U.S. patent application number 10/735865 was filed with the patent office on 2004-09-09 for neutrokine-alpha.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Ebner, Reinhard, Ni, Jian, Yu, Guo-Liang.
Application Number | 20040175801 10/735865 |
Document ID | / |
Family ID | 30772336 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175801 |
Kind Code |
A1 |
Yu, Guo-Liang ; et
al. |
September 9, 2004 |
Neutrokine-alpha
Abstract
The present invention relates to a novel Neutrokine-.alpha.
protein which is a member of the TNF protein family. In particular,
isolated nucleic acid molecules are provided encoding the human
Neutrokine-.alpha. protein including soluble forms of the
extracellular domain. Neutrokine-.alpha. polypeptides are also
provided as are vectors, host cells and recombinant methods for
producing the same. The invention further relates to screening
methods for identifying agonists and antagonists of
Neutrokine-.alpha. activity. Also provided are diagnostic methods
for detecting immune system-related disorders and therapeutic
methods for treating immune system-related disorders.
Inventors: |
Yu, Guo-Liang; (Berkeley,
CA) ; Ebner, Reinhard; (Gaithersburg, MD) ;
Ni, Jian; (Germantown, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
20850
|
Family ID: |
30772336 |
Appl. No.: |
10/735865 |
Filed: |
December 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10735865 |
Dec 16, 2003 |
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09005874 |
Jan 12, 1998 |
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6689579 |
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09005874 |
Jan 12, 1998 |
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PCT/US96/17957 |
Oct 25, 1996 |
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60036100 |
Jan 14, 1997 |
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Current U.S.
Class: |
435/69.5 ;
435/320.1; 435/325; 530/351; 536/23.5 |
Current CPC
Class: |
C07H 21/04 20130101;
A61K 38/00 20130101; C07K 14/52 20130101 |
Class at
Publication: |
435/069.5 ;
435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C07K 014/525; C07H
021/04; C12P 021/02 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding the Neutrokine-.alpha. polypeptide having the complete
amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide
sequence encoding the Neutrokine-.alpha. polypeptide having the
complete amino acid sequence encoded by the cDNA clone contained in
ATCC No. 97768 deposited on Oct. 22, 1996; (c) a nucleotide
sequence encoding the Neutrokine-.alpha. polypeptide extracellular
domain; (d) a nucleotide sequence encoding the Neutrokine-.alpha.
polypeptide transmembrane domain; (e) a nucleotide sequence
encoding the Neutrokine-.alpha. polypeptide intracellular domain;
(f) a nucleotide sequence encoding a soluble Neutrokine-.alpha.
polypeptide comprising the extracellular and intracellular domains
but lacking the transmembrane domain; and (g) a nucleotide sequence
complementary to any of the nucleotide sequences in (a), (b), (c),
(d), (e) or (f) above.
2. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence in FIG. 1 (SEQ ID NO:1).
3. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIG. 1 (SEQ ID NO:1) encoding the
Neutrokine-.alpha. polypeptide having the complete amino acid
sequence in FIG. 1 (SEQ ID NO:2).
4. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding a soluble Neutrokine-.alpha.
polypeptide comprising the extracellular domain shown in FIG. 1
(SEQ ID NO:2).
5. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide having the amino acid sequence consisting of
residues n-285 of SEQ ID NO:2, where n is an integer in the range
of 2-190 (b) a nucleotide sequence encoding a polypeptide having
the amino acid sequence consisting of residues 1-m of SEQ ID NO:2,
where m is an integer in the range of 274-284; (c) a nucleotide
sequence encoding a polypeptide having the amino acid sequence
consisting of residues n-m of SEQ ID NO:2, where n and m are
integers as defined respectively in (a) and (b) above; and (d) a
nucleotide sequence encoding a polypeptide consisting of a portion
of the complete Neutrokine-.alpha. amino acid sequence encoded by
the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
1996 wherein said portion excludes from 1 to 190 amino acids from
the amino terminus and from 1 to 11 amino acids from the C-terminus
of said complete amino acid sequence.
6. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence of the cDNA clone contained in
ATCC No. 97768 deposited on Oct. 22, 1996.
7. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the Neutrokine-.alpha.
polypeptide having the complete amino acid sequence encoded by the
cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
1996.
8. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding a soluble Neutrokine-.alpha.
polypeptide comprising the extracellular domain encoded by the cDNA
clone contained in ATCC No. 97768 deposited on Oct. 22, 1996.
9. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d), (e) or (f) of claim 1
wherein said polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues.
10. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a Neutrokine-.alpha. polypeptide having an amino acid sequence
in (a), (b), (c), (d), (e) or (f) of claim 1.
11. The isolated nucleic acid molecule of claim 10, which encodes
an epitope-bearing portion of a Neutrokine-.alpha. polypeptide
selected from the group consisting of: a polypeptide comprising
amino acid residues from about Phe-115 to about Leu-147 (SEQ ID
NO:2); a polypeptide comprising amino acid residues from about
Ile-150 to about Tyr-163 (SEQ ID NO:2); a polypeptide comprising
amino acid residues from about Ser-171 to about Phe-194 (SEQ ID
NO:2); a polypeptide comprising amino acid residues from about
Glu-223 to about Tyr-247 (SEQ ID NO:2); and a polypeptide
comprising amino acid residues from about Ser-271 to about Phe-278
(SEQ ID NO:2).
12. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
13. A recombinant vector produced by the method of claim 12.
14. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 13 into a host
cell.
15. A recombinant host cell produced by the method of claim 14.
16. A recombinant method for producing a Neutrokine-.alpha.
polypeptide, comprising culturing the recombinant host cell of
claim 15 under conditions such that said polypeptide is expressed
and recovering said polypeptide.
17. An isolated Neutrokine-.alpha. polypeptide comprising an amino
acid sequence at least 95% identical to a sequence selected from
the group consisting of: (a) the amino acid sequence of the
Neutrokine-.alpha. polypeptide having the complete amino acid
sequence in FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of
the Neutrokine-.alpha. polypeptide having the complete amino acid
sequence encoded by the cDNA clone contained in the ATCC No. 97768
deposited on Oct. 22, 1996; (c) the amino acid sequence of the
Neutrokine-.alpha. polypeptide extracellular domain; (d) the amino
acid sequence of the Neutrokine-.alpha. polypeptide transmembrane
domain; (e) the amino acid sequence of the Neutrokine-.alpha.
polypeptide intracellular domain; (f) the amino acid sequence of a
soluble Neutrokine-.alpha. polypeptide comprising the domain; and
(g) the amino acid sequence of an epitope-bearing portion of any
one of the polypeptides of (a), (b), (c), (d), (e) or (f).
18. An isolated polypeptide of claim 17 comprising an
epitope-bearing portion of the Neutrokine-.alpha. protein, wherein
said portion is selected from the group consisting of: a
polypeptide comprising amino acid residues from about Phe-115 to
about Leu-147 (SEQ ID NO:2); a polypeptide comprising amino acid
residues from about Ile-150 to about Tyr-163 (SEQ ID NO:2); a
polypeptide comprising amino acid residues from about Ser-171 to
about Phe-194 (SEQ ID NO:2); a polypeptide comprising amino acid
residues from about Glu-223 to about Tyr-247 (SEQ ID NO:2); a
polypeptide comprising amino acid residues from about Ser-271 to
about Phe-278 (SEQ ID NO:2).
19. An isolated antibody that binds specifically to a
Neutrokine-.alpha. polypeptide of claim 17.
20. A pharmaceutical composition comprising a polypeptide of claim
17 and a pharmaceutically acceptable carrier.
21. An isolated polynucleotide encoding a modified
Neutrokine-.alpha. protein, wherein, except for at least one
conservative amino acid substitution, said modified peptide has an
amino acid sequence that is identical to a member selected from the
group consisting of: (a) amino acids 1 to 285 of SEQ ID NO:2; (b)
amino acids 2 to 285 of SEQ ID NO:2; (c) amino acids 1 to 46 of SEQ
ID NO:2; (c) amino acids 47 to 72 of SEQ ID NO:2; and (c) amino
acids 73 to 286 of SEQ ID NO:2.
22. A modified Neutrokine-.alpha. polypeptide molecule, wherein,
except for at least one conservative amino acid substitution, said
modified peptide has an amino acid sequence that is identical to a
member selected from the group consisting of: (a) amino acids 1 to
285 of SEQ ID NO:2; (b) amino acids 2 to 285 of SEQ ID NO:2; (c)
amino acids 1 to 46 of SEQ ID NO:2; (c) amino acids 47 to 72 of SEQ
ID NO:2; and (c) amino acids 73 to 286 of SEQ ID NO:2.
23. An isolated nucleic acid molecule comprising a polynucleotide
having a sequence at least 95% identical to a sequence selected
selected from the group consisting of: (a) the nucleotide sequence
of SEQ ID NO:7; (b) the nucleotide sequence of SEQ ID NO:8; (c) the
nucleotide sequence of SEQ ID NO:9; (d) the nucleotide sequence of
a portion of the sequence shown in FIG. 1 (SEQ ID NO:1) wherein
said portion comprises at least 30 contiguous nucleotides from
nucleotide 1 to nucleotide 2442, excluding the sequence from
nucleotide 1387 to 1421, the sequence from nucleotide 9 to 382, the
sequence from nucleotide 1674 to 1996, the sequence from nucleotide
1401 to 1784, the sequence from nucleotide 900 to 1237, and any
fragments located within these sequences; and (e) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c) or (d) above.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/005,874 filed Jan. 12, 1998, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/036,100, filed Jan. 14, 1997 and which is also a
continuation-in-part of International Patent Application No.
PCT/US96/17957, filed Oct. 25, 1996, all of which are incorporated
by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel cytokine expressed
by neutrophils which has therefore been designated
Neutrokine-.alpha. protein ("Neutrokine-.alpha."). In particular,
isolated nucleic acid molecules are provided encoding the
Neutrokine-.alpha. protein. Neutrokine-.alpha. polypeptides are
also provided, as are vectors, host cells and recombinant methods
for producing the same.
[0003] Related Art
[0004] Human tumor necrosis factors (TNF-.alpha.) and (TNF-.beta.,
or lymphotoxin) are related members of a broad class of polypeptide
mediators, which includes the interferons, interleukins and growth
factors, collectively called cytokines (Beutler, B. and Cerami, A.,
Annu. Ret. Immunol., 7:625-655 (1989)). Sequence analysis of
cytokine receptors has defined several subfamilies of membrane
proteins (1) the Ig superfamily, (2) the hematopoietin (cytokine
receptor superfamily and (3) the tumor necrosis factor (TNF)/nerve
growth factor (NGF) receptor superfamily (for review of TNF
superfamily see, Gruss and Dower, Blood 85(12):3378-3404 (1995) and
Aggarwal and Natarajan, Eur. Cytokine Netw., 7(2):93-124 (1996)).
The TNF/NGF receptor superfamily contains at least 10 difference
proteins. Gruss and Dower, supra. Ligands for these receptors have
been identified and belong to at least two cytokine superfamilies.
Gruss and Dower, supra.
[0005] Tumor necrosis factor (a mixture of TNF-.alpha. and
TNF-.beta.) was originally discovered as a result of its anti-tumor
activity, however, now it is recognized as a pleiotropic cytokine
capable of numerous biological activities including apoptosis of
some transformed cell lines, mediation of cell activation and
proliferation and also as playing important roles in immune
regulation and inflammation.
[0006] To date, known members of the TNF-ligand superfamily include
TNF-.alpha., TNF-.beta. (lymphotoxin-.alpha.), LT-.beta., OX40L,
Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF
ligand superfamily are acidic, TNF-like molecules with
approximately 20% sequence homology in the extracellular domains
(range, 12%-36%) and exist mainly as membrane-bound forms with the
biologically active form being a trimeric/multimeric complex.
Soluble forms of the TNF ligand superfamily have only been
identified so far for TNF, LT-.beta., and Fas ligand (for a general
review, see Gruss, H. and Dower, S. K., Blood, 85(12):3378-3404
(1995)), which is hereby incorporated by reference in its entirety.
These proteins are involved in regulation of cell proliferation,
activation, and differentiation, including control of cell survival
or death by apoptosis or cytotoxicity (Armitage, R. J., Curr. Opin.
Immunol. 6:407 (1994) and Smith, C. A., Cell 75:959 (1994)).
[0007] Tumor necrosis factor-alpha (TNF-.alpha.; also termed
cachectin; hereinafter "TNF") is secreted primarily by monocytes
and macrophages in response to endotoxin or other stimuli as a
soluble homotrimer of 17 kD protein subunits (Smith, R. A. et al.,
J. Biol. Chem. 262:6951-6954 (1987)). A membrane-bound 26 kD
precursor form of TNF has also been described (Kriegler, M. et al.,
Cell 53:45-53 (1988)).
[0008] Accumulating evidence indicates that TNF is a regulatory
cytokine with pleiotropic biological activities. These activities
include: inhibition of lipoprotein lipase synthesis ("cachectin"
activity) (Beutler, B. et al., Nature 316:552 (1985)), activation
of polymorphonuclear leukocytes (Klebanoff, S. J. et al., J.
Immunol. 136:4220 (1986); Perussia, B., et al., J. Immunol. 138:765
(1987)), inhibition of cell growth or stimulation of cell growth
(Vilcek, J. et al., J. Exp. Med. 163:632 (1986); Sugarman, B. J. et
al., Science 230:943 (1985); Lachman, L. B. et al., J. Immunol.
