein y
represents an integer between 308 and 597, inclusive; and said
soluble OSM-R.beta. comprises amino acids -27 to x or 1 to x of SEQ
ID NO:6, wherein x is an integer between 432 and 714,
inclusive.
12. A composition comprising a receptor of claim 2 and a suitable
diluent or carrier.
13. An isolated DNA encoding an OSM-R.beta. polypeptide, wherein
said DNA is selected from the group consisting of: a) DNA
comprising the coding region of the nucleotide sequence presented
in SEQ ID NO:5; b) DNA capable of hybridizing under highly
stringent conditions to the DNA of (a), and encoding a biologically
active OSM-R.beta. polypeptide; and c) DNA that encodes the amino
acid sequence presented in SEQ ID NO:6.
14. An isolated DNA according to claim 13, wherein said OSM-R.beta.
comprises an amino acid sequence selected from the group consisting
of amino acids -27 to 952 and amino acids 1 to 952 of SEQ ID
NO:6.
15. An isolated DNA according to claim 13, wherein said DNA encodes
a soluble OSM-R.beta. polypeptide.
16. A DNA according to claim 15, where said soluble OSM-R.beta.
polypeptide comprises amino acids -27 to x or 1 to x of SEQ ID
NO:6, wherein x is an integer between 432 and 714, inclusive.
17. An isolated DNA encoding an OSM-R.beta. polypeptide, wherein
said OSM-R.beta. polypeptide is characterized by the N-terminal
amino acid sequence Glu Arg Leu Pro Leu Thr Pro Val Ser Leu Lys
Val.
18. An expression vector comprising a DNA according to claim
13.
19. An expression vector comprising a DNA according to claim
15.
20. A process for preparing an OSM-R.beta. polypeptide, comprising
culturing a host cell transformed with a vector according to claim
18 under conditions promoting expression of OSM-R.beta. and
recovering the OSM-R.beta. polypeptide from the culture.
21. A process for preparing an OSM-R.beta. polypeptide, comprising
culturing a host cell transformed with a vector according to claim
19 under conditions promoting expression of OSM-R.beta. and
recovering the OSM-R]3 polypeptide from the culture.
22. A purified OSM-R.beta. polypeptide encoded by a DNA according
to claim 13.
23. An OSM-R.beta. polypeptide according to claim 22, wherein said
OSM-R.beta. is a soluble OSM-R.beta. polypeptide.
24. A soluble OSM-R.beta. according to claim 23, comprising amino
acids -27 to x or 1 to x of SEQ ID NO:6, wherein x is an integer
between 432 and 714, inclusive.
25. A purified OSM-R.beta. polypeptide, wherein said OSM-R.beta.
polypeptide is characterized by the N-terminal amino acid sequence
Glu Arg Leu Pro Leu Thr Pro Val Ser Leu Lys Val.
26. An OSM-R.beta. polypeptide according to claim 25, comprising
amino acids 1 to 952 of SEQ ID NO:6.
27. An OSM-R.beta. polypeptide according to claim 22, wherein said
OSM-R.beta. is encoded by the OSM-R.beta. cDNA in the recombinant
vector deposited in strain ATCC 69675.
28. A soluble OSM-R.beta. polypeptide according to claim 15,
wherein said polypeptide additionally comprises an Fc polypeptide
fused to the C-terminus of said OSM-R.beta. polypeptide.
29. An antibody that is immunoreactive with an OSM-R.beta.
polypeptide according to claim 13.
30. An antibody according to claim 29, wherein said antibody is a
monoclonal antibody.
31. An isolated nucleic acid molecule comprising a sequence of at
least about 14 nucleotides of the coding region of the DNA sequence
presented in SEQ ID NO:5, or its DNA or RNA complement.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
08/308,881, filed Sep. 12, 1994, now allowed, which is a
continuation-in-part of application Ser. No. 08/249,553, filed May
26, 1994, currently pending.
BACKGROUND OF THE INVENTION
[0002] Oncostatin M is a secreted single-chain polypeptide cytokine
that regulates the growth of certain tumor-derived and normal cell
lines. A number of cell types have been found to bind the
oncostatin M protein. See, for example, Linsley et al., J. Biol.
Chem., 264: 4282 (1989). Oncostatin M has been shown to inhibit
proliferation of a number of tumor cell types (Linsley et al.
supra). In contrast, however, this protein has been implicated in
stimulating proliferation of Kaposi's sarcoma cells (Nair et al.,
Science 255:1430, 1992; Miles et al., Science 255:1432, 1992; and
Cai et al., Am. J. Pathol. 145:74, 1994).
[0003] Identifying and isolating oncostatin M-binding proteins,
such as cell surface oncostatin M receptors, is desirable for such
reasons as enabling study of the biological signal transduced via
the receptor. Such receptors in soluble form also could be used to
competitively inhibit a biological activity of oncostatin M in
various in vitro assays or in vivo procedures. A soluble form of
the receptor could be administered to bind oncostatin M in vivo,
thus inhibiting the binding of oncostatin M to endogenous cell
surface receptors, for example.
[0004] A protein known as gp130 has been found to bind oncostatin
M, but with relatively low affinity (Gearing et al., Science
255:1434, 1992). Heterodimeric receptors comprising a leukemia
inhibitory factor (LIF) receptor and gp130 bind oncostatin M with
higher affinity than does gp130 alone, but also bind LIF with high
affinity (Gearing et al., supra). For certain applications, a
receptor that binds oncostatin M with high affinity, but that does
not function as a high affinity LIF receptor, would be
advantageous. Prior to the present invention, no such receptor had
been identified or isolated.
SUMMARY OF THE INVENTION
[0005] The present invention provides a novel polypeptide that is
designated herein as the oncostatin M receptor B subunit (OSM-RB).
Also provided is a receptor comprising OSM-R.beta. linked
(preferably covalently) to an oncostatin M-binding protein known as
gp130. The gp130 polypeptide may be covalently linked to the
OSM-R.beta. polypeptide by any suitable means, such as via a
cross-linking reagent or a polypeptide linker. In one embodiment of
the invention, the receptor is a fusion protein produced by
recombinant DNA technology. This receptor comprising OSM-R.beta.
and gp130 binds oncostatin M at levels greater than does gp130
alone. Disorders mediated by oncostatin M may be treated by
administering a therapeutically effective amount of this inventive
receptor to a patient afflicted with such a disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 presents a Scatchard analysis generated from an assay
for binding of radioiodinated oncostatin M by cells expressing
recombinant gp130. The assay is described in example 2.
[0007] FIG. 2 presents a Scatchard analysis of the results of an
assay for binding of radioiodinated oncostatin M by cells
expressing both recombinant gp130 and recombinant OSM-R.beta.. As
described in example 2, the data in FIG. 2 demonstrate higher
affinity oncostatin M binding compared to the oncostatin M binding
by gp130 alone depicted in FIG. 1.
[0008] FIG. 3 is a bar graph representing binding of leukemia
inhibitory factor (LIF) and oncostatin M to various receptor
proteins, as described in example 5.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention provides a novel polypeptide
designated the oncostatin M receptor B subunit (OSM-R.beta.).
Isolated DNA encoding OSM-R.beta., expression vectors containing
OSM-R.beta. DNA, and host cells transformed with such expression
vectors are disclosed. Methods for production of recombinant
OSM-R.beta. polypeptides, including soluble forms of the protein,
are also disclosed. Antibodies immunoreactive with the novel
polypeptide are provided herein as well.
[0010] Another embodiment of the invention is directed to a
receptor capable of binding oncostatin M, wherein the receptor
comprises OSM-R.beta. and gp130. The receptor finds use in various
in vitro and in vivo procedures, including treatment of disorders
mediated by oncostatin M.
[0011] DNA and encoded amino acid sequences of the OSM-R.beta. cDNA
isolated in example 1 are presented in SEQ ID NO:5 and SEQ ID NO:6.
The encoded protein comprises (from N- to C-terminus) a signal
peptide (amino acids -27 to -1 of SEQ ID NO:6) followed by an
extracellular domain (amino acids 1 to 714), a transmembrane region
(amino acids 715 to 734) and a cytoplasmic domain (amino acids 735
to 952). E. coli cells transformed with a recombinant vector
comprising OSM-R.beta. cDNA in the cloning vector pBluescript.RTM.
SK.sup.- were deposited with the American Type Culture Collection,
Rockville, Md., U.S.A., on Aug. 16, 1994, and assigned accession
no. ATCC 69675.
[0012] The binding assay described in example 2 compared the
binding of oncostatin M by cells expressing either gp130 alone or
both gp130 and OSM-R.beta.. The cells expressing both gp130 and
OSM-R.beta. exhibited higher affinity oncostatin M binding than did
cells expressing gp130 alone. The assay described in example 5
demonstrates that OSM-R.beta. alone does not bind oncostatin M at a
detectable level. However, proteins expressed by cells
co-transfected with both a soluble OSM-R.beta./Fc fusion
protein-encoding vector and a soluble gp130/Fc fusion
protein-encoding vector bound oncostatin M at higher levels than
did proteins expressed by cells transfected with a soluble
gp130/Fc-encoding vector alone.
[0013] In one embodiment, a receptor of the present invention
comprises gp130 covalently linked to OSM-R.beta. by any suitable
means, such as via a cross-linking reagent or a polypeptide linker.
The gp130 and OSM-R.beta. proteins are covalently linked in a
manner that does not interfere with the resulting receptor's
ability to bind oncostatin M. In one embodiment, the receptor is a
fusion protein produced by recombinant DNA technology.
[0014] Alternatively, the receptor may comprise gp130
non-covalently complexed with OSM-R.beta.. Non-covalent bonding of
gp130 to OSM-R.beta. may be achieved by any suitable means that
does not interfere with the receptor's ability to bind oncostatin
M. In one approach, a first compound is attached to OSM-R.beta. and
a second compound that will non-covalently bond to the first
compound is attached to gp130. Examples of such compounds are
biotin and avidin. The receptor is thus formed through the
non-covalent interactions of biotin with avidin. In one embodiment
of the invention, OSM-R.beta. and gp130 are recombinant
polypeptides, each purified from recombinant cells and then
non-covalently bonded together to form the receptor. A host cell
may be transformed with two different expression vectors such that
both OSM-R.beta. and gp130 are produced by the recombinant host
cell. OSM-R.beta. and gp130 produced by such transformed host cells
may associate to form a complex through non-covalent interactions.
