U.S. patent application number 12/370897 was filed with the patent office on 2009-10-01 for human endokine alpha and methods of use.
This patent application is currently assigned to HUMAN GENOME SCIENCES, INC.. Invention is credited to JIAN NI, CRAIG A. ROSEN, GUO-LIANG YU.
Application Number | 20090246202 12/370897 |
Document ID | / |
Family ID | 27534053 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090246202 |
Kind Code |
A1 |
YU; GUO-LIANG ; et
al. |
October 1, 2009 |
Human Endokine Alpha and Methods of Use
Abstract
The present invention concerns a novel member of the tumor
necrosis factor (TNF) family of cytokines. In particular, isolated
nucleic acid molecules are provided encoding the endokine alpha
protein. Endokine alpha polypeptides are also provided, as are
vectors, host cells and recombinant methods for producing the same.
Also provided are diagnostic and therapeutic methods concerning TNF
family-related disorders.
Inventors: |
YU; GUO-LIANG; (BERKELEY,
CA) ; NI; JIAN; (GERMANTOWN, MD) ; ROSEN;
CRAIG A.; (LAYTONSVILLE, MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
HUMAN GENOME SCIENCES, INC.
ROCKVILLE
MD
|
Family ID: |
27534053 |
Appl. No.: |
12/370897 |
Filed: |
February 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11427956 |
Jun 30, 2006 |
7514081 |
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12370897 |
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10136511 |
May 2, 2002 |
7078027 |
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11427956 |
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09513584 |
Feb 25, 2000 |
6406867 |
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10136511 |
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09345790 |
Jul 1, 1999 |
6521742 |
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09513584 |
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08912227 |
Aug 15, 1997 |
5998171 |
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09345790 |
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60136788 |
May 28, 1999 |
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60122099 |
Feb 26, 1999 |
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60024058 |
Aug 16, 1996 |
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Current U.S.
Class: |
424/139.1 ;
435/320.1; 435/325; 435/440; 435/69.1; 514/1.1; 530/350; 530/387.9;
536/23.5 |
Current CPC
Class: |
C07K 2317/622 20130101;
A61P 35/02 20180101; C07K 14/5255 20130101; Y10S 514/885 20130101;
A61P 25/00 20180101; A61P 37/06 20180101; A61P 37/00 20180101; C07K
14/7151 20130101; C07K 16/24 20130101; C07K 2317/21 20130101; A61P
31/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
424/139.1 ;
536/23.5; 435/320.1; 435/440; 435/325; 435/69.1; 530/350;
530/387.9; 514/12 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/00 20060101 C07H021/00; C12N 15/63 20060101
C12N015/63; C12N 15/00 20060101 C12N015/00; C12N 5/00 20060101
C12N005/00; C12P 21/00 20060101 C12P021/00; C07K 14/525 20060101
C07K014/525; C07K 16/00 20060101 C07K016/00; A61K 38/16 20060101
A61K038/16; A61P 37/00 20060101 A61P037/00; A61P 31/00 20060101
A61P031/00; A61P 35/02 20060101 A61P035/02; A61P 37/06 20060101
A61P037/06; A61P 25/00 20060101 A61P025/00 |
Claims
1. An isolated nucleic acid molecule, comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about 1 to about
169 in SEQ ID NO:2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about 2 to about 169 in SEQ
ID NO:2; (c) a nucleotide sequence encoding a polypeptide having
the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97640; and (d) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), or (c).
2. An isolated nucleic acid molecule, comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), or (d) of claim 1 wherein
said polynucleotide which does not hybridize under stringent
hybridization conditions to a polynucleotide having a nucleotide
sequence consisting of only A residues or of only T residues.
3. An isolated nucleic acid molecule, comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of an endokine alpha polypeptide having an amino acid sequence in
(a), (b), (c), or (d) of claim 1.
4. An isolated nucleic acid molecule of claim 3, which encodes an
epitope-bearing portion of an endokine alpha polypeptide selected
from the group consisting of: a polypeptide comprising amino acid
residues from about 44 to about 158 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 44 to about 54 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about 57 to
about 68 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about 69 to about 78 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 94 to about 105 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about 108
to about 132 in SEQ ID NO:2; and a polypeptide comprising amino
acid residues from about 148 to about 158 in SEQ ID NO:2.
5. An isolated nucleic acid molecule, comprising a polynucleotide
having a sequence selected from the group consisting of: (a) a
nucleotide sequence of a fragment of the sequence shown in SEQ ID
NO: 1 wherein said fragment comprises at least 50 contiguous
nucleotides from SEQ ID NO: 1, provided that said isolated nucleic
acid molecule is not a fragment starting at nucleotide 26 and
ending at nucleotide 476 of SEQ ID NO: 1, or a subfragment thereof,
and (b) a nucleotide sequence complementary to a nucleotide
sequence in (a).