138:2913 (1987)), cytotoxic action on certain transformed cell
types (Lachman, L. B. et al., supra; Darzynkiewicz, Z. et al.,
Canc. Res. 44:83 (1984)), antiviral activity (Kohase, M. et al.,
Cell 45:659 (1986); Wong, G. H. W. et al., Nature 323:819 (1986)),
stimulation of bone resorption (Bertolini, D. R. et al., Nature
319:516 (1986); Saklatvala, J., Nature 322:547 (1986)), stimulation
of collagenase and prostaglandin E2 production (Dayer, J.-M. et
al., J. Exp. Med. 162:2163 (1985)); and immunoregulatory actions,
including activation of T cells (Yokota, S. et al., J. Immunol.
140:531 (1988)), B cells (Kehrl, J. H. et al., J. Exp. Med. 166:786
(1987)), monocytes (Philip, R. et al., Nature 323:86 (1986)),
thymocytes (Ranges, G. E. et al., J. Exp. Med. 167:1472 (1988)),
and stimulation of the cell-surface expression of major
histocompatibility complex (MHC) class I and class II molecules
(Collins, T. et al., Proc. Natl. Acad. Sci. USA 83:446 (1986);
Pujol-Borrel, R. et al., Nature 326:304 (1987)).
[0009] TNF is noted for its pro-inflammatory actions which result
in tissue injury, such as induction of procoagulant activity on
vascular endothelial cells (Pober, J. S. et al., J. Immunol.
136:1680 (1986)), increased adherence of neutrophils and
lymphocytes (Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and
stimulation of the release of platelet activating factor from
macrophages, neutrophils and vascular endothelial cells (Camussi,
G. et al., J. Exp. Med. 166:1390 (1987)).
[0010] Recent evidence implicates TNF in the pathogenesis of many
infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune
disorders, neoplastic pathology, e.g., in cachexia accompanying
some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and in
autoimmune pathologies and graft-versus host pathology (Piguet,
P.-F. et al., J. Exp. Med. 166:1280 (1987)). The association of TNF
with cancer and infectious pathologies is often related to the
host's catabolic state. A major problem in cancer patients is
weight loss, usually associated with anorexia. The extensive
wasting which results is known as "cachexia" (Kern, K. A. et al. J.
Parent. Enter. Nutr. 12:286-298 (1988)). Cachexia includes
progressive weight loss, anorexia, and persistent erosion of body
mass in response to a malignant growth. The cachectic state is thus
associated with significant morbidity and is responsible for the
majority of cancer mortality. A number of studies have suggested
that TNF is an important mediator of the cachexia in cancer,
infectious pathology, and in other catabolic states.
[0011] TNF is thought to play a central role in the
pathophysiological consequences of Gram-negative sepsis and
endotoxic shock (Michie, H. R. et al., Br. J. Surg. 76:670-671
(1989); Debets, J. M. H. et al., Second Vienna Shock Forum,
p.463-466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5:27-47
(1989)), including fever, malaise, anorexia, and cachexia.
Endotoxin is a potent monocyte/macrophage activator which
stimulates production and secretion of TNF (Kornbluth, S. K. et
al., J. Immunol. 137:2585-2591 (1986)) and other cytokines. Because
TNF could mimic many biological effects of endotoxin, it was
concluded to be a central mediator responsible for the clinical
manifestations of endotoxin-related illness. TNF and other
monocyte-derived cytokines mediate the metabolic and neurohormonal
responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med.
318:1481-1486 (1988)). Endotoxin administration to human volunteers
produces acute illness with flu-like symptoms including fever,
tachycardia, increased metabolic rate and stress hormone release
(Revhaug, A. et al., Arch. Surg. 123:162-170 (1988)). Elevated
levels of circulating TNF have also been found in patients
suffering from Gram-negative sepsis (Waage, A. et al., Lancet
1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum
p. 715-718 (1989); Debets, J. M. H. et al., Crit. Care Med.
17:489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161:982-987
(1990)).
[0012] Passive immunotherapy directed at neutralizing TNF may have
a beneficial effect in Gram-negative sepsis and endotoxemia, based
on the increased TNF production and elevated TNF levels in these
pathology states, as discussed above. Antibodies to a "modulator"
material which was characterized as cachectin (later found to be
identical to TNF) were disclosed by Cerami et al. (EPO Patent
Publication 0,212,489, Mar. 4, 1987). Such antibodies were said to
be useful in diagnostic immunoassays and in therapy of shock in
bacterial infections. Rubin et al. (EPO Patent Publication
0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human
TNF, the hybridomas secreting such antibodies, methods of producing
such antibodies, and the use of such antibodies in immunoassay of
TNF. Yone et al. (EPO Patent Publication 0,288,088, Oct. 26, 1988)
disclosed anti-TNF antibodies, including mAbs, and their utility in
immunoassay diagnosis of pathologies, in particular Kawasaki's
pathology and bacterial infection. The body fluids of patients with
Kawasaki's pathology (infantile acute febrile mucocutaneous lymph
node syndrome; Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T.,
Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated
TNF levels which were related to progress of the pathology (Yone et
al., supra).
[0013] Other investigators have described mAbs specific for
recombinant human TNF which had neutralizing activity in vitro
(Liang, C-M. et al. Biochem. Biophys. Res. Comm. 137:847-854
(1986); Meager, A. et al., Hybridoma 6:305-311 (1987); Fendly et
al., Hybridoma 6:359-369 (1987); Bringman, T S et al., Hybridoma
6:489-507 (1987); Hirai, M. et al., J. Immunol. Meth. 96:57-62
(1987); Moller, A. et al. (Cytokine 2:162-169 (1990)). Some of
these mAbs were used to map epitopes of human TNF and develop
enzyme immunoassays (Fendly et al., supra; Hirai et al., supra;
Moller et al., supra) and to assist in the purification of
recombinant TNF (Bringman et al., supra). However, these studies do
not provide a basis for producing TNF neutralizing antibodies that
can be used for in vivo diagnostic or therapeutic uses in humans,
due to immunogenicity, lack of specificity and/or pharmaceutical
suitability.
[0014] Neutralizing antisera or mAbs to TNF have been shown in
mammals other than man to abrogate adverse physiological changes
and prevent death after lethal challenge in experimental
endotoxemia and bacteremia. This effect has been demonstrated,
e.g., in rodent lethality assays and in primate pathology model
systems (Mathison, J. C. et al., J. Clin. Invest. 81:1925-1937
(1988); Beutler, B. et al., Science 229:869-871 (1985); Tracey, K.
J. et al., Nature 330:662-664 (1987); Shimamoto, Y. et al.,
Immunol. Lett. 17:311-318 (1988); Silva, A. T. et al., J. Infect.
Dis. 162:421-427 (1990); Opal, S. M. et al., J. Infect. Dis.
161:1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30:279-292
(1990)).
[0015] To date, experience with anti-TNF mAb therapy in humans has
been limited but shows beneficial therapeutic results, e.g., in
arthritis and sepsis. See, e.g., Elliott, M. J. et al., Baillieres
Clin. Rheumatol. 9:633-52 (1995); Feldmann M, et al., Ann. N.Y.
Acad. Sci. USA 766:272-8 (1995); van der Poll, T. et al., Shock
3:1-12 (1995); Wherry et al., Crit. Care. Med. 21:S436-40 (1993);
Tracey K. J., et al., Crit. Care Med. 21:S415-22 (1993).
[0016] Mammalian development is dependent on both the proliferation
and differentiation of cells as well as programmed cell death which
occurs through apoptosis (Walker, et al., Methods Achiev. Exp.
Pathol. 13:18 (1988). Apoptosis plays a critical role in the
destruction of immune thymocytes that recognize self antigens.
Failure of this normal elimination process may play a role in
autoimmune diseases (Gammon et al., Immunology Today 12:193
(1991)).
[0017] Itoh et al. (Cell 66:233 (1991)) described a cell surface
antigen, Fas/CD23 that mediates apoptosis and is involved in clonal
deletion of T-cells. Fas is expressed in activated T-cells,
B-cells, neutrophils and in thymus, liver, heart and lung and ovary
in adult mice (Watanabe-Fukunaga et al., J. Immunol. 148:1274
(1992)) in addition to activated T-cells, B-cells, neutorophils. In
experiments where a monoclonal Ab is cross-linked to Fas, apoptosis
is induced (Yonehara et al., J. Exp. Med. 169:1747 (1989); Trauth
et al., Science 245:301 (1989)). In addition, there is an example
where binding of a monoclonal Ab to Fas is stimulatory to T-cells
under certain conditions (Alderson et al., J. Exp. Med. 178:2231
(1993)).
[0018] Fas antigen is a cell surface protein of relative MW of 45
Kd. Both human and murine genes for Fas have been cloned by
Watanabe-Fukunaga et al., (J. Immunol. 148:1274 (1992)) and Itoh et
al. (Cell 66:233 (1991)). The proteins encoded by these genes are
both transmembrane proteins with structural homology to the Nerve
Growth Factor/Tumor Necrosis Factor receptor superfamily, which
includes two TNF receptors, the low affinity Nerve Growth Factor
receptor and CD40, CD27, CD30, and OX40.
[0019] Recently the Fas ligand has been described (Suda et al.,
Cell 75:1169 (1993)). The amino acid sequence indicates that Fas
ligand is a type II transmembrane protein belonging to the TNF
family. Thus, the Fas ligand polypeptide comprises three main
domains: a short intracellular domain at the amino terminal end and
a longer extracellular domain at the carboxy terminal end,
connected by a hydrophobic transmembrane domain. Fas ligand is
expressed in splenocytes and thymocytes, consistent with T-cell
mediated cytotoxicity. The purified Fas ligand has a MW of 40
kD.
[0020] Recently, it has been demonstrated that Fas/Fas ligand
interactions are required for apoptosis following the activation of
T-cells (Ju et al., Nature 373:444 (1995); Brunner et al., Nature
373:441 (1995)). Activation of T-cells induces both proteins on the
cell surface. Subsequent interaction between the ligand and
receptor results in apoptosis of the cells. This supports the
possible regulatory role for apoptosis induced by Fas/Fas ligand
interaction during normal immune responses.
[0021] Accordingly, there is a need to provide cytokines similar to
TNF that are involved in pathological conditions. Such novel
cytokines could be used to make novel antibodies or other
antagonists that bind these TNF-like cytokines for therapy of
disorders related to TNF-like cytokines.
SUMMARY OF THE INVENTION
[0022] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a cytokine that is
structurally similar to TNF and related cytokines and is believed
to have similar biological effects and activities. This cytokine is
named Neutrokine-.alpha. and the invention includes
Neutrokine-.alpha. polypeptides having at least a portion of the
amino acid sequence in FIGS. 1A and B (SEQ ID NO:2) or amino acid
sequence encoded by the cDNA clone deposited in a bacterial host on
Oct. 22, 1996 assigned ATCC number 97768. The nucleotide sequence
determined by sequencing the deposited Neutrokine-.alpha. clone,
which is shown in FIGS. 1A and B (SEQ ID NO:1), contains an open
reading frame encoding a complete polypeptide of 285 amino acid
residues including an N-terminal methionine, a predicted
intracellular domain of about 46 amino acid residues, a predicted
transmembrane domain of about 26 amino acids, a predicted
extracellular domain of about 213 amino acids, and a deduced
molecular weight for the complete protein of about 31 kDa. As for
other type II transmembrane proteins, soluble forms of
Neutrokine-.alpha. include all or a portion of the extracellular
domain cleaved from the transmembrane domain and a polypeptide
comprising the complete Neutrokine-.alpha. polypeptide lacking the
transmembrane domain, i.e., the extracellular domain linked to the
intracellular domain.
[0023] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding a full-length Neutrokine-.alpha.
polypeptide having the complete amino acid sequence in FIGS. 1A and
B (SEQ ID NO:2) or as encoded by the cDNA clone contained in the
ATCC Deposit of Oct. 22, 1996 ATCC Number 97768; (b) a nucleotide
sequence encoding the predicted extracellular domain of the
Neutrokine-.alpha. polypeptide having the amino acid sequence at
positions 73 to 285 in FIGS. 1A and B (SEQ ID NO:2) or as encoded
by the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
1996; (c) a nucleotide sequence encoding a polypeptide comprising
the Neutrokine-.alpha. intracellular domain (amino acid residues
from about 1 to about 46 in FIGS. 1A and B (SEQ ID NO:2)) or as
encoded by the cDNA clone contained in ATCC No. 97768 deposited on
Oct. 22, 1996; (d) a nucleotide sequence encoding a polypeptide
comprising the Neutrokine-.alpha. transmembrane domain (amino acid
residues from about 47 to about 72 in FIGS. 1A and B (SEQ ID NO:2)
or as encoded by the cDNA clone contained in ATCC No. 97768
deposited on Oct. 22, 1996; (e) a nucleotide sequence encoding a
soluble Neutrokine a polypeptide having the extracellular and
intracellular domains but lacking the transmembrane domain; and (f)
a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d) or (e) above.
[0024] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e) or (f) above, or a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e) or (f)
above. This polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues. An additional nucleic acid embodiment of the invention
relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of an
epitope-bearing portion of a Neutrokine-.alpha. polypeptide having
an amino acid sequence in (a), (b), (c), (d) or (e) above. A
further nucleic acid embodiment of the invention relates to an
isolated nucleic acid molecule comprising a polynucleotide which
encodes the amino acid sequence of a Neutrokine-.alpha. polypeptide
having an amino acid sequence which contains at least one amino
acid substitution, but not more than 50 amino acid substitutions,
even more preferably, not more than 40 amino acid substitutions,
still more preferably, not more than 30 amino acid substitutions,
and still even more preferably, not more than 20 amino acid
substitutions. Of course, in order of ever-increasing preference,
it is highly preferable for a polynucleotide which encodes the
amino acid sequence of a Neutrokine-.alpha. polypeptide to have an
amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5,
4, 3, 2 or 1 amino acid substitutions. Conservative substitutions
are preferable.