When such transformed cells express the membrane-bound forms of the
proteins, such cells are useful in various assays, including
competition assays.
[0015] The protein designated gp130 herein has been purified from
cellular sources that include placental tissue and a myeloma cell
line U266. A number of additional cell types have been found to
express gp130 mRNA, as reported by Hibi et al., in Cell 63:1149
(1990). gp130 has been reported to be involved in the formation of
high affinity interleukin-6 binding sites and in IL-6 signal
transduction (Hibi et al. supra). gp130 also serves as an affinity
converter for the LIF receptor (Gearing et al., Science 255:1434,
1992). The cloning and expression of cDNA encoding a full length
gp130 protein has been reported by Hibi et al., supra, which is
hereby incorporated by reference in its entirety.
[0016] As used herein, the terms OSM-R.beta. and gp130 include
variants and truncated forms of the native proteins that possess
the desired biological activity. Variants produced by adding,
substituting, or deleting amino acid(s) in the native sequence are
discussed in more detail below.
[0017] One example of an OSM-R.beta. polypeptide is that encoded by
the cDNA clone described in example 1 (i.e., encoded by the
OSM-R.beta. cDNA insert of the recombinant vector in deposited
strain ATCC 69675). Other OSM-R.beta. polypeptides include those
lacking all or part of the transmembrane region or the cytoplasmic
domain of the protein. Additional truncated OSM-R.beta.
polypeptides may be chosen with regard to sequences that are
conserved in the hematopoietin receptor family. The desirability of
including the signal sequence depends on such factors as the
position of the OSM-R.beta. in a fusion protein and the intended
host cells when the receptor is to be produced via recombinant DNA
technology.
[0018] One example of a suitable gp130 polypeptide is that
comprising the amino acid sequence presented in SEQ ID NO:2. E.
coli strain DH5.alpha. cells transformed with a gp130-encoding
recombinant vector designated B10G/pDC303 were deposited with the
American Type Culture Collection, Rockville, Md., on Nov. 14, 1991,
and assigned ATCC accession number 68827. The mammalian expression
vector pDC303 (into which the gp130 cDNA has been inserted to form
B10G/pDC303) is also known as SF CAV, and has been described in PCT
application WO 93/19777. The nucleotide sequence of the gp130 cDNA
contained in plasmid B10G/pDC303 and the amino acid sequence
encoded thereby are presented in SEQ ID NO: 1 and SEQ ID NO:2. The
protein comprises (in order from the N-terminus to the C-terminus)
a 22-amino acid signal sequence, complete extracellular domain
(amino acids 1-597), a transmembrane region (beginning with amino
acid 598), and a partial cytoplasmic domain (amino acids
621-686).
[0019] Alternatively, the gp130 protein disclosed by Hibi et al.
supra may be employed. The eighth amino acid of the signal peptide
is valine in the sequence reported by Hibi et al., but is leucine
in SEQ ID NO:2 (at position -15). This difference in amino acid
sequence may be attributable to genetic polymorphism (allelic
variation among individuals producing the protein). In addition,
the gp130 protein of SEQ ID NO:2 is truncated within the
cytoplasmic domain. terminating with the leucine residue found at
position 708 in the sequence presented in Hibi et al. supra.
Although truncated, the gp130 protein of SEQ ID NO:2 comprises the
extracellular domain responsible for oncostatin M binding, and thus
is suitable for use as a component of the receptors of the present
invention.
[0020] Regions of the gp130 protein corresponding to domains that
are conserved among certain receptors are discussed by Hibi et al,
supra, at page 1150, column 2, and page 1151, column 1. Other
truncated gp130 polypeptides chosen to include these conserved
regions may be employed.
[0021] Soluble OSM-R.beta. and gp130 polypeptides are preferred for
certain applications. In one embodiment of the present invention,
the receptor comprises soluble OSM-R.beta. covalently attached to
soluble gp130. "Soluble OSM-RB" as used in the context of the
present invention refers to polypeptides that are substantially
similar in amino acid sequence to all or part of the extracellular
region of a native OSM-R.beta. and that, due to the lack of a
transmembrane region that would cause retention of the polypeptide
on a cell membrane, are secreted upon expression. Suitable soluble
OSM-R.beta. polypeptides retain the desired biological activity.
Soluble OSM-R.beta. may also include part of the transmembrane
region or part of the cytoplasmic domain or other sequences,
provided that the soluble OSM-R.beta. protein is capable of being
secreted.
[0022] Likewise, the term "soluble gp130" as used herein refers to
proteins that are substantially similar in amino acid sequence to
all or part of the extracellular region of a native gp130 and are
secreted upon expression but retain the desired biological
activity. Soluble gp130 may include part of the transmembrane
region, cytoplasmic domain, or other sequences, as long as the
polypeptide is secreted.
[0023] In one embodiment, soluble OSM-R.beta. and gp130
polypeptides include the entire extracellular domain. To effect
secretion, the soluble polypeptides comprise the native signal
peptide or a heterologous signal peptide. Thus, examples of soluble
OSM-R.beta. polypeptides comprise amino acids -27 to 714 or 1 to
714 of SEQ ID NO:6. Examples of soluble gp130 polypeptides comprise
amino acids -22 to 597 or 1 to 597 of SEQ ID NO:2.
[0024] Additional examples of soluble gp130 polypeptides are those
lacking from one to all three of the fibronectin domains found
within the extracellular domain, as described in example 4 below.
These soluble gp130 polypeptides include those comprising amino
acids -22 to y or 1 to y of SEQ ID NO:2, wherein y is an integer
between 308 and 597, inclusive.
[0025] A soluble fusion protein comprising amino acids -97 through
432 of the OSM-R.beta. of SEQ ID NO:6 fused to an antibody Fc
region polypeptide is described in example 5. The OSM-R.beta.
moiety of the fusion protein, which is a fragment of the
OSM-R.beta. extracellular domain, retained the desired biological
activity. Thus, examples of soluble OSM-R.beta. polypeptides
comprise amino acids -27 to x, or 1 to x of SEQ ID NO:6, wherein x
is an integer between 432 and 714, inclusive.
[0026] Soluble OSM-R.beta. and soluble gp130 may be identified (and
distinguished from their non-soluble membrane-bound counterparts)
by separating intact cells which express the desired protein from
the culture medium, e.g., by centrifugation, and assaying the
medium (supernatant) for the presence of the desired protein. The
culture medium may be assayed using procedures which are similar or
identical to those described in the examples below. The presence of
OSM-R.beta. or gp130 in the medium indicates that the protein was
secreted from the cells and thus is a soluble form of the desired
protein. Soluble OSM-R.beta. and soluble gp130 may be
naturally-occurring forms of these proteins. Alternatively, soluble
fragments of OSM-R.beta. and gp130 proteins may be produced by
recombinant DNA technology or otherwise isolated, as described
below.
[0027] The use of soluble forms of OSM-R.beta. and gp130 is
advantageous for certain applications. Purification of the proteins
from recombinant host cells is facilitated, since the soluble
proteins are secreted from the cells. Further, a receptor of the
present invention comprising soluble OSM-R.beta. and gp130 proteins
is generally more suitable for intravenous administration.
[0028] With respect to the foregoing discussion of signal peptides
and the various domains of the gp130 and OSM-R.beta. proteins, the
skilled artisan will recognize that the above-described boundaries
of such regions of the proteins are approximate. For example,
although computer programs that predict the site of cleavage of a
signal peptide are available, cleavage can occur at sites other
than those predicted. Further, it is recognized that a protein
preparation can comprise a mixture of protein molecules having,
different N-terminal amino acids, due to cleavage of the signal
peptide at more than one site. In addition, the OSM-R.beta.
transmembrane region was identified by computer program prediction
in combination with homology to the transmembrane region of the LIF
receptor protein described by Gearing et al. (EMBO J. 10:2839,
1991). Thus, soluble OSM-R.beta. polypeptides comprising the
extracellular domain include those having a C-terminal amino acid
that may vary from that identified above as the C-terminus of the
extracellular domain. Further, post-translational processing that
can vary according to the particular expression system employed may
yield proteins having differing N-termini. Such variants that
retain the desired biological activities are encompassed by the
terms "OSM-R.beta. polypeptides" and "gp130 polypeptides" as used
herein.
[0029] Truncated OSM-R.beta. and gp130, including soluble
polypeptides, may be prepared by any of a number of conventional
techniques. In the case of recombinant proteins, a DNA fragment
encoding a desired fragment may be subcloned into an expression
vector. Alternatively, a desired DNA sequence may be chemically
synthesized using known techniques. DNA fragments also may be
produced by restriction endonuclease digestion of a full length
cloned DNA sequence, and isolated by electrophoresis on agarose
gels. Linkers containing restriction endonuclease cleavage site(s)
may be employed to insert the desired DNA fragment into an
expression vector, or the fragment may be digested at cleavage
sites naturally present therein. Oligonucleotides that reconstruct
the N- or C-terminus of a DNA fragment to a desired point may be
synthesized. The oligonucleotide may contain a restriction
endonuclease cleavage site upstream of the desired coding sequence
and position an initiation codon (ATG) at the N-terminus of the
coding sequence.
[0030] The well known polymerase chain reaction procedure also may
be employed to isolate a DNA sequence encoding a desired protein
fragment. Oligonucleotide primers comprising the desired termini of
the fragment are employed in such a polymerase chain reaction. Any
suitable PCR procedure may be employed. One such procedure is
described in Saiki et al., Science 239:487 (1988). Another is
described in Recombinant DNA Methodology, Wu et al., eds., Academic
Press Inc., San Diego (1989), pp. 189-196. In general, PCR
reactions involve combining the 5' and 3' oligonucleotide primers
with template DNA (in this case, OSM-R.beta. or gp130 DNA) and each
of the four deoxynucleoside triphosphates, in a suitable buffered
solution. The solution is heated, (e.g, from 95_ to 100_ C) to
denature the double-stranded DNA template and is then cooled before
addition of a DNA polymerase enzyme. Multiple cycles of the
reactions are carried out in order to amplify the desired DNA
fragment.