[0025] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of Neutrokine-.alpha. polypeptides or
peptides by recombinant techniques.
[0026] The invention further provides an isolated
Neutrokine-.alpha. polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
of the full-length Neutrokine-.alpha. polypeptide having the
complete amino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2)
or as encoded by the cDNA clone contained in ATCC No. 97768
deposited on Oct. 22, 1996; (b) the amino acid sequence of the
predicted extracellular domain of the Neutrokine-.alpha.
polypeptide having the amino acid sequence at positions 73 to 285
in FIGS. 1A and B (SEQ ID NO:2) or as encoded by the cDNA clone
contained in ATCC No. 97768 deposited on Oct. 22, 1996; (c) the
amino acid sequence of the Neutrokine-.alpha. intracellular domain
(amino acid residues from about 1 to about 46 in FIGS. 1A and B
(SEQ ID NO:2)) or as encoded by the cDNA clone contained in ATCC
No. 97768 deposited on Oct. 22, 1996; (d) the amino acid sequence
of the Neutrokine-.alpha. transmembrane domain (amino acid residues
from about 47 to about 72 in FIGS. 1A and B (SEQ ID NO:2)) or as
encoded by the cDNA clone contained in ATCC No. 97768 deposited on
Oct. 22, 1996; and (e) the amino acid sequence of the soluble
Neutrokine-.alpha. polypeptide having the extracellular and
intracellular domains but lacking the transmembrane domain, wherein
each of these domains is defined above.
[0027] The polypeptides of the present invention also include
polypeptides having an amino acid sequence with at least 90%
similarity, and more preferably at least 95% similarity to those
described in (a), (b), (c), (d) or (e) above, as well as
polypeptides having an amino acid sequence at least 80% identical,
more preferably at least 90% identical, and still more preferably
95%, 96%, 97%, 98% or 99% identical to those above.
[0028] An additional embodiment of this aspect of the invention
relates to a peptide or polypeptide which has the amino acid
sequence of an epitope-bearing portion of a Neutrokine-.alpha.
polypeptide having an amino acid sequence described in (a), (b),
(c), (d) or (e) above. Peptides or polypeptides having the amino
acid sequence of an epitope-bearing portion of a Neutrokine-.alpha.
polypeptide of the invention include portions of such polypeptides
with at least six or seven, preferably at least nine, and more
preferably at least about 30 amino acids to about 50 amino acids,
although epitope-bearing polypeptides of any length up to and
including the entire amino acid sequence of a polypeptide of the
invention described above also are included in the invention. In
another embodiment, the invention provides an isolated antibody
that binds specifically to an polypeptide having an amino acid
sequence described in (a), (b), (c), (d) or (e) above.
[0029] The invention further provides methods for isolating
antibodies that bind specifically to a Neutrokine-.alpha.
polypeptide having an amino acid sequence as described herein. Such
antibodies are useful diagnostically or therapeutically as
described below.
[0030] The invention also provides for pharmaceutical compositions
comprising soluble Neutrokine-.alpha. polypeptides, particularly
human Neutrokine-.alpha. polypeptides, which may be employed, for
instance, to treat tumor and tumor metastasis, infections by
bacteria, viruses and other parasites, immunodeficiencies,
inflammatory diseases, lymphadenopathy, autoimmune diseases, graft
versus host disease and to stimulate peripheral tolerance, destroy
some transformed cell lines, mediate cell activation and
proliferation, and are functionally linked as primary mediators of
immune regulation and inflammatory responses.
[0031] The invention further provides compositions comprising a
Neutrokine-.alpha. polynucleotide or a Neutrokine-.alpha.
polypeptide for administration to cells in vitro, to cells ex vivo
and to cells in vivo, or to a multicellular organism. In certain
particularly preferred embodiments of this aspect of the invention,
the compositions comprise a Neutrokine-.alpha. polynucleotide for
expression of a Neutrokine-.alpha. polypeptide in a host organism
for treatment of disease. Particularly preferred in this regard is
expression in a human patient for treatment of a dysfunction
associated with aberrant endogenous activity of a
Neutrokine-.alpha. gene.
[0032] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a cellular
response induced by Neutrokine-.alpha. which involves contacting
cells which express Neutrokine-.alpha. with the candidate compound,
assaying a cellular response, and comparing the cellular response
to a standard cellular response, the standard being assayed when
contact is made in absence of the candidate compound; whereby, an
increased cellular response over the standard indicates that the
compound is an agonist and a decreased cellular response over the
standard indicates that the compound is an antagonist.
[0033] In another aspect, a method for identifying
Neutrokine-.alpha. receptors is provided, as well as a screening
assay for agonists and antagonists using such receptors. This assay
involves determining the effect a candidate compound has on
Neutrokine-.alpha. binding to the Neutrokine-.alpha. receptor. In
particular, the method involves contacting a Neutrokine-.alpha.
receptor with a Neutrokine-.alpha. polypeptide and a candidate
compound and determining whether Neutrokine-.alpha. polypeptide
binding to the Neutrokine-.alpha. receptor is increased or
decreased due to the presence of the candidate compound. The
antagonists may be employed to prevent septic shock, inflammation,
cerebral malaria, activation of the HIV virus, graft-host
rejection, bone resorption, rheumatoid arthritis and cachexia
(wasting or malnutrition)
[0034] The present inventors have discovered that
Neutrokine-.alpha. is expressed not only in neutrophils, but also
in kidney, lung, peripheral leukocyte, bone marrow, T cell
lymphoma, B cell lymphoma, activated T cells, stomach cancer,
smooth muscle, macrophages, and cord blood tissue. For a number of
disorders of these tissues and cells, such as tumor and tumor
metastasis, infection of bacteria, viruses and other parasites,
immunodeficiencies, septic shock, inflammation, cerebral malaria,
activation of the HIV virus, graft-host rejection, bone resorption,
rheumatoid arthritis and cachexia (wasting or malnutrition, it is
believed that significantly higher or lower levels of
Neutrokine-.alpha. gene expression can be detected in certain
tissues (e.g., bone marrow) or bodily fluids (e.g., serum, plasma,
urine, synovial fluid or spinal fluid) taken from an individual
having such a disorder, relative to a "standard" Neutrokine-.alpha.
gene expression level, i.e., the Neutrokine-.alpha. expression
level in tissue or bodily fluids from an individual not having the
disorder. Thus, the invention provides a diagnostic method useful
during diagnosis of a disorder, which involves: (a) assaying
Neutrokine-.alpha. gene expression level in cells or body fluid of
an individual; (b) comparing the Neutrokine-.alpha. gene expression
level with a standard Neutrokine-.alpha. gene expression level,
whereby an increase or decrease in the assayed Neutrokine-.alpha.
gene expression level compared to the standard expression level is
indicative of a disorder.
[0035] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of
Neutrokine-.alpha. activity in the body comprising administering to
such an individual a composition comprising a therapeutically
effective amount of an isolated Neutrokine-.alpha. polypeptide of
the invention or an agonist thereof.
[0036] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
Neutrokine-.alpha. activity in the body comprising, administering
to such an individual a composition comprising a therapeutically
effective amount of an Neutrokine-.alpha. antagonist. Preferred
antagonists for use in the present invention are
Neutrokine-.alpha.-specific antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0037] FIGS. 1A and B show the nucleotide (SEQ ID NO:1) and deduced
amino acid (SEQ ID NO:2) sequences of the Neutrokine-.alpha.
protein. Amino acids 1 to 46 represent the intracellular domain,
amino acids 47 to 72 the transmembrane domain (the underlined
sequence), and amino acids 73 to 285, the extracellular domain (the
remaining sequence).
[0038] FIGS. 2A-C show the regions of identity between the amino
acid sequences of the Neutrokine-.alpha. protein (SEQ ID NO:2) and
TNF-.alpha. (SEQ ID NO:3), TNF-.beta. (lymphotoxin; SEQ ID NO:4)
and FAS ligand (SEQ ID NO:5), determined by the "Megalign" routine
which is part of the computer program called "DNAStar", Amino acid
residues that match the consensus sequence are shaded.
[0039] FIG. 3 shows an analysis of the Neutrokine-.alpha. amino
acid sequence (SEQ ID NO:2). Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible
regions; antigenic index and surface probability are shown. In the
"Antigenic Index--Jameson-Wolf" graph, the indicate location of the
highly antigenic regions of the Neutrokine-.alpha. protein, i.e.,
regions from which epitope-bearing peptides of the invention may be
obtained.
[0040] FIGS. 4A-C show the alignment of the Neutrokine-.alpha.
nucleotide sequence (SEQ ID NO:1) determined from the human cDNA
deposited in ATCC No. 97768 deposited on Oct. 22, 1996 with related
human cDNA clones of the invention which have been designated
HSOAD55 (SEQ ID NO:7), HSLAH84 (SEQ ID NO:8) and HLTBM08 (SEQ ID
NO:9).
DETAILED DESCRIPTION
[0041] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding Neutrokine-.alpha.
polypeptide having the amino acid sequence shown in FIGS. 1A and B
(SEQ ID NO:2), which was determined by sequencing a cloned cDNA
Neutrokine-.alpha.. The nucleotide sequence shown in FIGS. 1A and B
(SEQ ID NO:1) was obtained by sequencing the HNEDU15 clone, which
was deposited on Oct. 22, 1996 at the American Type Culture
Collection, 10801 University Drive, Manassas, Va. 20110-2209 and
assigned ATCC Deposit No. 97768. The deposited clone is contained
in the pBluescript SK(-) plasmid (Stratagene, La Jolla,
Calif.).
[0042] The Neutrokine-.alpha. protein of the present invention
shares sequence homology with the translation product of the human
mRNAs for TNF-.alpha., TNF-.beta. and Fas ligand (FIGS. 2A-C). As
noted above, TNF-.alpha. is thought to be an important cytokine
that plays a role in cytotoxicity, necrosis, apoptosis,
costimulation, proliferation, lymph node formation, immunoglobulin
class switch, differentiation, antiviral activity, regulation of
adhesion molecules and other cytokines and growth factors.
Nucleic Acid Molecules
[0043] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90%
identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced
DNA molecule. The actual sequence can be more precisely determined
by other approaches including manual DNA sequencing methods well
known in the art. As is also known in the art, a single insertion
or deletion in a determined nucleotide sequence compared to the
actual sequence will cause a frame shift in translation of the
nucleotide sequence such that the predicted amino acid sequence
encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the
sequenced DNA molecule, beginning at the point of such an insertion
or deletion.
[0044] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0045] Using the information provided herein, such as the
nucleotide sequence in FIGS. 1A and B, a nucleic acid molecule of
the present invention encoding a Neutrokine-.alpha. polypeptide may
be obtained using standard cloning and screening procedures, such
as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in FIGS. 1A and B (SEQ ID NO:1) was discovered in a cDNA library
derived from neutrophils. Expressed sequence tags corresponding to
a portion of the Neutrokine-.alpha. cDNA were also found in kidney,
lung, peripheral leukocyte, bone marrow, T cell lymphoma, B cell
lymphoma, activated T cells, stomach cancer, smooth muscle,
macrophages, and cord blood tissue.
[0046] The Neutrokine-.alpha. gene contains an open reading frame
encoding a protein of about 285 amino acid residues, an
intracellular domain of about 46 amino acids (amino acid residues
from about 1 to about 46 in FIG. 1 (SEQ ID NO:2)), a transmembrane
domain of about 26 amino acids (underlined amino acid residues from
about 47 to about 72 in FIGS. 1A and B (SEQ ID NO:2)), an
extracellular domain of about 213 amino acids (amino acid residues
from about 73 to about 285 in FIGS. 1A and B (SEQ ID NO:2)); and a
deduced molecular weight of about 31 kDa. The Neutrokine-.alpha.
protein shown in FIGS. 1A and B (SEQ ID NO: 2) is about 20% similar
and about 10% identical to human TNF-.alpha. which can be accessed
on GenBank as Accession No. 339764.
[0047] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, the actual
complete Neutrokine-.alpha. polypeptide encoded by the deposited
cDNA, which comprises about 285 amino acids, may be somewhat
shorter. In particular, the determined Neutrokine-.alpha. coding
sequence contains a second methionine codon which may serve as an
alternative start codon for translation of the open reading frame,
at nucleotide positions 210-213 in FIGS. 1A and B (SEQ ID NO:1).
More generally, the actual open reading frame may be anywhere in
the range of .+-.20 amino acids, more likely in the range of .+-.10
amino acids, of that predicted from either the first or second
methionine codon from the N-terminus shown in FIGS. 1A and B (SEQ
ID NO:1). It will further be appreciated that, depending on the
analytical criteria used for identifying various functional
domains, the exact "address" of the extracelluar, intracelluar and
transmembrane domains of the Neutrokine-.alpha. polypeptide may
differ slightly. For example, the exact location of the
Neutrokine-.alpha. extracellular domain in FIGS. 1A and B (SEQ ID
NO:2) may vary slightly (e.g., the address may "shift" by about 1
to about 20 residues, more likely about 1 to about 5 residues)
depending on the criteria used to define the domain. In this case,
the ends of the transmembrane domain and the beginning of the
extracellular domain were predicted on the basis of the
identification of the hydrophobic amino acid sequence in the above
indicated positions, as shown in FIG. 3. In any event, as discussed
further below, the invention further provides polypeptides having
various residues deleted from the N-terminus of the complete
polypeptide, including polypeptides lacking one or more amino acids
from the N-terminus of the extracellular domain described herein,
which constitute soluble forms of the extracellular domain of the
Neutrokine-.alpha. protein.
[0048] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0049] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0050] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) with
an initiation codon at positions 147-149 of the nucleotide sequence
shown in FIGS. 1A and B (SEQ ID NO:1). In addition, isolated
nucleic acid molecules of the invention include DNA molecules which
comprise a sequence substantially different from those described
above but which, due to the degeneracy of the genetic code, still
encode the Neutrokine-.alpha. protein. Of course, the genetic code
is well known in the art. Thus, it would be routine for one skilled
in the art to generate the degenerate variants described above. In
another aspect, the invention provides isolated nucleic acid
molecules encoding the Neutrokine-.alpha. polypeptide having an
amino acid sequence encoded by the cDNA contained in the plasmid
deposited on Oct. 22, 1996. Preferably, this nucleic acid molecule
will comprise a sequence encoding the extracellular domain of the
polypeptide encoded by the above-described deposited cDNA
clone.
[0051] The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in FIGS. 1A and B
(SEQ ID NO:1) or the nucleotide sequence of the Neutrokine-.alpha.
cDNA contained in the above-described deposited clone, or a nucleic
acid molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping, by in situ hybridization with
chromosomes, and for detecting expression of the Neutrokine-.alpha.
gene in human tissue, for instance, by Northern blot analysis.
[0052] The invention also provides nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1
which have been determined from the following related cDNA clones:
HSOAD55 (SEQ ID NO:7), HSLAH84 (SEQ ID NO:8), and HLTBM08 (SEQ ID
NO:9).
[0053] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. In particular, the invention provides a
polynucleotide having a nucleotide sequence representing the
portion of SEQ ID NO:1 which consists of positions 147-1001 of SEQ
ID NO:1.
[0054] Further, the invention includes a polynucleotide comprising
a sequence at least 95% identical to any portion of at least about
30 contiguous nucleotides, preferably at least about 50
nucleotides, of the sequence from nucleotide 1 to nucleotide 1082
in FIGS. 1A and B (SEQ ID NO:1), preferably excluding the
nucleotide sequences determined from the abovelisted cDNA clones
and the nucleotide sequences from nucleotide 797 to 1082, 810 to
1082, and 346 to 542. More preferably, the invention includes a
polynucleotide comprising nucleotide residues 147-500, 147-450,
147-400, 147, 350, 200-500, 200-450, 200-400, 200-350, 250-500,
250-450, 250-400, 250-350, 300-500, 300-450, 300-400, 300-350,
350-750, 350-700, 350-650, 350-600, 350-550, 400-750, 400-700,
400-650, 400-600, 400-550, 425-750, 425-700, 425-650, 425-600,
425-550, 450-1020, 450-1001, 450-950, 450-900, 450-850, 450-800,
450-775, 500-1001, 500-950, 500-900, 500-850, 500-800, 500-775,
550-1001, 550-950, 550-900, 550-850, 550-800, 550-775, 600-1001,
600-950, 600-900, 600-850, 600-800, 600-775, 650-1001, 650-950,
650-900, 650-850, 650-800, 650-775, 700-1001, 700-950, 700-900,
700-850, 700-800, 700-775, 825-1082, 850-1082, 875-1082, 900-1082,
925-1082, 950-1082, 975-1082, 1000-1082, 1025-1082, and
1050-1082.
[0055] More generally, by a fragment of an isolated nucleic acid
molecule having the nucleotide sequence of the deposited cDNA or
the nucleotide sequence shown in FIGS. 1A and B (SEQ ID NO:1) is
intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which are
useful as diagnostic probes and primers as discussed herein. Of
course, larger fragments 50-300 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in FIGS. 1A and B (SEQ ID NO:1). By a fragment at
least 20 nt in length, for example, is intended fragments which
include 20 or more contiguous bases from the nucleotide sequence of
the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A
and B (SEQ ID NO:1). Preferred nucleic acid fragments of the
present invention include nucleic acid molecules encoding
epitope-bearing portions of the Neutrokine-.alpha. polypeptide as
identified in FIGS. 1A and B and described in more detail
below.
[0056] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATCC No.
97768 deposited on Oct. 22, 1996. By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium cirate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0057] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below.
[0058] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A
and B (SEQ ID NO:1)). Of course, a polynucleotide which hybridizes
only to a poly A sequence (such as the 3' terminal poly(A) tract of
the Neutrokine-.alpha. cDNA shown in FIGS. 1A and B (SEQ ID NO:1)),
or to a complementary stretch of T (or U) residues, would not be
included in a polynucleotide of the invention used to hybridize to
a portion of a nucleic acid of the invention, since such a
polynucleotide would hybridize to any nucleic acid molecule
containing a poly(A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0059] As indicated, nucleic acid molecules of the present
invention which encode a Neutrokine-.alpha. polypeptide may
include, but are not limited to those encoding the amino acid
sequence of the extracellular domain of the polypeptide, by itself;
and the coding sequence for the extracellular domain of the
polypeptide and additional sequences, such as those encoding the
intracellular and transmembrane domain sequences, or a pre-, or
pro- or prepro-protein sequence; the coding sequence of the
extracellular domain of the polypeptide, with or without the
aforementioned additional coding sequences.
[0060] Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example, ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0061] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. The
"HA" tag is another peptide useful for purification which
corresponds to an epitope derived from the influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984). As discussed below, other such fusion proteins include the
Neutrokine-.alpha. fused to Fc at the N- or C-terminus.
[0062] Variant and Mutant Polynucleotides
[0063] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the Neutrokine-.alpha. protein.
Variants may occur naturally, such as a natural allelic variant. By
an "allelic variant" is intended one of several alternate forms of
a gene occupying a given locus on a chromosome of an organism.
Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0064] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the Neutrokine-.alpha. protein or portions thereof.
Also especially preferred in this regard are conservative
substitutions.
[0065] Most highly preferred are nucleic acid molecules encoding
the extracellular domain of the protein having the amino acid
sequence shown in FIGS. 1A and B (SEQ ID NO:2) or the extracellular
domain of the Neutrokine-.alpha. amino acid sequence encoded by the
deposited cDNA clone. Further embodiments include an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide
selected from the group consisting of: (a) a nucleotide sequence
encoding the Neutrokine-.alpha. polypeptide having the complete
amino acid sequence in FIGS. 1A and B (SEQ ID NO:2); (b) a
nucleotide sequence encoding the predicted extracellular domain of
the Neutrokine-.alpha. polypeptide having the amino acid sequence
at positions 73-285 in FIGS. 1A and B (SEQ ID NO:2); (c) a
nucleotide sequence encoding the Neutrokine-.alpha. polypeptide
having the complete amino acid sequence encoded by the cDNA clone
contained in ATCC No. 97768 deposited on Oct. 22, 1996; (d) a
nucleotide sequence encoding the extracellular domain of the
Neutrokine-.alpha. polypeptide having the amino acid sequence
encoded by the cDNA clone contained in ATCC No. 97768 deposited on
Oct. 22, 1996; and (e) a nucleotide sequence complementary to any
of the nucleotide sequences in (a), (b), (c) or (d) above.
[0066] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a Neutrokine-.alpha. polypeptide is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence encoding the Neutrokine-.alpha.
polypeptide. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence.
[0067] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in FIGS. 1A and B or to
the nucleotides sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0068] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIGS. 1A and B (SEQ ID NO:1) or to
the nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having Neutrokine-.alpha.
activity. This is because even where a particular nucleic acid
molecule does not encode a polypeptide having Neutrokine-.alpha.
activity, one of skill in the art would still know how to use the
nucleic acid molecule, for instance, as a hybridization probe or a
polymerase chain reaction (PCR) primer. Uses of the nucleic acid
molecules of the present invention that do not encode a polypeptide
having Neutrokine-.alpha. activity include, inter alia, (1)
isolating the Neutrokine-.alpha. gene or allelic variants thereof
in a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal spreads to provide precise chromosomal
location of the Neutrokine-.alpha. gene, as described in Verma et
al., Human Chromosomes: A Manual of Basic Techniques, Pergamon
Press, New York (1988); and Northern Blot analysis for detecting
Neutrokine-.alpha. mRNA expression in specific tissues.
[0069] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIGS. 1A and B (SEQ ID NO:1) or to
the nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having Neutrokine-.alpha. protein activity. By
"a polypeptide having Neutrokine-.alpha. activity" is intended
polypeptides exhibiting activity similar, but not necessarily
identical, to an activity of the extracellular domain or of the
full-length Neutrokine-.alpha. protein of the invention, as
measured in a particular biological assay. For example, the
Neutrokine-.alpha. protein of the present invention modulates cell
proliferation, cytotoxicity and cell death. An in vitro cell
proliferation, cytotoxicity and cell death assay for measuring the
effect of a protein on certain cells can be performed by using
reagents well known and commonly available in the art for detecting
cell replication and/or death. For instance, numerous such assays
for TNF-related protein activities are described in the various
references in the Background section of this disclosure, above.
Briefly, such an assay involves collecting human or animal (e.g.,
mouse) cells and mixing with (1) transfected host cell-supernatant
containing Neutrokine-.alpha. protein (or a candidate polypeptide)
or (2) nontransfected host cell-supernatant control, and measuring
the effect on cell numbers or viability after incubation of certain
period of time. Such cell proliferation modulation activities as
can be measure in this type of assay are useful for treating tumor,
tumor metastasis, infections, autoimmune diseases inflammation and
other immune-related diseases.
[0070] Neutrokine-.alpha. modulates cell proliferation and
differentiation in a dose-dependent manner in the above-described
assay. Thus, "a polypeptide having Neutrokine a protein activity"
includes polypeptides that also exhibit any of the same cell
modulatory (particularly immunomodulatory) activities in the
above-described assays in a dose-dependent manner. Although the
degree of dose-dependent activity need not be identical to that of
the Neutrokine-.alpha. protein, preferably, "a polypeptide having
Neutrokine-.alpha. protein activity" will exhibit substantially
similar dose-dependence in a given activity as compared to the
Neutrokine-.alpha. protein (i.e., the candidate polypeptide will
exhibit greater activity or not more than about 25-fold less and,
preferably, not more than about tenfold less activity relative to
the reference Neutrokine-.alpha. protein).
[0071] Like other members of TNF family, Neutrokine-.alpha.
exhibits activity on leukocytes including for example monocytes,
lymphocytes and neutrophils. For this reason Neutrokine-.alpha. is
active in directing the proliferation, differentiation and
migration of these cell types. Such activity is useful for immune
enhancement or suppression, myeloprotection, stem cell
mobilization, acute and chronic inflammatory control and treatment
of leukemia. Assays for measuring such activity are known in the
art. For example, see Peters et al., Immun. Today 17:273 (1996);
Young et al., J. Exp. Med. 182:1111 (1995); Caux et al., Nature
390:258 (1992); and Santiago-Schwarz et al., Adv. Exp. Med. Biol.
378:7 (1995)."
[0072] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIG. 1 (SEQ ID NO:1) will encode a polypeptide "having
Neutrokine-.alpha. protein activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having Neutrokine-.alpha. protein activity. This is
because the skilled artisan is fully aware of amino acid
substitutions that are either less likely or not likely to
significantly effect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid), as
further described below. A further nucleic acid embodiment of the
invention relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of a
Neutrokine-.alpha. polypeptide having an amino acid sequence which
contains at least one conservative amino acid substitution, but not
more than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably, not more than 30 conservative amino acid
substitutions, and still even more preferably, not more than 20
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a
polynucleotide which encodes the amino acid sequence of a
Neutrokine-.alpha. polypeptide to have an amino acid sequence which
contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative
amino acid substitutions.
[0073] Vectors and Host Cells
[0074] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of Neutrokine-.alpha. polypeptides or fragments
thereof by recombinant techniques. The vector may be, for example,
a phage, plasmid, viral or retroviral vector. Retroviral vectors
may be replication competent or replication defective. In the
latter case, viral propagation generally will occur only in
complementing host cells. The polynucleotides may be joined to a
vector containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as
a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it may be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0075] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the
extracellular domain of the transcripts expressed by the constructs
will preferably include a translation initiating at the beginning
and a termination codon (UAA, UGA or UAG) appropriately positioned
at the end of the polypeptide to be translated.
[0076] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0077] Among vectors preferred for use in bacteria include pHE4-5
(ATCC Accession No. 209311; and variations thereof), pQE70, pQE60
and pQE-9, available from QIAGEN, Inc., supra; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic
vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from
Pharmacia. Other suitable vectors will be readily apparent to the
skilled artisan.
[0078] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0079] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to stabilize
and purify proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5 has been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0080] The Neutrokine-.alpha. protein can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
[0081] Neutrokine-.alpha. Polypeptides and Fragments
[0082] The invention further provides an isolated
Neutrokine-.alpha. polypeptide having the amino acid sequence
encoded by the deposited cDNA, or the amino acid sequence in FIGS.
1A and B (SEQ ID NO:2), or a peptide or polypeptide comprising a
portion of the above polypeptides.