[0031] The gp130 polypeptide is attached to the OSM-R.beta.
polypeptide through a covalent or non-covalent linkage. Covalent
attachment is preferred for certain applications, e.g. in vivo use,
in view of the enhanced stability generally conferred by covalent,
as opposed to non-covalent, bonds. In constructing the receptor of
the present invention, covalent linkage may be accomplished via
cross-linking reagents, peptide linkers, or any other suitable
technique.
[0032] Numerous reagents useful for cross-linking one protein
molecule to another are known. Heterobifunctional and
homobifunctional linkers are available for this purpose from Pierce
Chemical Company, Rockford, Ill., for example. Such linkers contain
two functional groups (e.g., esters and/or maleimides) that will
react with certain functional groups on amino acid side chains,
thus linking one polypeptide to another.
[0033] One type of peptide linker that may be employed in the
present invention separates gp130 and OSM-R.beta. domains by a
distance sufficient to ensure that each domain properly folds into
the secondary and tertiary structures necessary for the desired
biological activity. The linker also should allow the extracellular
domains of gp130 and OSM-R.beta. to assume the proper spatial
orientation to form the binding site for oncostatin M.
[0034] Suitable peptide linkers are known in the art, and may be
employed according to conventional techniques. Among the suitable
peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and
4,935,233, which are hereby incorporated by reference. A peptide
linker may be attached to gp130 and to OSM-R.beta. by any of the
conventional procedures used to attach one polypeptide to another.
The cross-linking reagents available from Pierce Chemical Company
as described above are among those that may be employed. Amino
acids having side chains reactive with such reagents may be
included in the peptide linker, e.g., at the termini thereof.
Preferably, a fusion protein comprising gp130 joined to OSM-R.beta.
via a peptide linker is prepared by recombinant DNA technology.
[0035] In one embodiment of the invention, OSM-R.beta. and gp130
are linked via polypeptides derived from immunoglobulins.
Preparation of fusion proteins comprising heterologous polypeptides
fused to various portions of antibody-derived polypeptides
(including the Fc domain) has been described, e.g., by Ashkenazi et
al. (PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677,
1990). As one example, a polypeptide derived from the Fc region of
an antibody may be attached to the C-terminus of OSM-R.beta.. A
separate Fc polypeptide is attached to the C-terminus of gp130.
Disulfide bonds form between the two Fc polypeptides (e.g., in the
so-called hinge region, where interchain disulfide bonds are
normally present in antibody molecules), producing a heterodimer
comprising the gp130 and to OSM-R.beta./Fc fusion protein linked to
the gp130/Fc fusion protein. Advantageously, host cells are
co-transfected with two different expression vectors, one encoding
soluble OSM-R.beta./Fc and the other encoding soluble gp130/Fc. The
heterodimer is believed to form intracellularly or during
secretion.
[0036] The term "Fc polypeptide" as used herein includes native and
mutein forms, as well as truncated Fc polypeptides containing the
hinge region that promotes dimerization. cDNA encoding a single
chain polypeptide derived from the Fc region of a human IgG1
antibody has been cloned into the pBluescript SK.RTM. cloning
vector (Stratagene Cloning, Systems, La Jolla, Calif.) to produce a
recombinant vector designated hIgG1Fc. A unique BglII site is
positioned near the 5' end of the inserted Fc encoding sequence. An
SpeI site is immediately downstream of the stop codon. The DNA and
encoded amino acid sequences of the cloned Fc cDNA are presented in
SEQ ID NO:3 and SEQ ID NO:4. The Fc polypeptide encoded by the cDNA
extends from the N-terminal hinge region to the native C-terminus,
i.e., is an essentially full-length antibody Fc region. One
suitable mutein of this Fc polypeptide is described in U.S. patent
application Ser. No. 08/097,827, hereby incorporated by reference.
The mutein exhibits reduced affinity for Fc receptors.
[0037] Homodimers comprising two OSM-RB.beta.Fc polypeptides or two
gp130/Fc polypeptides linked via disulfide bonds are also produced
by certain of the transfected host cells disclosed herein. The
homodimers may be separated from each other and from the
heterodimer by virtue of differences in size (e.g., by gel
electrophoresis). The heterodimer also may be purified by
sequential immunoaffinity chromatography (described below).
[0038] In an alternative embodiment, a first fusion polypeptide
comprising gp130 (or a fragment thereof) upstream of the constant
region of an antibody light chain (or a fragment thereof) is
prepared. A second fusion polypeptide comprises OSM-R.beta.
upstream of the constant region of an antibody heavy chain (or a
heavy chain fragment, the N-terminus of which extends at least
through the CHI region. Disulfide bond(s) form between the
gp130-light chain fusion polypeptide and the OSM-R.beta.-heavy
chain fusion polypeptide, thus producing a receptor of the present
invention. As a further alternative, an OSM-R.beta.-antibody light
chain fusion polypeptide is prepared and combined with (disulfide
bonded to) a fusion polypeptide comprising gp130 fused to an
antibody heavy chain. When two of the foregoing disulfide bonded
molecules are combined, additional disulfide bonds form between the
two Fc regions. The resulting receptor of the present invention
comprising four fusion polypeptides resembles an antibody in
structure and displays the oncostatin M binding site
bivalently.
[0039] The gp130 and OSM-R.beta. polypeptides may be separately
purified from cellular sources, and then linked together.
Alternatively, the receptor of the present invention may be
produced using recombinant DNA technology. The gp130 and
OSM-R.beta. polypeptides may be produced separately and purified
from transformed host cells for subsequent covalent linkage. In one
embodiment of the present invention, a host cell is
transformed/transfected with foreign DNA that encodes gp130 and
OSM-R.beta. as separate polypeptides. The two polypeptides may be
encoded by the same expression vector with start and stop codons
for each of the two genes, or the recombinant cells may be
co-transfected with two separate expression vectors. In another
embodiment, the receptor is produced as a fusion protein in
recombinant cells.
[0040] In one embodiment of the present invention, the receptor
protein is a recombinant fusion protein of the formula:
R.sub.1-L-R.sub.2 or R.sub.2-L-R.sub.1
[0041] wherein R.sub.1 represents gp130 or a gp130 fragment;
R.sub.2 represents OSM-R.beta. or an OSM-R.beta. fragment; and L
represents a peptide linker.
[0042] The fusion proteins of the present invention include
constructs in which the C-terminal portion of gp130 is fused to the
linker which is fused to the N-terminal portion of OSM-R.beta., and
also constructs in which the C-terminal portion of OSM-R.beta. is
fused to the linker which is fused to the N-terminal portion of
gp130. gp130 is covalently linked to OSM-R.beta. in such a manner
as to produce a single protein which retains the desired biological
activities of gp130 and OSM-R.beta.. The components of the fusion
protein are listed in their order of occurrence (i.e., the
N-terminal polypeptide is listed first, followed by the linker and
then the C-terminal polypeptide).
[0043] A DNA sequence encoding a fusion protein is constructed
using recombinant DNA techniques to insert separate DNA fragments
encoding gp130 and OSM-R.beta. into an appropriate expression
vector. The 3' end of a DNA fragment encoding gp130 is ligated (via
the linker) to the 5' end of the DNA fragment encoding OSM-R.beta.
with the reading frames of the sequences in phase to permit
translation of the mRNA into a single biologically active fusion
protein. Alternatively, the 3' end of a DNA fragment encoding
OSM-R.beta. may be ligated (via the linker) to the 5' end of the
DNA fragment encoding gp130, with the reading frames of the
sequences in phase to permit translation of the mRNA into a single
biologically active fusion protein. A DNA sequence encoding an
N-terminal signal sequence may be retained on the DNA sequence
encoding the N-terminal polypeptide, while stop codons, which would
prevent read-through to the second (C-terminal) DNA sequence, are
eliminated. Conversely, a stop codon required to end translation is
retained on the second DNA sequence. DNA encoding a signal sequence
is preferably removed from the DNA sequence encoding the C-terminal
polypeptide.
[0044] A DNA sequence encoding a desired polypeptide linker may be
inserted between, and in the same reading frame as, the DNA
sequences encoding gp130 and OSM-R.beta. using any suitable
conventional technique. For example, a chemically synthesized
oligonucleotide encoding the linker and containing appropriate
restriction endonuclease cleavage sites may be ligated between the
sequences encoding gp130 and OSM-R.beta..
[0045] Alternatively, a chemically synthesized DNA sequence may
contain a sequence complementary to the 3' terminus (without the
stop codon) of either gp130 or OSM-R.beta., followed by a
linker-encoding sequence which is followed by a sequence
complementary to the 5' terminus of the other of gp130 and
OSM-R.beta.. Oligonucleotide directed mutagenesis is then employed
to insert the linker-encoding sequence into a vector containing a
direct fusion of gp130 and OSM-R.beta..
[0046] The present invention provides isolated DNA sequences
encoding the above-described fusion proteins comprising gp130,
OSM-R.beta., and a peptide linker. DNA encoding the novel
OSM-R.beta. polypeptides disclosed herein is also provided, as is
DNA encoding OSM-R.beta. polypeptides fused to immunoglobin-derived
polypeptides. OSM-R.beta.-encoding DNA encompassed by the present
invention includes, for example, cDNA, chemically synthesized DNA,
DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic
OSM-R.beta. DNA may be isolated using the cDNA isolated in Example
1, or fragments thereof, as a probe using standard techniques.
[0047] Also provided herein are recombinant expression vectors
containing the isolated DNA sequences. "Expression vector" refers
to a replicable DNA construct used to express DNA which encodes the
desired protein and which includes a transcriptional unit
comprising an assembly of (1) genetic element(s) having a
regulatory role in gene expression, for example, promoters,
operators, or enhancers, operatively linked to (2) a DNA sequence
encoding a desired protein which is transcribed into mRNA and
translated into protein, and (3) appropriate transcription and
translation initiation and termination sequences. The choice of
promoter and other regulatory elements generally varies according
to the intended host cell.