[0083] Variant and Mutant Polypeptides
[0084] To improve or alter the characteristics of
Neutrokine-.alpha. polypeptides, protein engineering may be
employed. Recombinant DNA technology known to those skilled in the
art can be used to create novel mutant proteins or "muteins
including single or multiple amino acid substitutions, deletions,
additions or fusion proteins. Such modified polypeptides can show,
e.g., enhanced activity or increased stability. In addition, they
may be purified in higher yields and show better solubility than
the corresponding natural polypeptide, at least under certain
purification and storage conditions.
[0085] N-Terminal and C-Terminal Deletion Mutants
[0086] For instance, for many proteins, including the extracellular
domain or the mature form(s) of a secreted protein, it is known in
the art that one or more amino acids may be deleted from the
N-terminus or C-terminus without substantial loss of biological
function. For instance, Ron et al., J. Biol. Chem., 268:2984-2988
(1993) reported modified KGF proteins that had heparin binding
activity even if 3, 8, or 27 amino-terminal amino acid residues
were missing.
[0087] In the present case, since the protein of the invention is a
member of the TNF polypeptide family, deletions of N-terminal amino
acids up to the Gly (G) residue at position 191 in FIGS. 1A and B
(SEQ ID NO:2) may retain some biological activity such as
cytotoxicity to appropriate target cells. Polypeptides having
further N-terminal deletions including the Gly (G) residue would
not be expected to retain such biological activities because it is
known that this residue in TNF-related polypeptides is in the
beginning of the conserved domain required for biological
activities. However, even if deletion of one or more amino acids
from the N-terminus of a protein results in modification of loss of
one or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or extracellular domain of the protein
generally will be retained when less than the majority of the
residues of the complete or extracellular domain of the protein are
removed from the N-terminus. Whether a particular polypeptide
lacking N-terminal residues of a complete protein retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0088] Accordingly, the present invention further provides
polypeptides having one or more residues from the amino terminus of
the amino acid sequence of the Neutrokine-.alpha. shown in FIG. 1
(SEQ ID NO:2), up to the glycine residue at position 191 (Gly-191
residue from the amino terminus), and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides having the amino acid sequence of residues n-285 of
SEQ ID NO:2, where n is an integer in the range of 2-190 and 191 is
the position of the first residue from the N-terminus of the
complete Neutrokine-.alpha. polypeptide (shown in SEQ ID NO:2)
believed to be required for activity of the Neutrokine-.alpha.
protein. More in particular, the invention provides polynucleotides
encoding polypeptides having the amino acid sequence of residues
2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285,
11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285,
19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285,
27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285,
35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285,
43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285,
51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285,
59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285,
67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285,
75-285, 76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285,
83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285,
91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285,
99-285, 100-285, 101-285, 102-285, 103-285, 104-285, 105-285,
106-285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285,
113-285, 114-285, 115-285, 116-285, 117-285, 118-285, 119-285,
120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285,
127-285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285,
134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285,
141-285, 142-285, 143-285, 144-285, 145-285, 146-285, 147-285,
148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-285,
155-285, 156-285, 157-285, 158-285, 159-285, 160-285, 161-285,
162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285,
169-285, 170-285, 171-285, 172-285, 173-285, 174-285, 175-285,
176-285, 177-285, 178-285, 179-285, 180-285, 181-285, 182-285,
183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and
190-285 of SEQ ID NO:2. Polynucleotides encoding these polypeptides
also are provided.
[0089] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein
(Dobeli et al., J. Biotechnology 7:199-216 (1988). Since the
present protein is a member of the TNF polypeptide family,
deletions of C-terminal amino acids up to the leucine residue at
position 284 are expected to retain most if not all biological
activity such as receptor binding and modulation of cell
replication. Polypeptides having deletions of up to about 10
additional C-terminal residues (i.e., up to the glycine residue at
position 273) also may retain some activity such as receptor
binding, although such polypeptides would lack a portion of the
conserved TNF domain beginning at about Leu-284. However, even if
deletion of one or more amino acids from the C-terminus of a
protein results in modification of loss of one or more biological
functions of the protein, other biological activities may still be
retained. Thus, the ability of the shortened protein to induce
and/or bind to antibodies which recognize the complete or mature
protein generally will be retained when less than the majority of
the residues of the complete or mature protein are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art.
[0090] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the Neutrokine-.alpha. shown in FIGS.
1A and B (SEQ ID NO:2), up to the glycine residue at position 274
(Gly-274) and polynucleotides encoding such polypeptides. In
particular, the present invention provides polypeptides having the
amino acid sequence of residues 1-m of the amino acid sequence in
SEQ ID NO:2, where m is any integer in the range of 274 to 284.
More in particular, the invention provides polynucleotides encoding
polypeptides having the amino acid sequence of residues 1-274,
1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283 and
1-284 of SEQ ID NO:2. Polynucleotides encoding these polypeptides
also are provided.
[0091] Also provided are polypeptides having one or more amino
acids deleted from both the amino and the carboxyl termini, which
may be described generally as having residues n-m of SEQ ID NO:2,
where n and m are integers as described above. Also included are a
nucleotide sequence encoding a polypeptide consisting of a portion
of the complete Neutrokine-.alpha. amino acid sequence encoded by
the cDNA clone contained in ATCC No. 97768 deposited on Oct. 22,
1996 where this portion excludes from 1 to 190 amino acids from the
amino terminus or from 1 to 11 amino acids from the C-terminus of
the complete amino acid sequence (or any combination of these
N-terminal and C-terminal deletions) encoded by the cDNA clone in
the deposited clone. Polynucleotides encoding all of the above
deletion polypeptides also are provided.
[0092] Other Mutants
[0093] In addition to terminal deletion forms of the protein
discussed above, it will be recognized by one of ordinary skill in
the art that some amino acid sequences of the Neutrokine-.alpha.
polypeptide can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0094] Thus, the invention further includes variations of the
Neutrokine-.alpha. polypeptide which show substantial
Neutrokine-.alpha. polypeptide activity or which include regions of
Neutrokine-.alpha. protein such as the protein portions discussed
below. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions selected according to general rules
known in the art so as have little effect on activity. For example,
guidance concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main approaches for studying the
tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0095] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0096] Thus, the fragment, derivative or analog of the polypeptide
of FIGS. 1A and B (SEQ ID NO:2), or that encoded by the deposited
cDNA, may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
extracellular domain of the polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or (iv) one in
which the additional amino acids are fused to the extracellular
domain of the polypeptide, such as an IgG Fc fusion region peptide
or leader or secretory sequence or a sequence which is employed for
purification of the extracellular domain of the polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein
[0097] Thus, the Neutrokine-.alpha. of the present invention may
include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation. As
indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0098] Amino acids in the Neutrokine-.alpha. protein of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
receptor binding or in vitro or in vitro proliferative
activity.
[0099] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
[0100] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors. For example,
Ostade et al., Nature 361:266-268 (1993) describes certain
mutations resulting in selective binding of TNF-.alpha. to only one
of the two known types of TNF receptors. Since Neutrokine-.alpha.
is a member of the TNF polypeptide family, mutations similar to
those in TNF-.alpha. are likely to have similar effects in
Neutrokine-.alpha..
[0101] Sites that are critical for ligand-receptor binding can also
be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science
255:306-312 (1992)). Since Neutrokine-.alpha. is a member of the
TNF-related protein family, to modulate rather than completely
eliminate biological activities of Neutrokine-.alpha., preferably
mutations are made in sequences encoding amino acids in the TNF
conserved domain, i.e., in positions 191-284 of FIGS. 1A and B (SEQ
ID NO:2), more preferably in residues within this region which are
not conserved in all members of the TGF family. By making a
specific mutation in Neutrokine-.alpha. in the position where such
a conserved amino acid is typically found in related TNFs,
Neutrokine-.alpha. will act as an antagonist, thus possessing
angiogenic activity. Accordingly, polypeptides of the present
invention include Neutrokine-.alpha. mutants. Such
Neutrokine-.alpha. mutants are comprised of the full-length or
preferably the extracellular domain of the Neutrokine-.alpha. amino
acid sequence shown in FIGS. 1A and B (SEQ ID NO:2). Also forming
part of the present invention are isolated polynucleotides
comprising nucleic acid sequences which encode the above
Neutrokine-.alpha. mutants.
[0102] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the
Neutrokine-.alpha. polypeptide can be substantially purified by the
one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
[0103] The polypeptides of the present invention include the
complete polypeptide encoded by the deposited cDNA including the
intracellular, transmembrane and extracellular domains of the
polypeptide encoded by the deposited cDNA, the extracellular domain
minus the intracellular and transmembrane domains of the protein,
the complete polypeptide of FIGS. 1A and B (SEQ ID NO:2), the
extracellular domain of FIGS. 1A and B (SEQ ID NO:2) minus the
intracellular and transmembrane domains, as well as polypeptides
which have at least 90% similarity, more preferably at least 95%
similarity, and still more preferably at least 96%, 97%, 98% or 99%
similarity to those described above.
[0104] Further polypeptides of the present invention include
polypeptides at least 80% identical, more preferably at least 90%
or 95% identical, still more preferably at least 96%, 97%, 98% or
99% identical to the polypeptide encoded by the deposited cDNA or
to the polypeptide of FIGS. 1A and B (SEQ ID NO:2), and also
include portions of such polypeptides with at least 30 amino acids
and more preferably at least 50 amino acids.
[0105] A further embodiment of the invention relates to a peptide
or polypeptide which comprises the amino acid sequence of a
Neutrokine-.alpha. polypeptide having an amino acid sequence which
contains at least one conservative amino acid substitution, but not
more than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably, not more than 30 conservative amino acid
substitutions, and still even more preferably, not more than 20
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a peptide
or polypeptide to have an amino acid sequence which comprises the
amino acid sequence of a Neutrokine-.alpha. polypeptide, which
contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2
or 1 conservative amino acid substitutions.
[0106] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0107] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
Neutrokine-.alpha. polypeptide is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of the Neutrokine-.alpha. polypeptide. In other words, to
obtain a polypeptide having an amino acid sequence at least 95%
identical to a reference amino acid sequence, up to 5% of the amino
acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up
to 5% of the total amino acid residues in the reference sequence
may be inserted into the reference sequence. These alterations of
the reference sequence may occur at the amino or carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0108] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2) or to
the amino acid sequence encoded by deposited cDNA clone can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0109] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0110] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting
Neutrokine-.alpha. protein expression as described below or as
agonists and antagonists capable of enhancing or inhibiting
Neutrokine-.alpha. protein function. Further, such polypeptides can
be used in the yeast two-hybrid system to "capture"
Neutrokine-.alpha. protein binding proteins which are also
candidate agonists and antagonists according to the present
invention. The yeast two hybrid system is described in Fields and
Song, Nature 340:245-246 (1989).
[0111] Epitope-Bearing Portions
[0112] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0113] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) "Antibodies that react with predetermined sites on
proteins", Science, 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0114] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate
Neutrokine-.alpha. specific antibodies include: a polypeptide
comprising amino acid residues from about Phe-115 to about Leu-147
in FIGS. 1A and B (SEQ ID NO:2); a polypeptide comprising amino
acid residues from about Ile-150 to about Tyr-163 in FIGS. 1A and B
(SEQ ID NO:2); a polypeptide comprising amino acid residues from
about Ser-171 to about Phe-194 in FIGS. 1A and B (SEQ ID NO:2); a
polypeptide comprising amino acid residues from about Glu-223 to
about Tyr-247 in FIGS. 1A and B (SEQ ID NO:2); a polypeptide
comprising amino acid residues from about Ser-271 to about Phe-278
in FIGS. 1A and B (SEQ ID NO:2); These polypeptide fragments have
been determined to bear antigenic epitopes of the
Neutrokine-.alpha. protein by the analysis of the Jameson-Wolf
antigenic index, as shown in FIG. 3, above.
[0115] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See, e.g.,
Houghten, R. A. (1985) General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids. Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
[0116] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art. See, for instance, Geysen et al., supra. Further still,
U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method
of detecting or determining the sequence of monomers (amino acids
or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0117] Fusion Proteins
[0118] As one of skill in the art will appreciate,
Neutrokine-.alpha. polypeptides of the present invention and the
epitope-bearing fragments thereof described above can be combined
with parts of the constant domain of immunoglobulins (IgG),
resulting in chimeric polypeptides. These fusion proteins
facilitate purification and show an increased half-life in vivo.
This has been shown, e.g., for chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84-86
(1988)). Fusion proteins that have a disulfide-linked dimeric
structure due to the IgG part can also be more efficient in binding
and neutralizing other molecules than the monomeric
Neutrokine-.alpha. protein or protein fragment alone (Fountoulakis
et al., J. Biochem. 270:3958-3964 (1995)).
Immune System-Related Disorder Diagnosis
[0119] The present inventors have discovered that
Neutrokine-.alpha. is expressed in various tissues and particularly
in neutrophils. For a number of immune system-related disorders,
substantially altered (increased or decreased) levels of
Neutrokine-.alpha. gene expression can be detected in immune system
tissue or other cells or bodily fluids (e.g., sera, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to a "standard" Neutrokine-.alpha. gene
expression level, that is, the Neutrokine-.alpha. expression level
in immune system tissues or bodily fluids from an individual not
having the immune system disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of an system disorder,
which involves measuring the expression level of the gene encoding
the Neutrokine-.alpha. protein in immune system tissue or other
cells or body fluid from an individual and comparing the measured
gene expression level with a standard Neutrokine-.alpha. gene
expression level, whereby an increase or decrease in the gene
expression level compared to the standard is indicative of an
immune system disorder.
[0120] In particular, it is believed that certain tissues in
mammals with cancer of the immune express significantly enhanced or
reduced levels of the Neutrokine-.alpha. protein and mRNA encoding
the Neutrokine-.alpha. protein when compared to a corresponding
"standard" level. Further, it is believed that enhanced or
depressed levels of the Neutrokine-.alpha. protein can be detected
in certain body fluids (e.g., sera, plasma, urine, and spinal
fluid) from mammals with such a cancer when compared to sera from
mammals of the same species not having the cancer.
[0121] Thus, the invention provides a diagnostic method useful
during diagnosis of a immune system disorder, including cancers of
this system, which involves measuring the expression level of the
gene encoding the Neutrokine-.alpha. protein in immune system
tissue or other cells or body fluid from an individual and
comparing the measured gene expression level with a standard
Neutrokine-.alpha. gene expression level, whereby an increase or
decrease in the gene expression level compared to the standard is
indicative of an immune system disorder.
[0122] Where a diagnosis of a disorder in the immune system,
including diagnosis of a tumor, has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting enhanced or
depressed Neutrokine-.alpha. gene expression will experience a
worse clinical outcome relative to patients expressing the gene at
a level nearer the standard level.
[0123] By "assaying the expression level of the gene encoding the
Neutrokine-.alpha. protein" is intended qualitatively or
quantitatively measuring or estimating the level of the
Neutrokine-.alpha. protein or the level of the mRNA encoding the
Neutrokine-.alpha. protein in a first biological sample either
directly (e.g., by determining or estimating absolute protein level
or mRNA level) or relatively (e.g., by comparing to the
Neutrokine-.alpha. protein level or mRNA level in a second
biological sample). Preferably, the Neutrokine-.alpha. protein
level or mRNA level in the first biological sample is measured or
estimated and compared to a standard Neutrokine-.alpha. protein
level or mRNA level, the standard being taken from a second
biological sample obtained from an individual not having the
disorder or being determined by averaging levels from a population
of individuals not having a disorder of the immune system. As will
be appreciated in the art, once a standard Neutrokine-.alpha.
protein level or mRNA level is known, it can be used repeatedly as
a standard for comparison.
[0124] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains Neutrokine-.alpha. protein or mRNA.
As indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain free
extracellular domains of the Neutrokine-.alpha. protein, immune
system tissue, and other tissue sources found to express complete
or free extracellular domain of the Neutrokine-.alpha. or a
Neutrokine-.alpha. receptor. Methods for obtaining tissue biopsies
and body fluids from mammals are well known in the art. Where the
biological sample is to include mRNA, a tissue biopsy is the
preferred source.
[0125] The present invention is useful for diagnosis or treatment
of various immune system-related disorders in mammals, preferably
humans. Such disorders include but are not limited to tumors and
tumor metastasis, infections by bacteria, viruses and other
parasites, immunodeficiencies, inflammatory diseases,
lymphadenopathy, autoimmune diseases, and graft versus host
disease.
[0126] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-ph- enol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the Neutrokine-.alpha. protein are then assayed
using any appropriate method. These include Northern blot analysis,
S1 nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0127] Assaying Neutrokine-.alpha. protein levels in a biological
sample can occur using antibody-based techniques. For example,
Neutrokine-.alpha. protein expression in tissues can be studied
with classical immunohistological methods (Jalkanen, M., et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting Neutrokine .alpha. protein gene expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). Suitable antibody assay labels are
known in the art and include enzyme labels, such as, glucose
oxidase, and radioisotopes, such as iodine (.sup.125I, .sup.121I),
carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0128] In addition to assaying Neutrokine-.alpha. protein levels in
a biological sample obtained from an individual, Neutrokine-.alpha.
protein can also be detected in vivo by imaging. Antibody labels or
markers for in vivo imaging of Neutrokine-.alpha. protein include
those detectable by X-radiography, NMR or ESR. For X-radiography,
suitable labels include radioisotopes such as barium or cesium,
which emit detectable radiation but are not overtly harmful to the
subject. Suitable markers for NMR and ESR include those with a
detectable characteristic spin, such as deuterium, which may be
incorporated into the antibody by labeling of nutrients for the
relevant hybridoma.
[0129] A Neutrokine-.alpha. protein-specific antibody or antibody
fragment which has been labeled with an appropriate detectable
imaging moiety, such as a radioisotope (for example, .sup.131I,
.sup.112In, .sup.99mTc), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (for
example, parenterally, subcutaneously or intraperitoneally) into
the mammal to be examined for immune system disorder. It will be
understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to
produce diagnostic images. In the case of a radioisotope moiety,
for a human subject, the quantity of radioactivity injected will
normally range from about 5 to 20 millicuries of .sup.99mTc. The
labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain
Neutrokine-.alpha. protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982)).
[0130] Antibodies
[0131] Neutrokine-.alpha.-protein specific antibodies for use in
the present invention can be raised against the intact
Neutrokine-.alpha. protein or an antigenic polypeptide fragment
thereof, which may be presented together with a carrier protein,
such as an albumin, to an animal system (such as rabbit or mouse)
or, if it is long enough (at least about 25 amino acids), without a
carrier.
[0132] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab')2
fragments) which are capable of specifically binding to
Neutrokine-.alpha. protein. Fab and F(ab')2 fragments lack the Fc
fragment of intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding of an
intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Thus, these fragments are preferred.
[0133] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
Neutrokine-.alpha. protein or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of Neutrokine-.alpha. protein is prepared and purified
to render it substantially free of natural contaminants. Such a
preparation is then introduced into an animal in order to produce
polyclonal antisera of greater specific activity.
[0134] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or Neutrokine-.alpha. protein
binding fragments thereof). Such monoclonal antibodies can be
prepared using hybridoma technology (Kohler et al., Nature 256:495
(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et
al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:
Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981)
pp. 563-681). In general, such procedures involve immunizing an
animal (preferably a mouse) with a Neutrokine-.alpha. protein
antigen or, more preferably, with a Neutrokine-.alpha.
protein-expressing cell. Suitable cells can be recognized by their
capacity to bind anti-Neutrokine-.alpha. protein antibody. Such
cells may be cultured in any suitable tissue culture medium;
however, it is preferable to culture cells in Earle's modified
Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C.), and supplemented with about
10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin,
and about 100 .mu.g/ml of streptomycin. The splenocytes of such
mice are extracted and fused with a suitable myeloma cell line. Any
suitable myeloma cell line may be employed in accordance with the
present invention; however, it is preferable to employ the parent
myeloma cell line (SP2O), available from the American Type Culture
Collection, Rockville, Md. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al. (Gastroenterology
80:225-232 (1981)). The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding the Neutrokine-.alpha. protein
antigen.
[0135] Alternatively, additional antibodies capable of binding to
the Neutrokine-.alpha. protein antigen may be produced in a
two-step procedure through the use of anti-idiotypic antibodies.
Such a method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
Neutrokine-.alpha.-protein specific antibodies are used to immunize
an animal, preferably a mouse. The splenocytes of such an animal
are then used to produce hybridoma cells, and the hybridoma cells
are screened to identify clones which produce an antibody whose
ability to bind to the Neutrokine-.alpha. protein-specific antibody
can be blocked by the Neutrokine-.alpha. protein antigen. Such
antibodies comprise anti-idiotypic antibodies to the
Neutrokine-.alpha. protein-specific antibody and can be used to
immunize an animal to induce formation of further
Neutrokine-.alpha. protein-specific antibodies.
[0136] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, Neutrokine-.alpha. protein-binding
fragments can be produced through the application of recombinant
DNA technology or through synthetic chemistry.
[0137] Where in vivo imaging is used to detect enhanced levels of
Neutrokine-.alpha. protein for diagnosis in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).
Treatment of Immune System-Related Disorders
[0138] As noted above, Neutrokine-.alpha. polynucleotides and
polypeptides are useful for diagnosis of conditions involving
abnormally high or low expression of Neutrokine-.alpha. activities.
Given the cells and tissues where Neutrokine-.alpha. is expressed
as well as the activities modulated by Neutrokine-.alpha., it is
readily apparent that a substantially altered (increased or
decreased) level of expression of Neutrokine-.alpha. in an
individual compared to the standard or "normal" level produces
pathological conditions related to the bodily system(s) in which
Neutrokine-.alpha. is expressed and/or is active.
[0139] It will also be appreciated by one of ordinary skill that,
since the Neutrokine-.alpha. protein of the invention is a member
of the TNF family, the extracellular domain of the protein may be
released in soluble form from the cells which express
Neutrokine-.alpha. by proteolytic cleavage and therefore, when
Neutrokine-.alpha. protein (particularly a soluble form of the
extracellular domain) is added from an exogenous source to cells,
tissues or the body of an individual, the protein will exert its
modulating activities on any of its target cells of that
individual. Also, cells expressing this type II transmembrane
protein may be added to cells, tissues or the body of an individual
whereby the added cells will bind to cells expressing receptor for
Neutrokine-.alpha. whereby the cells expressing Neutrokine-.alpha.
can cause actions (e.g., cytotoxicity) on the receptor-bearing
target cells.
[0140] Therefore, it will be appreciated that conditions caused by
a decrease in the standard or normal level of Neutrokine-.alpha.
activity in an individual, particularly disorders of the immune
system, can be treated by administration of Neutrokine-.alpha.
protein (in the form of soluble extracellular domain or cells
expressing the complete protein). Thus, the invention also provides
a method of treatment of an individual in need of an increased
level of Neutrokine-.alpha. activity comprising administering to
such an individual a pharmaceutical composition comprising an
amount of an isolated Neutrokine-.alpha. polypeptide of the
invention, effective to increase the Neutrokine-.alpha. activity
level in such an individual.
[0141] Since Neutrokine-.alpha. belongs to the TNF superfamily, it
also should also modulate angiogenesis. In addition, since
Neutrokine-.alpha. inhibits immune cell functions, it will have a
wide range of anti-inflammatory activities. Neutrokine-.alpha. may
be employed as an anti-neovascularizing agent to treat solid tumors
by stimulating the invasion and activation of host defense cells,
e.g., cytotoxic T cells and macrophages and by inhibiting the
angiogenesis of tumors. Those of skill in the art will recognize
other non-cancer indications where blood vessel proliferation is
not wanted. They may also be employed to enhance host defenses
against resistant chronic and acute infections, for example,
myobacterial infections via the attraction and activation of
microbicidal leukocytes. Neutrokine-.alpha. may also be employed to
inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis
for the treatment of T-cell mediated auto-immune diseases and
lymphocytic leukemias. Neutrokine-.alpha. may also be employed to
stimulate wound healing, both via the recruitment of debris
clearing and connective tissue promoting inflammatory cells. In
this same manner, Neutrokine-.alpha. may also be employed to treat
other fibrotic disorders, including liver cirrhosis, osteoarthritis
and pulmonary fibrosis. Neutrokine-.alpha. also increases the
presence of eosinophils which have the distinctive function of
killing the larvae of parasites that invade tissues, as in
schistosomiasis, trichinosis and ascariasis. It may also be
employed to regulate hematopoiesis, by regulating the activation
and differentiation of various hematopoietic progenitor cells, for
example, to release mature leukocytes from the bone marrow
following chemotherapy, i.e., in stem cell mobilization.
Neutrokine-.alpha. may also be employed to treat sepsis.
[0142] Formulations
[0143] The Neutrokine-.alpha. polypeptide composition (preferably
containing a polypeptide which is a soluble form of the
extracellular domain) will be formulated and dosed in a fashion
consistent with good medical practice, taking into account the
clinical condition of the individual patient (especially the side
effects of treatment with Neutrokine-.alpha. polypeptide alone),
the site of delivery of the Neutrokine-.alpha. polypeptide
composition, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" of Neutrokine-.alpha. polypeptide for purposes
herein is thus determined by such considerations.
[0144] As a general proposition, the total pharmaceutically
effective amount of Neutrokine-.alpha. polypeptide administered
parenterally per dose will be in the range of about 1 .mu.g/kg/day
to 10 mg/kg/day of patient body weight, although, as noted above,
this will be subject to therapeutic discretion. More preferably,
this dose is at least 0.01 mg/kg/day, and most preferably for
humans between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the Neutrokine-.alpha. polypeptide is typically
administered at a dose rate of about 1 .mu.g/kg/hour to about 50
.mu.g/kg/hour, either by 1-4 injections per day or by continuous
subcutaneous infusions, for example, using a mini-pump. An
intravenous bag solution may also be employed. The length of
treatment needed to observe changes and the interval following
treatment for responses to occur appears to vary depending on the
desired effect.
[0145] Pharmaceutical compositions containing the
Neutrokine-.alpha. of the invention may be administered orally,
rectally, parenterally, intracistemally, intravaginally,
intraperitoneally, topically (as by powders, ointments, drops or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically acceptable carrier" is meant a non-toxic solid,
semisolid or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type. The term "parenteral" as used
herein refers to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
[0146] The Neutrokine-.alpha. polypeptide is also suitably
administered by sustained-release systems. Suitable examples of
sustained-release compositions include semi-permeable polymer
matrices in the form of shaped articles, e.g., films, or
mirocapsules. Sustained-release matrices include polylactides (U.S.
Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556
(1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.)
or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
Neutrokine-.alpha. polypeptide compositions also include
liposomally entrapped Neutrokine-.alpha. polypeptide. Liposomes
containing Neutrokine-.alpha. polypeptide are prepared by methods
known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
(USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.
(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP
143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes
are of the small (about 200-800 Angstroms) unilamellar type in
which the lipid content is greater than about 30 mol. percent
cholesterol, the selected proportion being adjusted for the optimal
Neutrokine-.alpha. polypeptide therapy.