[0048] In the expression vectors, regulatory elements controlling
transcription or translation are generally derived from mammalian,
microbial, viral or insect genes. 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. Vectors derived from retroviruses
also may be employed.
[0049] DNA regions are operably linked when they are functionally
related to each other. For example, DNA encoding a signal peptide
(secretory leader) is operably linked to DNA for a polypeptide if
the polypeptide is expressed as a precursor that is secreted
through the host cell membrane; a promoter is operably linked to a
coding sequence if it controls the transcription of the sequence;
and 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, continuous and in reading frame.
[0050] Transformed host cells are cells which have been transformed
or transfected with foreign DNA using recombinant DNA techniques.
In the context of the present invention, the foreign DNA includes a
sequence encoding the inventive proteins. Host cells may be
transformed for purposes of cloning or amplifying the foreign DNA,
or may be transformed with an expression vector for production of
the protein. Suitable host cells include prokaryotes, yeast or
higher eukaryotic cells. 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, N.Y., 1985), the relevant disclosure of which is
hereby incorporated by reference.
[0051] Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. 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. Examples of suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium, and various species within the genera Pseudomonas,
Streptomyces, and Staphlylococcus, although others may also be
employed as a matter of choice.
[0052] 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 (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 this provides simple means for identifying transformed
cells.
[0053] Promoters commonly used in recombinant microbial expression
vectors include the .beta.-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 thermoinducible repressor. Plasmid vectors
available from the American Type Culture Collection which
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).
[0054] The recombinant receptor protein may also be expressed in
yeast hosts, preferably from 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 21 .mu.m yeast plasmid or an
autonomously replicating sequence (ARS), a promoter, DNA encoding
the receptor fusion protein, sequences for polyadenylation and
transcription termination and a selection gene. Preferably, yeast
vectors will include an origin of replication and selectable
markers permitting transformation of both yeast and E. coli, e.g.,
the ampicillin resistance gene of E. coli and the S. cerevisiae
trp1 gene, which 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
lesion in the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan.
[0055] 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), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase and glucokinase. Suitable
vectors and promoters for use in yeast expression are further
described in R. Hitzeman et al., EPA 73,657.
[0056] Preferred yeast vectors can be assembled using DNA sequences
from pBR322 for selection and replication in E. coli (Ampr 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, which 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
30:922, 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.
[0057] Suitable yeast transformation protocols are known to those
of skill in the art. An exemplary technique is described by Hinnen
et al., Proc. Natl Acad. Sci. USA 75:1929, (1978), selecting for
Trp+ transformants in a selective medium consisting of 0.67% yeast
nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml adenine
and 20 .mu.g/ml uracil.
[0058] 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% 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.
[0059] Various mammalian or insect cell culture systems can be
employed to express recombinant protein. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, Bio/Technology 6:47 (1988). Examples of
suitable mammalian host cell lines include L cells, C127, 3T3,
Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Additional
suitable mammalian host cells include CV-1 cells (ATCC CCL70) and
COS-7 cells (ATCC CRL 1651; described by Gluzman, Cell 23:175,
1981), both derived from monkey kidney. Another monkey kidney cell
line, CV-1/EBNA (ATCC CRL 10478), was derived by transfection of
the CV-1 cell line with a gene encoding Epstein-Barr virus nuclear
antigen-1 (EBNA-1) and with a vector containing CMV regulatory
sequences (McMahan et al., EMBO J. 10:2821, 1991). The EBNA-1 gene
allows for episomal replication of expression vectors, such as
HAV-EO or pDC406, that contain the EBV origin of replication.
[0060] Mammalian expression vectors may comprise non-transcribed
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
poly-adenylation site, splice donor and acceptor sites, and
transcriptional termination sequences. 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 which
also contains the SV40 viral origin or 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.
[0061] Exemplary vectors can be constructed as disclosed by Okayama
and Berg (Mol. Cell. Biol. 3:280, 1983). One 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). Vectors
derived from retroviruses also may be employed.
[0062] When secretion of the OSM-R.beta. protein from the host cell
is desired, the expression vector may comprise DNA encoding a
signal or leader peptide. In place of the native signal sequence, a
heterologous signal sequence may be added, such as the signal
sequence for interleukin-7 (IL-7) described in U.S. Pat. No.
4,965,195; the signal sequence for interleukin-2 receptor described
in Cosman et al., Nature 312:768 (1984); the interleukin-4 signal
peptide described in EP 367,566; the type I interleukin-1 receptor
signal peptide described in U.S. Pat. No. 4,968,607; and the type
II interleukin-1 receptor signal peptide described in EP
460,846.
[0063] The present invention provides a process for preparing the
recombinant proteins of the present invention, comprising culturing
a host cell transformed with an expression vector comprising a DNA
sequence that encodes said protein under conditions that promote
expression. The desired protein is then purified from culture media
or cell extracts. The desired protein may be OSM-R.beta. or the
heterodimeric receptor, for example. Cell-free translation systems
could also be employed to produce the desired protein using RNA
derived from the novel DNA of the present invention.
[0064] As one example, supernatants from expression systems that
secrete recombinant protein into the culture medium can be first
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. Following the concentration step, the
concentrate can be applied to a suitable purification matrix. For
example, a suitable affinity matrix can comprise oncostatin M. An
oncostatin M affinity matrix may be prepared by coupling
recombinant human oncostatin M to cyanogen bromide-activated
Sepharose (Pharmacia) or Hydrazide Affigel (Biorad), according to
manufacturer's recommendations. Sequential immunopurification using
antibodies bound to a suitable support is preferred. Proteins
binding to an antibody specific for OSM-R.beta. are recovered and
contacted with antibody specific for gp130 on an insoluble support.
Proteins immunoreactive with both antibodies may thus be identified
and isolated.
[0065] Alternatively, an anion exchange resin can be employed, for
example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dextran,
cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable
cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Sulfopropyl groups are
preferred. One or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a fusion protein.
[0066] Some or all of the foregoing purification steps, in various
combinations, can be employed to provide an essentially homogeneous
recombinant protein. Recombinant cell culture enables the
production of the fusion protein free of those contaminating
proteins which may be normally associated with gp130 or OSM-R.beta.
as they are found in nature in their respective species of origin,
e.g., on the surface of certain cell types.
[0067] The foregoing purification procedures are among those that
may be employed to purify non-recombinant receptors of the present
invention as well. When linking procedures that may produce
homodimers (gp130-linker-gp130 and OSM-R.beta.-linker-OSM-R.beta.)
are employed, purification procedures that separate the heterodimer
from such homodimers are employed. An example of such a procedure
is sequential immunopurification as discussed above. In one
embodiment, OSM-R.beta. (recombinant or non-recombinant) is
purified such that no bands corresponding to other (contaminating)
proteins are detectable by SDS-PAGE.
[0068] Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of recombinant fusion
proteins can disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0069] Fermentation of yeast which express fusion proteins as a
secreted protein greatly simplifies purification. Secreted
recombinant protein resulting from a large-scale fermentation can
be purified by methods analogous to those disclosed by Urdal et al.
(J. Chromatog. 296:171, 1984), involving two sequential,
reversed-phase HPLC steps for purification of a recombinant protein
on a preparative HPLC column.
[0070] The DNA or amino acid sequences of gp130 and OSM-R.beta. may
vary from those presented in SEQ ID NO:1 and SEQ ID NO:5,
respectively. Due to the known degeneracy of the genetic code,
there can be considerable variation in nucleotide sequences
encoding the same amino acid sequence. In addition, DNA sequences
capable of hybridizing to the native DNA sequence of SEQ ID NO: 1
or SEQ ID NO:5 under moderately stringent or highly stringent
conditions, and which encode a biologically active gp130 or
OSM-R.beta. polypeptide, respectively, are also considered to be
gp130-encoding or OSM-R.beta.-encoding DNA sequences, in the
context of the present invention. Such hybridizing sequences
include but are not limited to variant sequences such as those
described below, and DNA derived from other mammalian species.
Human OSM-R.beta. is within the scope of the present invention, as
are OSM-R.beta. proteins derived from other mammalian species,
including but not limited to rat, bovine, porcine, or various
non-human primates.
[0071] Moderately stingent conditions include conditions described
in, for example, Sambrook et al, Molecular Cloning: A Laboratory
Manual, 2nd ed., Vol. 1, pp 1.101-104, Cold Spring Harbor
Laboratory Press, 1989. Conditions of moderate stingency, as
defined by Sambrook et al., include use of a prewashing solution of
5.times. SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization
conditions of about 55_C, 5.times. SSC, overnight. Highly stringent
conditions include higher temperatures of hybridization and
washing. The skilled artisan will recognize that the temperature
and wash solution salt concentration may be adjusted as necessary
according to factors such as the length of the probe. One
embodiment of the invention is directed to DNA sequences that will
hybridize to the OSM-R.beta. DNA of SEQ ID NO:5 under highly
stringent conditions, wherein said conditions include hybridization
at 68.degree. C. followed by washing in 0.1.times.SSC/0.1% SDS at
63-68.degree. C. In another embodiment, the present invention
provides a heterodimeric receptor comprising OSM-R.beta. and gp130,
wherein said OSM-R.beta. and gp130 are encoded by DNA that
hybridizes to the DNA of SEQ ID NO:5 or SEQ ID NO: 1, respectively,
under moderately or highly stringent conditions.
[0072] Further, certain mutations in a nucleotide sequence which
encodes OSM-R.beta. or gp130 will not be expressed in the final
protein product. For example, nucleotide substitutions may be made
to enhance expression, primarily to avoid secondary structure loops
in the transcribed mRNA (see EP 75,444A). Other alterations of the
nucleotide sequence may be made 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.
[0073] The amino acid sequence of native gp130 or OSM-R.beta. may
be varied by substituting, deleting, adding, or inserting one or
more amino acids to produce a gp130 or OSM-R.beta. variant.