[0147] For parenteral administration, in one embodiment, the
Neutrokine-.alpha. polypeptide is formulated generally by mixing it
at the desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0148] Generally, the formulations are prepared by contacting the
Neutrokine .alpha. polypeptide uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired formulation.
Preferably the carrier is a parenteral carrier, more preferably a
solution that is isotonic with the blood of the recipient. Examples
of such carrier vehicles include water, saline, Ringer's solution,
and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl oleate are also useful herein, as well as liposomes.
[0149] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0150] The Neutrokine-.alpha. polypeptide is typically formulated
in such vehicles at a concentration of about 0.1 mg/ml to 100
mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
Neutrokine-.alpha. polypeptide salts.
[0151] Neutrokine-.alpha. polypeptide to be used for therapeutic
administration must be sterile. Sterility is readily accomplished
by filtration through sterile filtration membranes (e.g., 0.2
micron membranes). Therapeutic Neutrokine-.alpha. polypeptide
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0152] Neutrokine-.alpha. polypeptide ordinarily will be stored in
unit or multi-dose containers, for example, sealed ampoules or
vials, as an aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
Neutrokine-.alpha. polypeptide solution, and the resulting mixture
is lyophilized. The infusion solution is prepared by reconstituting
the lyophilized Neutrokine-.alpha. polypeptide using bacteriostatic
Water-for-Injection.
[0153] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
[0154] Agonists and Antagonists--Assays and Molecules
[0155] The invention also provides a method of screening compounds
to identify those which enhance or block the action of
Neutrokine-.alpha. on cells, such as its interaction with
Neutrokine-.alpha. binding molecules such as receptor molecules. An
agonist is a compound which increases the natural biological
functions of Neutrokine-.alpha. or which functions in a manner
similar to Neutrokine while antagonists decrease or eliminate such
functions.
[0156] In another aspect of this embodiment the invention provides
a method for identifying a receptor protein or other ligand-binding
protein which binds specifically to a Neutrokine-.alpha.
polypeptide. For example, a cellular compartment, such as a
membrane or a preparation thereof, may be prepared from a cell that
expresses a molecule that binds Neutrokine-.alpha.. The preparation
is incubated with labeled Neutrokine-.alpha. and complexes of
Neutrokine-.alpha. bound to the receptor or other binding protein
are isolated and characterized according to routine methods known
in the art. Alternatively, the Neutrokine-.alpha. polypeptide may
be bound to a solid support so that binding molecules solubilized
from cells are bound to the column and then eluted and
characterized according to routine methods.
[0157] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
Neutrokine-.alpha. such as a molecule of a signaling or regulatory
pathway modulated by Neutrokine-.alpha.. The preparation is
incubated with labeled Neutrokine-.alpha. in the absence or the
presence of a candidate molecule which may be a Neutrokine-.alpha.
agonist or antagonist. The ability of the candidate molecule to
bind the binding molecule is reflected in decreased binding of the
labeled ligand. Molecules which bind gratuitously, i.e., without
inducing the effects of Neutrokine-.alpha. on binding the
Neutrokine-.alpha. binding molecule, are most likely to be good
antagonists. Molecules that bind well and elicit effects that are
the same as or closely related to Neutrokine-.alpha. are
agonists.
[0158] Neutrokine-.alpha.-like effects of potential agonists and
antagonists may by measured, for instance, by determining activity
of a second messenger system following interaction of the candidate
molecule with a cell or appropriate cell preparation, and comparing
the effect with that of Neutrokine-.alpha. or molecules that elicit
the same effects as Neutrokine-.alpha.. Second messenger systems
that may be useful in this regard include but are not limited to
AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis
second messenger systems.
[0159] Another example of an assay for Neutrokine-.alpha.
antagonists is a competitive assay that combines Neutrokine-.alpha.
and a potential antagonist with membrane-bound receptor molecules
or recombinant Neutrokine-.alpha. receptor molecules under
appropriate conditions for a competitive inhibition assay.
Neutrokine-.alpha. can be labeled, such as by radioactivity, such
that the number of Neutrokine-.alpha. molecules bound to a receptor
molecule can be determined accurately to assess the effectiveness
of the potential antagonist.
[0160] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing Neutrokine-.alpha. induced
activities, thereby preventing the action of Neutrokine-.alpha. by
excluding Neutrokine-.alpha. from binding.
[0161] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix
formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and Dervan et al., Science 251: 1360 (1991). The methods are based
on binding of a polynucleotide to a complementary DNA or RNA. For
example, the 5' coding portion of a polynucleotide that encodes the
extracellular domain of the polypeptide of the present invention
may be used to design an antisense RNA oligonucleotide of from
about 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription thereby preventing transcription and the production
of Neutrokine-.alpha.. The antisense RNA oligonucleotide hybridizes
to the mRNA in vivo and blocks translation of the mRNA molecule
into Neutrokine-.alpha. polypeptide. The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of
Neutrokine-.alpha..
[0162] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described above.
[0163] The antagonists may be employed for instance to inhibit
Neutrokine-.alpha. the chemotaxis and activation of macrophages and
their precursors, and of neutrophils, basophils, B lymphocytes and
some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and
natural killer cells, in certain auto-immune and chronic
inflammatory and infective diseases. Examples of auto-immune
diseases include multiple sclerosis, and insulin-dependent
diabetes. The antagonists may also be employed to treat infectious
diseases including silicosis, sarcoidosis, idiopathic pulmonary
fibrosis by preventing the recruitment and activation of
mononuclear phagocytes. They may also be employed to treat
idiopathic hyper-eosinophilic syndrome by preventing eosinophil
production and migration. Endotoxic shock may also be treated by
the antagonists by preventing the migration of macrophages and
their production of the human chemokine polypeptides of the present
invention. The antagonists may also be employed for treating
atherosclerosis, by preventing monocyte infiltration in the artery
wall. The antagonists may also be employed to treat
histamine-mediated allergic reactions and immunological disorders
including late phase allergic reactions, chronic urticaria, and
atopic dermatitis by inhibiting chemokine-induced mast cell and
basophil degranulation and release of histamine. IgE-mediated
allergic reactions such as allergic asthma, rhinitis, and eczema
may also be treated. The antagonists may also be employed to treat
chronic and acute inflammation by preventing the attraction of
monocytes to a wound area. They may also be employed to regulate
normal pulmonary macrophage populations, since chronic and acute
inflammatory pulmonary diseases are associated with sequestration
of mononuclear phagocytes in the lung. Antagonists may also be
employed to treat rheumatoid arthritis by preventing the attraction
of monocytes into synovial fluid in the joints of patients.
Monocyte influx and activation plays a significant role in the
pathogenesis of both degenerative and inflammatory arthropathies.
The antagonists may be employed to interfere with the deleterious
cascades attributed primarily to IL-1 and TNF, which prevents the
biosynthesis of other inflammatory cytokines. In this way, the
antagonists may be employed to prevent inflammation. The
antagonists may also be employed to inhibit
prostaglandin-independent fever induced by chemokines. The
antagonists may also be employed to treat cases of bone marrow
failure, for example, aplastic anemia and myelodysplastic syndrome.
The antagonists may also be employed to treat asthma and allergy by
preventing eosinophil accumulation in the lung. The antagonists may
also be employed to treat subepithelial basement membrane fibrosis
which is a prominent feature of the asthmatic lung.
[0164] Antibodies against Neutrokine-.alpha. may be employed to
bind to and inhibit Neutrokine-.alpha. activity to treat ARDS, by
preventing infiltration of neutrophils into the lung after injury.
The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0165] Chromosome Assays
[0166] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0167] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a
Neutrokine-.alpha. protein gene. This can be accomplished using a
variety of well known techniques and libraries, which generally are
available commercially. The genomic DNA then is used for in situ
chromosome mapping using well known techniques for this
purpose.
[0168] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Fluorescence
in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from
the cDNA as short as 50 or 60 bp. For a review of this technique,
see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,
Pergamon Press, New York (1988).
[0169] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0170] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0171] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1a
Expression and Purification of "His-Tagged" Neutrokine-.alpha. in
E. coli
[0172] The bacterial expression vector pQE9 (PD10) is used for
bacterial expression in this example. (QIAGEN, Inc., supra). pQE9
encodes ampicillin antibiotic resistance ("Ampr") and contains a
bacterial origin of replication ("ori"), an IPTG inducible
promoter, a ribosome binding site ("RBS"), six codons encoding
histidine residues that allow affinity purification using
nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by
QIAGEN, Inc., supra, and suitable single restriction enzyme
cleavage sites. These elements are arranged such that an inserted
DNA fragment encoding a polypeptide expresses that polypeptide with
the six His residues (i.e., a "6.times. His tag") covalently linked
to the amino terminus of that polypeptide.
[0173] The DNA sequence encoding the desired portion of the
Neurokine-.alpha. protein comprising the extracellular domain
sequence is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the Neurokine-.alpha. protein
and to sequences in the deposited construct 3' to the cDNA coding
sequence. Additional nucleotides containing restriction sites to
facilitate cloning in the pQE9 vector are added to the 5' and 3'
primer sequences, respectively.
[0174] For cloning the extracellular domain of the protein, the 5'
primer has the sequence 5' GTG GGA TCC AGC CTC CGG GCA GAG CTG 3'
(SEQ ID NO:10) containing the underlined Bam HI restriction site
followed by 18 nucleotides of the amino terminal coding sequence of
the extracellular domain of the Neurokine-.alpha. sequence in FIGS.
1A and B. One of ordinary skill in the art would appreciate, of
course, that the point in the protein coding sequence where the 5'
primer begins may be varied to amplify a DNA segment encoding any
desired portion of the complete Neutrokine .alpha. protein shorter
or longer than the extracellular domain of the form. The 3' primer
has the sequence 5'-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC ACC-3'
(SEQ ID NO:11) containing the underlined Hind III restriction site
followed by two stop codons and 18 nucleotides complementary to the
3' end of the coding sequence of the Neurokine-.alpha. DNA sequence
in FIGS. 1A and B.
[0175] The amplified Neurokine-.alpha. DNA fragment and the vector
pQE9 are digested with Bam HI and Hind III and the digested DNAs
are then ligated together. Insertion of the Neurokine-.alpha. DNA
into the restricted pQE9 vector places the Neurokine-.alpha.
protein coding region downstream from the IPTG-inducible promoter
and in-frame with an initiating AUG and the six histidine
codons.
[0176] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E.
coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing Neurokine-.alpha. protein, is
available commercially from QIAGEN, Inc., supra. Transformants are
identified by their ability to grow on LB plates in the presence of
ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing. Clones containing the
desired constructs are grown overnight ("O/N") in liquid culture in
LB media supplemented with both ampicillin (100 .mu.g/ml) and
kanamycin (25 .mu.g/ml). The O/N culture is used to inoculate a
large culture, at a dilution of approximately 1:25 to 1:250. The
cells are grown to an optical density at 600 nm ("OD600") of
between 0.4 and 0.6. Isopropyl-.beta.-D-thiogalactopyranoside
("IPTG") is then added to a final concentration of 1 mM to induce
transcription from the lac repressor sensitive promoter, by
inactivating the lacI repressor. Cells subsequently are incubated
further for 3 to 4 hours. Cells then are harvested by
centrifugation.
[0177] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the
Neurokine-.alpha. is loaded onto a nickel-nitrilo-tri-acetic acid
("Ni-NTA") affinity resin column (available from QIAGEN, Inc.,
supra). Proteins with a 6.times. His tag bind to the Ni-NTA resin
with high affinity and can be purified in a simple one-step
procedure (for details see: The QIAexpressionist, 1995, QIAGEN,
Inc., supra). Briefly the supernatant is loaded onto the column in
6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes
of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M
guanidine-HCl pH 6, and finally the Neurokine-.alpha. is eluted
with 6 M guanidine-HCl, pH 5.
[0178] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. C.
Example 1b
Expression and Purification of Neutrokine-.alpha. in E. coli
[0179] The bacterial expression vector pQE60 is used for bacterial
expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic
resistance ("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin sold by QIAGEN, Inc., supra, and suitable single
restriction enzyme cleavage sites. These elements are arranged such
that a DNA fragment encoding a polypeptide may be inserted in such
as way as to produce that polypeptide with the six His residues
(i.e., a "6.times. His tag") covalently linked to the carboxyl
terminus of that polypeptide. However, in this example, the
polypeptide coding sequence is inserted such that translation of
the six His codons is prevented and, therefore, the polypeptide is
produced with no 6.times. His tag.
[0180] The DNA sequence encoding the desired portion of the
Neurokine-.alpha. protein comprising the extracellular domain
sequence is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the Neurokine-.alpha. protein
and to sequences in the deposited construct 3' to the cDNA coding
sequence. Additional nucleotides containing restriction sites to
facilitate cloning in the pQE60 vector are added to the 5' and 3'
sequences, respectively.
[0181] For cloning the extracellular domain of the protein, the 5'
primer has the sequence 5' GTG TCA TGA GCC TCC GGG CAG AGC TG 3'
(SEQ ID NO:12) containing the underlined Bsp HI restriction site
followed by 17 nucleotides of the amino terminal coding sequence of
the extracellular domain of the Neurokine-.alpha. sequence in FIGS.