Variants that possess the desired biological activity of the native
gp130 and OSM-R.beta. proteins may be employed in the receptor of
the present invention. Assays by which the biological activity of
variant proteins may be analyzed are described in the examples
below. Biologically active gp130 polypeptides are capable of
binding oncostatin M. The desired biological activity of the
OSM-R.beta. polypeptides disclosed herein is the ability to enhance
the binding of oncostatin M when OSM-R.beta. is joined to gp130,
compared to the level of oncostatin M binding to gp130 alone.
[0074] Alterations to the native amino acid sequence may be
accomplished by any of a number of known techniques. For example,
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.
[0075] 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. Exemplary 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); Craig
(BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); U.S. Pat.
No. 4,518,584, and U.S. Pat. No. 4,737,462, which are incorporated
by reference herein.
[0076] Bioequivalent variants of OSM-R.beta. and gp130 may be
constructed by, for example, making various substitutions of amino
acid residues or deleting terminal or internal amino acids not
needed for biological activity. In one embodiment of the invention,
the variant amino acid sequence is at least 80% identical,
preferably at least 90% identical, to the native sequence. Percent
similarity may be determined, for example, by comparing sequence
information using the GAP computer program, version 6.0, available
from the University of Wisconsin Genetics Computer Group (UWGCG).
The GAP program utilizes 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). Briefly, the GAP program
defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are similar, divided by the total
number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unary comparison matrix (containing a value of 1 for identities and
0 for non-identities) for nucleotides, and the weighted comparison
matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
[0077] Generally, substitutions should be made conservatively;
i.e., the most preferred substitute amino acids are those having
physiochemical characteristics resembling those of the residue to
be replaced. 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.
[0078] Cysteine residues can be deleted or replaced with other
amino acids to prevent formation of unnecessary or incorrect
intramolecular disulfide bridges upon renaturation. Hydrophilic
amino acids may be substituted for hydrophobic amino acids in the
transmembrane region and/or intracellular domain of gp130 and
OSM-R.beta. to enhance water solubility of the proteins.
[0079] Adjacent dibasic amino acid residues may be modified to
enhance expression in yeast systems in which KEX2 protease activity
is present. EP 212,914 discloses the use of site-specific
mutagenesis to inactivate KEX2 protease processing sites in a
protein. KEX2 protease processing sites are inactivated by
deleting, adding or substituting residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these
adjacent basic residues. These amino acid pairs, which constitute
KEX2 proteases processing sites, are found at residues 290-291,
291-292, 580-581, and 797-798 of the OSM-R.beta. protein of SEQ ID
NO:6. These KEX2 sites are found at positions 153-154 and 621-622
of the gp130 protein of SEQ ID NO:2. Lys-Lys pairings are
considerably less susceptible to KEX2 cleavage, and conversion of
Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and
preferred approach to inactivating KEX2 sites.
[0080] The present invention also includes proteins with or without
associated native-pattern glycosylation. Expression of DNAs
encoding the fusion proteins in bacteria such as E. coli provides
non-glycosylated molecules. Functional mutant analogs 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 A1
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.
[0081] The OSM-R.beta. amino acid sequence in SEQ ID NO:6 contains
16 such N-glycosylation sites, all found in the extracellular
domain, at amino acids 15-17, 57-59, 104-106, 136-138, 149-151,
194-196, 280-282, 299-301, 318-320, 334-336, 353-355, 395-397,
419-421, 464-466, 482-484, and 553-555 of SEQ ID NO:6. The
extracellular domain of gp130 comprises N-glycosylation sites at
positions 21-23, 61-63, 109-111, 135-137, 205-207, 224-226,
357-359, 361-363, 368-370, 531-533, and 542-544 of SEQ ID NO:2.
Such a site 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 Al and Z, or an amino acid other than Asn
between Asn and Al. Known procedures for inactivating
N-glycosylation sites in proteins include those described in U.S.
Pat. No. 5,071,972 and EP 276,846.
[0082] Variants of the receptor proteins of the present invention
also include various structural forms of the primary protein which
retain biological activity. Due to the presence of ionizable amino
and carboxyl groups, for example, a receptor 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.
[0083] The primary amino acid structure also may be modified by
forming covalent or aggregative conjugates with other chemical
moieties, such as glycosyl groups, lipids, phosphate, acetyl groups
and the like. Covalent derivatives are prepared by linking
particular functional groups to amino acid side chains or at the N-
or C-termini. Other derivatives of the receptor protein within the
scope of this invention include covalent or aggregative conjugates
of the receptor protein with other proteins or polypeptides, such
as by synthesis in recombinant culture as N- or C-terminal fusions.
For example, the conjugated polypeptide may be a signal (or leader)
polypeptide sequence at the N-terminal region of the protein which
co-translationally or post-translationally directs transfer of the
protein from its site of synthesis to its site of function inside
or outside of the cell membrane or wall (e.g., the yeast a-factor
leader).
[0084] Peptides may be fused to the desired protein (e.g., via
recombinant DNA techniques) to facilitate purification or
identification. Examples include poly-His or the Flag.RTM. peptide
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:7) (Hopp et al.,
Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912). The
Flag.RTM. peptide is highly antigenic and provides an epitope
reversibly bound by a specific monoclonal antibody, enabling rapid
assay and facile purification of expressed recombinant protein.
Expression systems useful for fusing the Flag.RTM. octapeptide to
the N- or C-terminus of a given protein are available from Eastman
Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.,
as are monoclonal antibodies that bind the octapeptide.
[0085] Encompassed by the present invention are OSM-R.beta.
polypeptides in the form of oligomers, such as dimers or trimers.
Such oligomers may be naturally occurring or produced by
recombinant DNA technology. The present invention provides
oligomers of OSM-R.beta. (preferably the extracellular domain or a
fragment thereof), linked by disulfide bonds or expressed as fusion
proteins with or without peptide linkers. Oligomers may be formed
by disulfide bonds between cysteine residues on different
OSM-R.beta. polypeptides, for example. In another embodiment,
OSM-R.beta. oligomers may be prepared using polypeptides derived
from immunoglobulins, as described above.
[0086] Naturally occurring OSM-R.beta. variants are also
encompassed by the present invention. Examples of such variants are
proteins that result from alternative mRNA splicing events or from
proteolytic cleavage of the OSM-R.beta. protein, wherein the
desired biological activity is retained. Alternative splicing of
mRNA may yield a truncated but biologically active OSM-R.beta.,
protein, such as a naturally occurring soluble form of the protein,
for example. 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 OSM-R.beta. protein (generally
from 1-5 terminal amino acids). Naturally occurring gp130 variants
may be employed in the inventive receptors.
[0087] The present invention also provides a pharmaceutical
composition comprising a receptor protein of the present invention
with a physiologically acceptable carrier or diluent. Such carriers
and diluents will be nontoxic to recipients at the dosages and
concentrations employed. Such compositions may, for example,
comprise the receptor protein in a buffered solution, to which may
be added antioxidants such as ascorbic acid, low molecular weight
(less than about ten residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients. The receptor of the present invention may be
administered by any suitable method in a manner appropriate to the
indication, such as intravenous injection, local administration,
continuous infusion, sustained release from implants, etc.
[0088] The heterodimeric receptor of the present invention
(comprising gp130 and OSM-R.beta.) is useful as an oncostatin M
binding reagent. This receptor, which preferably comprises soluble
gp130 and soluble OSM-R.beta., has applications both in vitro and
in vivo. The receptors may be employed in in vitro assays, e.g., in
studies of the mechanism of transduction of the biological signal
that is initiated by binding of oncostatin M to this receptor on a
cell. Such receptors also could be used to inhibit a biological
activity of oncostatin M in various in vitro assays or in vivo
procedures. In one embodiment of the invention, the inventive
receptor is administered to bind oncostatin M, thus inhibiting
binding of the oncostatin M to endogenous cell surface receptors.
Biological activity mediated by such binding of oncostatin M to the
cells thus is also inhibited. gp130 alone binds oncostatin M, but
with relatively low affinity (Gearing et al., Science 255:1434,
1992). Heterodimeric receptors comprising a leukemia inhibitory
factor (LIF) receptor and gp130 bind oncostatin M with higher
affinity than does gp130 alone, but also bind LIF with high
affinity (Gearing et al., supra). Receptors of the present
invention, produced by cells co-transfected with OSM-R.beta.- and
gp130-encodinc, DNA, for example, bind oncostatin M with high
affinity but do not function as a high affinity LIF receptors. Such
receptors of the present invention may be employed when inhibition
of an oncostatin M-mediated activity, but not a LIF-mediated
activity, is desired, for example. Oncostatin M shares certain
properties with LIF, but exhibits other activities that are not
exhibited by LIF. In addition, use of the receptors of the present
invention in vitro assays offers the advantage of allowing one to
determine that the assay results are attributable to binding of
oncostain M, but not LIF, by the receptor.
[0089] In one embodiment of the invention, a heterodimeric receptor
comprising OSM-R.beta. and gp130 is administered in vivo to inhibit
a biological activity of oncostatin M. Oncostatin M has exhibited
growth modulating activity on a variety of different cell types,
and has been reported to stimulate hematopoiesis, stimulate
epithelial cell proliferation, increase plasmin activity (thereby
inducing fibrinolysis), inhibit angiogenesis and supress expression
of major histocompatibility complex antigens on endothelial cells.
See PCT application WO 9109057 and European patent application no.
422,186. When these or other biological effects of oncostatin M are
undesirable, a receptor of the present invention may be
administered to bind oncostatin M.
[0090] The inventive receptor may be administered to a patient in a
therapeutically effective amount to treat a disorder mediated by
oncostatin M. A disorder is said to be mediated by oncostatin M
when oncostatin M causes (directly or indirectly) or exacerbates
the disorder. Soluble receptor proteins can be used to
competitively bind to oncostatin M, thereby inhibiting binding of
oncostatin M to endogenous cell surface receptors. Oncostatin M is
believed to stimulate production of the cytokine interleukin-6
(IL-6), as reported by Brown et al., J. Immunol. 147:2175 (1991).