1A and B. One of ordinary skill in the art would appreciate, of
course, that the point in the protein coding sequence where the 5'
primer begins may be varied to amplify a desired portion of the
complete protein shorter or longer than the extracellular domain of
the form. The 3' primer has the sequence 5'-GTG AAG CTT TTA TTA CAG
CAG TTT CAA TGC ACC 3' (SEQ 13) containing the underlined Hind III
restriction site followed by two stop codons 8 nucleotides
complementary to the 3' end of the coding sequence in the
Neurokine-.alpha. DNA sequence in FIGS. 1A and B.
[0182] The amplified Neurokine-.alpha. DNA fragments and the vector
pQE60 are digested with Bsp HI and Hind III and the digested DNAs
are then ligated together. Insertion of the Neurokine-.alpha. DNA
into the restricted pQE60 vector places the Neurokine-.alpha.
protein coding region including its associated stop codon
downstream from the IPTG-inducible promoter and in-frame with an
initiating AUG. The associated stop codon prevents translation of
the six histidine codons downstream of the insertion point.
[0183] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E.
coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kanr"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing Neurokine-.alpha. protein, is
available commercially from QIAGEN, Inc., supra. Transformants are
identified by their ability to grow on LB plates in the presence of
ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
[0184] One of ordinary skill in the art recognizes that any of a
number of bacterial expression vectors may be useful in place of
pQE9 and pQE60 in the expression protocols presented in this
example. For example, the novel pHE4 series of bacterial expression
vectors, in particular, the pHE4-5 vector may be used for bacterial
expression in this example (ATCC Accession No. 209311; and
variations thereof). The plasmid DNA designated
pHE4-5/MPIF.DELTA.23 in ATCC Deposit No. 209311 is vector plasmid
DNA which contains an insert which encodes another ORF. The
construct was deposited with the American Type Culture Collection,
12301 Park Lawn Drive, Rockville, Md. 20852, on Sep. 30, 1997.
Using the Nde I and Asp 718 restriction sites flanking the
irrelevant MPIF ORF insert, one of ordinary skill in the art could
easily use current molecular biological techniques to replace the
irrelevant ORF in the pHE4-5 vector with the Neutrokine-.alpha. ORF
of the present invention.
[0185] The pHE4-5 bacterial expression vector includes a neomycin
phosphotransferase gene for selection, an E. coli origin of
replication, a T5 phage promoter sequence, two lac operator
sequences, a Shine-Delgarno sequence, and the lactose operon
repressor gene (lacIq). These elements are arranged such that an
inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times. His tag")
covalently linked to the amino terminus of that polypeptide. The
promoter and operator sequences of the pHE4-5 vector were made
synthetically. Synthetic production of nucleic acid sequences is
well known in the art (CLONETECH 95/96 Catalog, pages 215-216,
CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).
[0186] Clones containing the desired Neutrokine-.alpha. constructs
are grown overnight ("O/N") in liquid culture in LB media
supplemented with both ampicillin (100 .mu.g/ml) and kanamycin (25
.mu.g/ml). The O/N culture is used to inoculate a large culture, at
a dilution of approximately 1:25 to 1:250. The cells are grown to
an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-b-D-thiogalactopyranos- ide ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0187] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the Neutrokine
.alpha. is dialyzed against 50 mM Na-acetate buffer pH 6,
supplemented with 200 mM NaCl. Alternatively, the protein can be
successfully refolded by dialyzing it against 500 mM NaCl, 20%
glycerol, 25 mM Tris/HCl pH 7.4, containing protease inhibitors.
After renaturation the protein can be purified by ion exchange,
hydrophobic interaction and size exclusion chromatography.
Alternatively, an affinity chromatography step such as an antibody
column can be used to obtain pure Neurokine-.alpha. protein. The
purified protein is stored at 4.degree. C. or frozen at -80.degree.
C.
Example 2
Cloning and Expression of Neutrokine-.alpha. Protein in a
Baculovirus Expression System
[0188] In this illustrative example, the plasmid shuttle vector
pA2GP is used to insert the cloned DNA encoding the extracellular
domain of the protein, lacking its naturally associated
intracellular and transmembrane sequences, into a baculovirus to
express the extracellular domain of the Neurokine-.alpha. protein,
using a baculovirus leader and standard methods as described in
Summers et al., A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures, Texas Agricultural Experimental
Station Bulletin No. 1555 (1987). This expression vector contains
the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by the secretory
signal peptide (leader) of the baculovirus gp67 protein and
convenient restriction sites such as Bam HI, Xba I and Asp 718. The
polyadenylation site of the simian virus 40 ("SV40") is used for
efficient polyadenylation. For easy selection of recombinant virus,
the plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate viable virus that expresses the cloned polynucleotide.
[0189] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0190] The cDNA sequence encoding the extracellular domain of the
Neurokine-.alpha. protein in the deposited clone, lacking the AUG
initiation codon and the naturally associated intracellular and
transmembrane domain sequences shown in FIGS. 1A and B (SEQ ID NO:2
is amplified using PCR oligonucleotide primers corresponding to the
5' and 3' sequences of the gene. The 5' primer has the sequence
5'-GTG GGA TCC CCG GGC AGA GCT GCA GGG C-3' (SEQ ID NO:14)
containing the underlined Bam HI restriction enzyme site followed
by 18 nucleotides of the sequence of the extracellular domain of
the Neurokine-.alpha. protein shown in FIGS. 1A and B, beginning
with the indicated N-terminus of the extracellular domain of the
protein. The 3' primer has the sequence 5'-GTG GGA TCC TTA TTA CAG
CAG TTT CAA TGC ACC-3' (SEQ ID NO:15) containing the underlined Bam
HI restriction site followed by two stop codons and 18 nucleotides
complementary to the 3' coding sequence in FIGS. 1A and B.
[0191] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with Bam HI and again
is purified on a 1% agarose gel. This fragment is designated herein
F1.
[0192] The plasmid is digested with the restriction enzymes Bam HI
and optionally, can be dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein "V1".
[0193] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human Neurokine-.alpha. gene by digesting DNA from
individual colonies using Bam HI and then analyzing the digestion
product by gel electrophoresis. The sequence of the cloned fragment
is confirmed by DNA sequencing. This plasmid is designated herein
pA2GP-Neutrokine-.alpha..
[0194] Five .mu.g of the plasmid pA2GP-Neutrokine-.alpha. is
co-transfected with 1.0 .mu.g of a commercially available
linearized baculovirus DNA ("BaculoGold.TM. baculovirus DNA",
Pharmingen, San Diego, Calif.), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:
7413-7417 (1987). One .mu.g of BaculoGold.TM. virus DNA and 5 .mu.g
of the plasmid pA2GP Neurokine-.alpha. are mixed in a sterile well
of a microtiter plate containing 50 .mu.l of serum-free Grace's
medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10
.mu.l Lipofectin plus 90 .mu.l Grace's medium are added, mixed and
incubated for 15 minutes at room temperature. Then the transfection
mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711)
seeded in a 35 mm tissue culture plate with 1 ml Grace's medium
without serum. The plate is then incubated for 5 hours at
27.degree. C. The transfection solution is then removed from the
plate and 1 ml of Grace's insect medium supplemented with 10% fetal
calf serum is added. Cultivation is then continued at 27.degree. C.
for four days.
[0195] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10). After appropriate incubation, blue stained plaques are
picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 .mu.l of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at
4.degree. C. The recombinant virus is called
V-Neurokine-.alpha..
[0196] To verify the expression of the Neurokine-.alpha. gene Sf9
cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-Neurokine-.alpha. at a multiplicity of infection
("MOI") of about 2. If radiolabeled proteins are desired, 6 hours
later the medium is removed and is replaced with SF900 II medium
minus methionine and cysteine (available from Life Technologies
Inc., Rockville, Md.). After 42 hours, 5 .mu.Ci of
.sup.35S-methionine and 5 .mu.Ci .sup.35S-cysteine (available from
Amersham) are added. The cells are further incubated for 16 hours
and then are harvested by centrifugation. The proteins in the
supernatant as well as the intracellular proteins are analyzed by
SDS-PAGE followed by autoradiography (if radiolabeled).
[0197] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the extracellular domain of the protein and
thus the cleavage point and length of the secretory signal
peptide.
Example 3
Cloning and Expression of Neutrokine-.alpha. in Mammalian Cells
[0198] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and
pBC12MI (ATCC 67109). Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[0199] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0200] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
[0201] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites Bam
HI, Xba I and Asp 718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[0202] The expression plasmid, pNeurokine-.alpha.-HA, is made by
cloning a portion of the deposited cDNA encoding the extracellular
domain of the Neurokine-.alpha. protein into the expression vector
pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen,
Inc.). To produce a soluble, secreted form of the polypeptide, the
extracellular domain is fused to the secretory leader sequence of
the human IL-6 gene.
[0203] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[0204] A DNA fragment encoding the extracellular domain of the
Neurokine-.alpha. polypeptide is cloned into the polylinker region
of the vector so that recombinant protein expression is directed by
the CMV promoter. The plasmid construction strategy is as follows.
The Neurokine-.alpha. cDNA of the deposited clone is amplified
using primers that contain convenient restriction sites, much as
described above for construction of vectors for expression of
Neurokine-.alpha. in E. coli. Suitable primers include the
following, which are used in this example. The 5' primer,
containing the underlined Bam HI site, a Kozak sequence, an AUG
start codon, a sequence encoding the secretory leader peptide from
the human IL-6 gene, and 18 nucleotides of the 5' coding region of
the extracellular domain of Neurokine-.alpha. protein, has the
following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA
AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT
GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3' (SEQ
ID NO:16). The 3' primer, containing the underlined Bam HI
restriction site and 18 of nucleotides complementary to the 3'
coding sequence immediately before the stop codon, has the
following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3'
(SEQ ID NO:17).
[0205] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with Bam HI and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037), and the transformed culture is plated on ampicillin media
plates which then are incubated to allow growth of ampicillin
resistant colonies. Plasmid DNA is isolated from resistant colonies
and examined by restriction analysis or other means for the
presence of the fragment encoding the Neutrokine-.alpha.
extracellular domain.
[0206] For expression of recombinant Neurokine-.alpha., COS cells
are transfected with an expression vector, as described above,
using DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of Neurokine-.alpha. by the vector.
[0207] Expression of the Neurokine-.alpha.-HA fusion protein is
detected by radiolabeling and immunoprecipitation, using methods
described in, for example Harlow et al., Antibodies: A Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988). To this end, two days after transfection, the
cells are labeled by incubation in media containing
.sup.35S-cysteine for 8 hours. The cells and the media are
collected, and the cells are washed and the lysed with
detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS,
1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et
al. cited above. Proteins are precipitated from the cell lysate and
from the culture media using an HA-specific monoclonal antibody.
The precipitated proteins then are analyzed by SDS-PAGE and
autoradiography. An expression product of the expected size is seen
in the cell lysate, which is not seen in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0208] The vector pC4 is used for the expression of
Neurokine-.alpha. protein. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC Accession No. 37146). To produce a soluble,
secreted form of the Neurokine-.alpha. polypeptide, the portion of
the deposited cDNA encoding the extracellular domain is fused to
the secretory leader sequence of the human IL-6 gene. The vector
plasmid contains the mouse DHFR gene under control of the SV40
early promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids can
be selected by growing the cells in a selective medium (alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see,
e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R.
T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C.
1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and
Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells grown in
increasing concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[0209] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rouse
Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology,
March 1985:438-447) plus a fragment isolated from the enhancer of
the immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, Xba I, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar systems can be used to express the Neurokine-.alpha. in a
regulated way in mammalian cells (Gossen, M., & Bujard, H.
1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551). For the
polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell
lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more
than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[0210] The plasmid pC4 is digested with the restriction enzymes Bam
HI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0211] The DNA sequence encoding the extracellular domain of the
Neutrokine-.alpha. protein is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene. The
5' primer, containing the underlined Bam HI site, a Kozak sequence,
an AUG start codon, a sequence encoding the secretory leader
peptide from the human IL-6 gene, and 18 nucleotides of the 5'
coding region of the extracellular domain of Neurokine-.alpha.
protein, has the following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC
TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC
CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG
GCC AGC-3' (SEQ ID NO:16). The 3' primer, containing the underlined
Bam HI and 18 of nucleotides complementary to the 3' coding
sequence immediately before the stop codon, has the following
sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3' (SEQ ID
NO:17).
[0212] The amplified fragment is digested with the endonuclease Bam
HI and then purified again on a 1% agarose gel. The isolated
fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed
and bacteria are identified that contain the fragment inserted into
plasmid pC4 using, for instance, restriction enzyme analysis.
[0213] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using lipofectin
(Felgner et al., supra). The plasmid pSV2-neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are trypsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10
.mu.M, 20 .mu.M). The same procedure is repeated until clones are
obtained which grow at a concentration of 100-200 .mu.M. Expression
of the desired gene product is analyzed, for instance, by SDS-PAGE
and Western blot or by reversed phase HPLC analysis.
Example 4
Tissue Distribution of Neutrokine-.alpha. mRNA Expression
[0214] Northern blot analysis is carried out to examine
Neutrokine-.alpha. gene expression in human tissues, using methods
described by, among others, Sambrook et al., cited above. A cDNA
probe containing the entire nucleotide sequence of the
Neutrokine-.alpha. protein (SEQ ID NO:1) is labeled with .sup.32P
using the rediprime.TM. DNA labeling system (Amersham Life
Science), according to manufacturer's instructions. After labeling,
the probe is purified using a CHROMA SPIN-100.TM. column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The purified labeled probe is then used to examine
various human tissues for Neutrokine-.alpha. mRNA.
[0215] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from Clontech and are examined with the labeled probe using
ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
[0216] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0217] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
* * * * *