Oncostatin M therefore may indirectly mediate disorders associated
with the presence of IL-6. IL-6 has been reported to be involved in
the pathogenesis of AIDS-associated Kaposi's sarcoma (dewit et al.,
J. Intern. Med. [England] 229:539, 1991). Oncostatin M has been
reported to play a role in stimulating proliferation of Kaposi's
sarcoma cells (Nair et al., Science 255:1430, 1992, and Miles et
al., Science 255:1432, 1992). Binding of oncostatin M by a receptor
of the present invention (preferably a soluble form thereof) thus
may be useful in treating Kaposi's sarcoma.
[0091] Heterodimeric receptors comprising OSM-R.beta. linked to
gp130 also find use in assays for biological activity of oncostatin
M proteins, which biological activity is measured in terms of
binding affinity for the receptor. To illustrate, the receptor may
be employed in a binding assay to measure the biological activity
of an oncostatin M fragment, variant, or mutein. The receptor is
useful for determining whether biological activity of oncostatin M
is retained after modification of an oncostatin M protein (e g.,
chemical modification, mutation, etc.). The binding affinity of the
modified oncostatin M protein for the receptor is compared to that
of an unmodified oncostatin M protein to detect any adverse impact
of the modification on biological activity. Biological activity
thus can be assessed before the modified protein is used in a
research study or assay, for example.
[0092] The heterodimeric receptors also find use as reagents that
may be employed by those conducting "quality assurance" studies,
e.g., to monitor shelf life and stability of oncostatin M proteins
under different conditions. The receptors may be used to confirm
biological activity (in terms of binding affinity for the receptor)
in oncostatin M proteins (hat have been stored at different
temperatures, for different periods of time, or which have been
produced in different types of recombinant expression systems, for
example.
[0093] The present invention further provides fragments of the
OSM-R.RTM. nucleotide sequences presented herein. Such fragments
desirably comprise at least about 14 nucleotides of the sequence
presented in SEQ ID NO:5. DNA and RNA complements of said fragments
are provided herein, along with both single-stranded and
double-stranded forms of the OSM-R.beta. DNA.
[0094] Among the uses of such nucleic acid fragments is use as a
probe. Such probes may be employed in cross-species hybridization
procedures to isolate OSM-R.beta. DNA from additional mammalian
species. As one example, a probe corresponding to the extracellular
domain of OSM-R.beta. may be employed. The probes also find use in
detecting the presence of OSM-R.beta. nucleic acids in in vitro
assays and in such procedures as Northern and Southern blots. Cell
types expressing OSM-R.beta. can be identified. Such procedures are
well known, and the skilled artisan can choose a probe of suitable
length, depending on the particular intended application. The
probes may be labeled (e.g., with .sup.32P) by conventional
techniques.
[0095] Other useful fragments of the OSM-R.beta. nucleic acids are
antisense or sense oligonucleotides comprising a single-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target OSM-R.beta. mRNA (sense) or OSM-R.beta. DNA (antisense)
sequences. Antisense or sense oligonucleotides, according to the
present invention, may comprise a fragment of the coding region of
OSM-R.beta. cDNA. Such a fragment generally comprises at least
about 14 nucleotides, preferably from about 14 to about 30
nucleotides. The ability to create an antisense or a 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.
[0096] 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
antisense oligonucleotides thus may be used to block expression of
OSM-R.beta. proteins.
[0097] Antisense or sense oligonucleotides further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or
other sugar linkages, such as those described in WO91/06629) and
wherein such sugar linkages are resistant to endogenous nucleases.
Such oligonucleotides with resistant sugar linkages are stable in
vivo (i.e., capable of resisting enzymatic degradation) but retain
sequence specificity to be able to bind to target nucleotide
sequences. Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10448, and 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.
[0098] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by insertion of the antisense or sense
oligonucleotide into a suitable retroviral vector, then contacting
the cell with the retroviral vector containing the inserted
sequence, either in vivo or ex vivo. Suitable retroviral vectors
include, but are not limited to, those derived from the murine
retrovirus M-MuLV, N2 (a retroviri's derived from M-MuLV), or the
double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT
Application US 90/02656).
[0099] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface
receptors.
[0100] 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.
[0101] The following examples are provided to illustrate certain
embodiments of the invention, and are not to b& construed as
limiting the scope of the invention.
EXAMPLES
Example 1
Isolation of DNA Encoding OSM-R.beta.
[0102] DNA encoding the 1 subunit of the oncostatin M receptor was
isolated as follows. The procedure began with preparation of
oligonucleotides degenerate to amino acid sequences that are
conserved among proteins of the hematopoictin receptor family.
[0103] Alignment of the amino acid sequences of three proteins in
the hematopoietin receptor family (gp130, LIF receptor, and G-CSF
receptor) reveals several highly conserved regions. Such conserved
regions are identified and discussed by Gearing et al. in
Polyfunctional Cytokines: IL-6 and LIF, Bock et al., Eds., John
Wiley & Sons, Chichester, UK, 1992, page 245. After including
homologous sequences from the .gamma. chain of the IL-2 receptor as
well (Takeshita et al. Science 257:379, 1992), oligonucleotides
degenerate to certain of the conserved regions (i.e., sets of
oligonucleotides that include all possible DNA sequences that can
encode the amino acid sequences in the conserved regions) were
prepared by conventional techniques.
[0104] Two sets of degenerate oligonucleotides were used as primers
in a polymerase chain reaction (PCR). 5' primers were degenerate to
the amino acid sequence PheArgXArgCys (SEQ ID NO:9), which is found
at positions 275-279 of the gp130 sequence of SEQ ID NO:2, wherein
X represents lie (found at that position in gp130 and LIF-R) or Val
(for IL-2R.gamma.). Additional 5' primers degenerate to the
sequence LeuGlnIleArgCys (SEQ ID NO: 10), which is found at the
corresponding position in G-CSF-R, were employed as well. The 3'
primers were degenerate to the amino acid sequence TrpSerXTrpSer
(SEQ ID NO:11), which is found at positions 288-292 of the gp130
sequence of SEQ ID NO:2, wherein X represents Asp (found at that
position in gp130 and G-CSF-R), Lys (for LIF-R), or Glu (for
IL-2R.gamma.).
[0105] To test the viability of this approach, PCR was conducted
using the above-described primers with LIF-R, gp130, G-CSF-R, or
IL-2R.gamma. DNA as the template. The reactions were conducted by
conventional techniques, and the reaction products were analyzed by
gel electrophoresis. For each reaction, a band about 50 base-pairs
in size was seen on the gel, indicating successful amplification of
a DNA fragment of the expected size.
[0106] PCR was then conducted using genomic human DNA as the
template. The reaction products were analyzed by gel
electrophoresis, and a 50 bp band was visualized. This band was
excised from the gel, and the DNA was eluted therefrom. The DNA was
subcloned into the cloning vector pBLUESCRIPT.RTM. SK, which is
available from Stratagene Cloning Systems, La Jolla, Calif. E. coli
cells were transformed with the resulting recombinant vectors, and
individual colonies of the transformants were cultivated in 96-well
plates.
[0107] Twelve colonies were chosen at random, and the recombinant
vectors were isolated therefrom. The nucleotide sequences of the
DNA inserts of the vectors were determined. Seven of these inserts
were identified by their sequence as gp130 DNA, two were LIF-R, one
contained a stop codon and did not appear to be of interest, and
two contained a novel sequence (the same sequence, in both
orientations). An oligonucleotide probe containing this novel
sequence (the portion of the insert that is between the two primer
sequences) was prepared and labeled with .sup.32P by standard
techniques.
[0108] The .sup.32P-labeled probe was used to screen two different
cDNA libraries, one derived from human placenta and the other from
a cell line designated IMTLH-1. The placental library was chosen
because placenta is a rich source of growth and differentiation
factors. The IMTLH cells, obtained by transformation of human bone
marrow stromal cells with pSV-neo, were chosen because they were
found to bind oncostatin M but not LIF (Thoma et al., J. Biol.
Chem. 269:6215, 1994). In addition, an RNA band of about 5.5-6.0 kb
was detected on Northern blots of RNA derived from IMTLH-1 cells
and placenta, probed with the above-identified .sup.32P-labeled
probe.
[0109] Positive clones were isolated from both libraries and
determined by DNA sequencing to contain various portions of the
novel DNA of interest. Although an initiator codon (indicating the
5' end of a coding region) was identified, none of the clones
appeared to contain the stop codon that would represent the 3' end
of the coding region.
[0110] An oligonucleotide probe corresponding to sequence found
near the 3' end of several of the clones was synthesized and
labeled with .sup.32P by standard techniques. The probe was used to
screen a cDNA library derived from the SV40-transformed human lung
fibroblast cell line WI-26 VA4. This library was constructed as
described in example 2 of U.S. Pat. No. 5,264,416, which is hereby
incorporated by reference. Clones comprising additional coding
sequence at the 3' end (compared to the previously-identified
clones above) were isolated.
[0111] An expression vector was constructed, containing a DNA
fragment comprising this 3' end of the novel sequence ligated to
DNA fragments from the above-described clones containing the 5' end
of the novel sequence. The nucleotide sequence of the human
OSM-R.beta. DNA in the resulting recombinant vector is presented in
SEQ ID NO:5. The protein encoded by the isolated DNA is presented
in SEQ ID NO:6.
[0112] The vector was a mammalian expression vector designated
pDC409. This vector is similar to pDC406, described in McMahan et
al., (EMBO J. 10:2821, 1991). A Bgl II site outside the multiple
cloning site (mcs) in pDC406 has been deleted so that the BglII
site in the mcs of pDC409 is unique. The pDC409 multiple cloning
site (mcs) differs from that of pDC406 in that it contains
additional restriction sites and three stop codons (one in each
reading frame). A T7 polymerase promoter downstream of the mcs
facilitates sequencing of DNA inserted into the mcs.
[0113] The OSM-R.beta. cDNA insert was excised from an expression
vector using restriction enzymes that cleave within the 5' and 3'
non-coding regions of the cDNA. The excised cDNA was ligated into
the EcoRV site of the cloning vector pBluescript.RTM. SK.sup.-
(Stratagene Cloning Systems, LaJolla, Calif.). The Eco RV site,
found in the multiple cloning site of the vector, was destroyed by
insertion of the cDNA. E. coli cells transformed with the resulting
recombinant vector were deposited with the American Type Culture
Collection, Rockville, Md., U.S.A., on Aug. 16, 1994, and assigned
accession no. ATCC 69675. The deposit was made under the terms of
the Budapest Treaty.
[0114] The encoded OSM-R.beta. amino acid sequence presented in SEQ
ID NO:6 comprises an N-terminal signal peptide (amino acids -97 to
-1) followed by an extracellular domain (amino acids 1 to 714), a
transmembrane region (amino acids 715 to 734) and a cytoplasmic
domain (amino acids 735 to 952). The OSM-R.beta. amino acid
sequence is approximately 30% identical to that of the LIF receptor
protein described in Gearing et al. (EMBO J. 10:2839, 1991) and in
U.S. Pat. No. 5,284,755, hereby incorporated by reference. The DNA
sequence of the coding region of OSM-R.beta. is about 48% identical
to the portion of LIF-R DNA that aligns with the OSM-R.beta. coding
region when the above-described GAP computer program is
employed.
Example 2
Assay to Detect Binding of Oncostatin M
[0115] An assay for binding of oncostatin M by cells expressing
both recombinant gp130 and recombinant OSM-R.beta. was conducted as
follows. An assay for oncostatin M binding by cells expressing
gp130 alone was also conducted for purposes of comparison.
[0116] Oncostatin M may be purified from cells in which the protein
is naturally found, or from cells transformed with an expression
vector encoding oncostatin M. One source of oncostatin M is phorbol
ester-treated U937 cells, as described by Zarling et al., PNAS
U.S.A. 83:9739 (1986). Purification of recombinant oncostatin M is
described by Linsley et al. (J. Biol. Chem. 264:4282-4289, 1989)
and Gearing et al. (EMBO J. 10:2839, 1991).
[0117] Oncostatin M (OSM) may be radiolabeled using any suitable
conventional procedure. Radioiodination of oncostatin M has been
described by Linsley et al., supra., for example. In one suitable
procedure, OSM is radiolabeled using a commercially available
enzymobead radioiodination reagent (BioRad) according to
manufacturer's instructions. The resulting .sup.125I-OSM is diluted
to a working stock solution in binding medium, which is RPMI 1640
medium containing 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v)
sodium azide, and 20 mM Hepes, pH 7.4.
[0118] CV1-EBNA-1 cells in 150 mm dishes (3.6.times.10.sup.6
cells/dish) were transfected with a gp130-encoding expression
vector, or were co-transfected with the gp130-encoding vector and
an OSM-R.beta.-encoding vector. All cells were additionally
co-transfected with a mammalian expression vector designated
pDC410, described below.
[0119] The OSM-R.beta.-encoding vector was the recombinant vector
described in example 1, comprising full length OSM-R.beta. DNA in
mammalian expression vector pDC409. The gp130-encoding vector
comprised the human gp130 DNA sequence of SEQ ID NO: 1 in a
mammalian expression vector designated pDC304. A similar
recombinant vector, comprising the same gp130-encoding DNA in
mammalian expression vector pDC303, was deposited in E. coli strain
DHS.alpha. host cells with the American Type Culture Collection,
Rockville, Md. These transformed cells were deposited under the
name B10G/pDC303 (DH5.alpha.) on Nov. 14, 1991 and assigned ATCC
Accession No. 68827. The deposit was made under the terms of the
Budapest Treaty.
[0120] pDC304 comprises a NotI site in its multiple cloning site,
but is otherwise identical to pDC303. pDC304 also is essentially
identical to pCAV/INOT, described in PCT application WO 90/05183,
except that a segment of the adenovirus-2 tripartite leader (TPL)
containing a cryptic promoter functional in bacteria has been
deleted. Protein expression from the cryptic promoter is
potentially disadvantageous for preparing and isolating a desired
recombinant plasmid in bacterial cells.
[0121] The pDC410 vector is identical to the pDC409 vector
described in example 1, except that the EBV origin of replication
of pDC409 is replaced by DNA encoding the SV40 large T antigen
driven from the SV40 promoter in pDC410. Co-transfecting the cells
with this vector provides the SV40 T-antigen that drives high level
DNA replication of the other plasmid vectors, which contain the
SV40 origin of replication. pDC410 thus is important for episomal
replication of the co-transfected vectors in CVI-EBNA-1 cells.
[0122] The transfected cells were cultured for 24 hours,
trypsinized and replated, then cultured another 24 hours to permit
expression of the encoded proteins, which were retained on the cell
membrane. The adherent cells were dislodged using 5 nM EDTA in PBS,
then washed twice with binding medium (RPMI 1640 medium containing
25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, and 20 mM
HEPES, pH 7.2). The cells then were incubated with various
concentrations of .sup.125I-labeled oncostatin M in binding medium
for 1 hour at 37.degree. C. with gentle agitation.
[0123] Free and cell-bound .sup.125I-oncostatin M were separated
using the phthalate oil separation method of Dower et al. (J.
Immunol. 132:751, 1984), essentially as described by Park et al.
(J. Biol. Chem. 261:4177, 1986, and Proc. Natl. Acad. Sci. USA
84:5267, 1987). The free and cell-bound .sup.125I-oncostatin M were
quantified on a Packard Autogamma Counter. Affinity calculations
(Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) were generated on
RS/1 (BBN Software, Boston, Mass.) run on a Microvax computer.
[0124] The results are presented in FIGS. 1 and 2, in the form of
Scatchard analyses. FIG. 1 presents the results for cells
expressing gp130 alone. These transfected cells exhibited a single
affinity class of binding, with approximately 29,310 receptor sites
per cell, and an affinity constant (Ka) of 2.64.times.10.sup.8.
FIG. 2 presents the results for cells expressing gp130 and
OSM-R.beta.. A biphasic pattern can be seen, indicating two binding
components. The first component (approximately 2196 receptor sites
per cell) exhibited an affinity constant of 7.18.times.10.sup.9.
The second component (approximately 36,471 receptor sites per cell)
exhibited an affinity constant of 2.34.times.10.sup.8. Thus, a
relatively high affinity binding component is seen in the cells
expressing both gp130 and OSM-R.beta.. These high affinity binding
sites were absent in the cells expressing gp130 alone.
[0125] The cells co-transfected with both OSM-RB- and
gp130-encoding expression vectors expressed a receptor protein of
the present invention. The receptor binds oncostatin M with higher
affinity than does the gp130 protein expressed on cells transfected
with the gp130-encoding vector alone.
Example 3
Preparation of Monoclonal Antibodies Directed Against
OSM-R.beta.
[0126] Purified OSM-R.beta. polypeptides of the present invention
are employed as immunogens to generate monoclonal antibodies
immunoreactive therewith using conventional techniques, for
example, those disclosed in U.S. Pat. No. 4,411,993. Suitable
immunogens include, but are not limited to, full length recombinant
OSM-R.beta. or fragments thereof, such as the extracellular domain.
To immunize mice, the immunogen is emulsified in complete Freund's
adjuvant and injected subcutaneously in amounts ranging from
10-100.mu.g into Balb/c mice. Ten to twelve 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.
[0127] 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 to a murine myeloma cell line,
e.g., NS 1 or, preferably, P3x63Ag8.653 (ATCC CRL 1580). Hybridoma
cell lines generated by this procedure are plated in multiple
microtiter plates in a HAT selective medium (hypoxantine,
aminopterin, and thymidine) to inhibit proliferation of non-fused
cells, myeloma hybrids, and spleen cell hybrids.
[0128] Hybridoma clones thus generated can be screened by ELISA for
reactivity with the receptor protein, for example, by adaptations
of the techniques disclosed by Engvall et al., Immunochem 8.871
(1971) and in U.S. Pat. No. 4,704,004. A preferred screening
technique is the antibody capture technique described in Beckmann
et al. (J. Immunol. 144:4212, 1990). Positive clones are then
injected into the peritoneal cavities of syngeneic Balb/c mice to
produce ascites containing high concentrations (greater than 1
mg/ml) of anti-OSM-R.beta. 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 4
Receptors Comprising gp130 Polypeptides Lacking FNIII Domains
[0129] DNA sequences encoding soluble gp130 proteins lacking
fibronectin type III (FNIII) domains were isolated and fused to an
Fc-encoding sequence. Deleting the FNIII domains affords the
advantage of reducing the size of the gp130/Fc fusion protein.
gp130 contains three FNIII domains, comprising amino acids 300
(Tyr) to 399 (Phe), 400 (Gln) to 496 (Pro), and 497 (Pro) to 597
(Glu), respectively, of SEQ ID NO:2. From one to all three of the
FNIII domains may be removed from gp130 to reduce the size of the
protein.
[0130] The FNIII domains of gp130 were removed by digesting a
recombinant gp13O/Fc-encoding expression vector with BstX1, then
blunting the overhang using T4 DNA polymerase according to
conventional procedures. The recognition site for BstX1 spans
nucleotides 1231-1242 of SEQ ID NO:1,(gp130), cleaving within the
codons for amino acids 10-11 of the first FNIII domain of gp130.
The cleaved vector was then digested with EcoR5, which cleaves
within the polylinker of the vector upstream of the Fc sequence and
generates blunt ends. The (BstXl)/EcoR5 fragment comprising a
sequence encoding the 5' end of gp130 (lacking the FNIII domains),
the vector sequences, the Fc sequence, and a portion of the
polylinker, was ligated to recircularize the vector.
[0131] E. coli cells were transformed with the ligation mixture,
plasmids were isolated therefrom, and the desired recombinant
plasmid was identified by restriction analysis. The fusion protein
encoded by the construct comprises (from N- to C-terminus) amino
acids -22 to 308 of SEQ ID NO:2 (gp130), a four amino acid spacer
peptide -Asn-Arg-Tyr-Val-encode- d by the polylinker segment, and
amino acids 1-232 of SEQ ID NO:3 (Fc). The gp130 polypeptide moiety
contains the first 9 amino acids of the first FNIII domain, but
lacks the remainder of the first FNIII domain and all of the second
and third FNIII domains.
[0132] A heterodimeric receptor of the present invention may
comprise OSM-R.beta. and the foregoing truncated gp130 polypeptide
lacking FNIII domains. COS-7 cells or other suitable host cells are
co-transfected with OSM-R.beta.-encoding and truncated
gp130-encoding expression vectors. The co-transfected cells are
cultured to express the heterodimeric receptor.
EXAMPLE 5
Assay for Binding of Oncostatin M and LIF by Receptors
[0133] An assay for binding of oncostatin M or leukemia inhibitory
factor (LIF) by various receptor proteins was conducted as follows.
The receptor proteins included soluble OSM-R.beta./Fc, gp130/Fc,
LIF-R/Fc, and combinations thereof. Results of the assay are
presented in FIG. 3.
[0134] An expression vector encoding a soluble OSM-R.beta./Fc
fusion protein, which comprised a truncated extracellular domain of
OSM-R.beta. fused to the N-terminus of an Fc region polypeptide
derived from an antibody, was constructed as follows. The
recombinant expression vector prepared in example 1, comprising
OSM-R.beta. DNA in vector pDC409, was digested with the restriction
enzyme SphI, treated with T4 DNA polymerase to remove the 3'
overhangs (generating blunt ends), then digested with Sal I, which
cleaves upstream of the OSM-R.beta. coding region. The desired
fragment, which includes the 5' end of the OSM-R.beta. DNA,
terminating at nucleotide 1744 of SEQ ID NO:5, was isolated by
conventional techniques.
[0135] A recombinant vector designated hIgG1Fc comprises the Fc
polypeptide-encoding cDNA of SEQ ID NO:3, as described above.
Vector hIgG1Fc was digested with the restriction enzymes Sna B1 and
NotI, which cleave in the polylinker region of the vector, upstream
and downstream, respectively, of the Fc polypeptide-encoding,
cDNA.
[0136] The thus-isolated Fc polypeptide-encoding DNA fragment and
the OSM-R.beta.-encoding DNA fragment isolated above were ligated
into a SalI/NotI-digested expression vector pDC304 such that the Fc
polypeptide DNA was fused to the 3' end of the OSM-R.beta. DNA. The
mammalian expression vector pDC304 is described in example 2. The
resulting expression vector encoded a fusion protein comprising
amino acids -27 through 432 of the OSM-R.beta. sequence of SEQ ID
NO:6, followed by a valine residue encoded by a vector polylinker
segment, followed by amino acids 1 through 232 of the Fc
polypeptide sequence of SEQ ID NO:4.
[0137] An expression vector encoding a soluble human gp130/Fc
fusion protein was constructed as follows. Recombinant vector
B10G/pDC303 (ATCC 68827) comprising human gp130 cDNA was digested
with EcoR1, and the resulting 5' overhang was rendered blunt using
T4 DNA polymerase. The recognition site for EcoR1 comprises
nucleotides 2056-2061 of SEQ ID NO:1. The EcoR1-digested vector was
then cleaved with XhoI, which cleaves in the vector upstream of the
gp130 cDNA insert.
[0138] Vector hIgG1Fc, comprising Fc polypeptide-encoding, cDNA as
described above, was digested with StuI (a blunt cutter) and NotI,
which cleave upstream and downstream, respectively, of the inserted
Fc cDNA. The XhoI/(EcoR1) gp130 fragment isolated above was ligated
to the Fc-containing fragment and to XhoI/NotI-digested mammalian
expression vector pDC304.
[0139] E. coli cells were transformed with the ligation mixture,
plasmids were isolated therefrom by conventional procedures, and
the desired recombinant vector was identified by restriction
analysis. The gp130/Fc fusion protein encoded by the recombinant
vector comprises (from N- to C-terminus) amino acids -22 to 582 of
SEQ ID NO:2 (gp130), followed by 7 amino acids constituting a
peptide linker encoded by the polylinker segment of plasmid
hIgG1Fc, followed by amino acids 1-232 of SEQ ID NO:4 (Fc).
[0140] An expression vector encoding a soluble human LIF-R/Fc
fusion protein was constructed as described in example 5 of U.S.
Pat. No. 5,984,755, hereby incorporated by reference. Briefly, a
recombinant vector designated pHLIF-R-65 contains human LIF-R cDNA
(a partial clone encoding a complete signal peptide, extracellular
domain, and transmembrane region, and a partial cytoplasmic domain)
in vector pDC303. The mammalian expression vector pDC303 is
described in PCT application WO 93/19777. E. coli cells transformed
with pHLIF-R-65 were deposited with the American Type Culture
Collection, Rockville, Md., on Dec. 11, 1990, and assigned
accession no. 68491. DNA encoding the LIF-R signal peptide and
extracellular domain (truncated at the C-terminus) was isolated and
fused to DNA encoding an antibody Fc region polypeptide in
pBluescript.RTM.SK.sup.-. The gene fusion was excised from the
cloning vector and inserted into the above-described mammalian
expression vector pDC304. The resulting recombinant expression
vector encoded a LIF-R/Fc fusion protein comprising amino acids -44
through 702 of the LIF-R sequence presented in U.S. Pat. No.
5,284,755, followed by a linker comprising six amino acids encoded
by a vector polylinker segment, followed by amino acids 1 through
232 of the Fc amino acid sequence of SEQ ID NO:4.
[0141] CV-1-EBNA cells were transfected with one of the three
recombinant expression vectors prepared above, or co-transfected
with two of the vectors, as follows:
1 Experiment Cells transfected with vector(s) encoding: A empty
expression vector (control) B gp130/Fc C LIF-R/Fc D OSM-R.beta./Fc
E OSM-R.beta./Fc and LIF-R/Fc F OSM-R.beta./Fc and gp130/Fc G
gp130/Fc and LIF-R/Fc
[0142] The transfected cells were cultured to allow expression and
secretion of the fusion proteins into the culture medium.
Cross-linked agarose beads bearing Protein A (Protein A Sepharose
CL-4B, Pharmacia Biotech, Inc., Piscataway, N.J.) were added to the
culture supernatants, whereupon the fusion proteins bound to the
beads via the interaction of the Fc moiety with the Protein A.
Radioiodinated oncostatin M or radioiodinated LIF was also added to
the culture supernatants. Preparation of .sup.125I-oncostatin M is
described in example 2 above. Among the known procedures for
purifying and radioiodinating LIF are those described in example 1
of U.S. Pat. No. 5,284,755. The .sup.125I-LIF employed in this
assay was recombinant human LIF labeled with .sup.125I using the
enzymobead reagent (BioRad).
[0143] The culture supernatants were incubated with the Protein A
beads and .sup.125I-LIF or .sup.125I-oncostatin M for 18 hours at
4.degree. C. Free and cell-bound .sup.125I-LIF or
.sup.125I-oncostatin M were separated by low speed centrifugation
through a single step density gradient of 3% glucose in PBS. The
bead-bound radioiodinated proteins were quantified on a Packard
Autogamma counter.
[0144] The results are presented in FIG. 3. The bar graph in FIG. 3
represents the binding of oncostatin M or LIF to the proteins
expressed by cells transfected as described above for experiments A
to G. The expressed proteins are bound to the Protein A beads.
[0145] Experiment A (control) revealed no significant binding of
LIF or oncostatin M to proteins expressed by cells transfected with
the empty expression vector pDC304. The soluble gp130/Fc protein
bound oncostatin M, but no significant binding of LIF was
demonstrated (experiment B). The soluble LIF-R/Fc protein bound
LIF, but not oncostatin M (experiment C). No detectable binding of
LIF or oncostatin M by the soluble OSM-R.beta./Fc protein was
demonstrated (experiment D).
[0146] Proteins expressed by cells co-transfected with soluble
LIF-R/Fc and OSM-R.beta. encoding vectors did not bind detectable
quantities of oncostatin M, but bound LIF (experiment E). Proteins
expressed by cells co-transfected with soluble OSM-R.beta./Fc and
soluble gp130/Fc encoding vectors bound oncostatin M, but did not
bind detectable quantities of LIF (experiment F). The binding of
oncostatin M in experiment F could be inhibited by including
unlabeled (cold) oncostatin M in the assay. The proteins expressed
by cells co-transfected with expression vectors encoding soluble
gp130/Fc and LIF-R/Fc (experiment G) bound both oncostatin M and
LIF. The LIF binding in experiment G was inhibited by adding cold
LIF to the assay.
[0147] The proteins expressed when cells are co-transfected with
vectors encoding soluble OSM-R/Fc and soluble gp130/Fc, in
accordance with the present invention, thus bind oncostatin M but
not LIF. This is advantageous when binding of oncostatin M (e.g.,
to inhibit or study a biological activity thereof) is desired, but
binding, of LIF is not desired. The proteins expressed by cells
co-transfected with soluble gp130/Fc and soluble LIF-R/Fc encoding
vectors bind both oncostatin M and LIF, and thus do not offer this
advantageous property. In addition, cells expressing both soluble
OSM-R.beta./Fc and soluble gp130/Fc bound oncostatin M at a higher
level than did cells expressing soluble gp130/Fc alone.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0148] SEQ ID NO:1 and SEQ ID NO:2 present the DNA sequence and
encoded amino acid sequence for cloned cDNA encoding an N-terminal
fragment of gp130.
[0149] SEQ ID NO:3 and SEQ ID NO:4 present the DNA sequence and
encoded amino acid sequence for cloned cDNA encoding a polypeptide
that corresponds to the Fc region of an IgG1 antibody.
[0150] SEQ ID NO:5 and SEQ ID NO:6 present the DNA and encoded
amino acid sequence for cloned cDNA encoding the oncostatin M
receptor .beta. subunit of the present invention.
[0151] SEQ ID NO:7 presents the amino acid sequence of a peptide
that may be employed to facilitate purification of polypeptides
fused thereto.
[0152] SEQ ID NO:8 presents a spacer peptide encoded by a
polylinker in an expression vector, as described in example 4.
[0153] SEQ ID NOS:9, 10, and 11 are peptides that correspond to
conserved sequences, as described in example 1.
Sequence CWU 1
1
* * * * *