6. A method for making a recombinant vector, comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
7. A recombinant vector produced by the method of claim 6.
8. A method of making a recombinant host cell, comprising
introducing the recombinant vector of claim 7 into a host cell.
9. A recombinant host cell produced by the method of claim 8.
10. A recombinant method for producing an endokine alpha
polypeptide, comprising culturing the recombinant host cell of
claim 9 under conditions such that said polypeptide is expressed
and recovering said polypeptide.
11. An isolated endokine alpha polypeptide having an amino acid
sequence at least 95% identical to a sequence selected from the
group consisting of: (a) amino acids from about 1 to about 169 in
SEQ ID NO:2; (b) amino acids from about 2 to about 169 in SEQ ID
NO:2; (c) the amino acid sequence of the endokine alpha polypeptide
having the amino acid sequence encoded by the cDNA clone contained
in ATCC Deposit No. 97640; and (d) the amino acid sequence of an
epitope-bearing portion of any one of the polypeptides of (a), (b),
or (c).
12. An isolated polypeptide of claim 11, comprising an
epitope-bearing portion of endokine alpha, wherein said portion is
selected from the group consisting of: a polypeptide comprising
amino acid residues from about 44 to about 158 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 44 to about
54 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 57 to about 68 in SEQ ID NO:2; a polypeptide comprising
amino acid residues from about 69 to about 78 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 94 to about
105 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 108 to about 132 in SEQ ID NO:2; and a polypeptide
comprising amino acid residues from about 148 to about 158 in SEQ
ID NO:2.
13. An isolated polypeptide of claim 11, which is produced or
contained in a recombinant host cell.
14. An isolated polypeptide of claim 13, wherein said recombinant
host cell is mammalian.
15. An isolated nucleic acid molecule, comprising a polynucleotide
encoding an endokine alpha polypeptide wherein, except for one to
fifty conservative amino acid substitutions, said polypeptide has a
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide comprising amino acids from about 1
to about 169 in SEQ ID NO:2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about 2 to about 169 in SEQ
ID NO:2; (c) a nucleotide sequence encoding a polypeptide having
the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97640; and (d) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), or (c).
16. An isolated endokine alpha polypeptide wherein, except for one
to fifty conservative amino acid substitutions, said polypeptide
has a sequence selected from the group consisting of: (a) amino
acids from about 1 to about 169 in SEQ ID NO:2; (b) amino acids
from about 2 to about 169 in SEQ ID NO:2; (c) the amino acid
sequence of the endokine alpha polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97640; and (d) the amino acid sequence of an epitope-bearing
portion of any one of the polypeptides of (a), (b), or (c).
17. An isolated antibody or antibody fragment that binds
specifically to an endokine alpha polypeptide of claim 11.
18. A method for treating an individual in need of a decreased
level of endokine alpha activity, comprising administering to said
individual a composition comprising the isolated antibody or
antibody fragment of claim 17.
19. A method of treating an individual having a disorder selected
from the group consisting of: AIDS, chronic lymphocyte disorder,
common variable immunodeficiency, a tumor, parasitic disease,
autoimmune diseases, lupus, arthritis, idiopathic thrombocytopenic
purpura, multiple sclerosis, chronic inflammation, acute
inflammation, acute allograft rejection, graft versus host disease,
transplant rejection, fetal resorption, fecal peritonitis, skin
allergies, bowel disease, a wound, sepsis, ALL, Hodgkins disease,
non-Hodgkins lymphoma, chronic lymphocyte leukemia, plasmacytomas,
multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases,
chronic myelogenous leukemia, chronic hypergammaglobulinemeia,
autoimmune hematological disorders, polychondritis, scleroderma,
Wegener granulomatosis, dermatomyositis, chronic active hepatitis,
myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic
sprue, autoimmune thyroiditis, idiopathic Addison's disease,
vitilogo, gluten-sensitive enteropathy, autoimmune neutropenias,
pemphigus vulgaris, Goodpasture's disease, bullous pemphigoid,
discoid lupus, dense deposit disease, endocrine opthalmopathy, IBD,
asthma, Graves disease, sarcoidosis, cirrhosis, juvenile diabetes,
insulin dependent diabetes mellitus, uveitis, autoimmune gastritis,
lymphopenias, polyarteritis nodosa, Sjogren's syndrome, Bechet's
disease, Hashimoto's disease, primary myxedema, polymyositis, mixed
connective tissue disease, keratoconjunctivitis sicca, vernal
keratoconjunctivitis, interstitial lung fibrosis,
glomerulonephritis, hepatitis, autoimmune hemolytic anemia, contact
sensitivity disease, monophasic EAE, SCIDS, Alzheimer's disease,
Parkinson's disease and primary lateral sclerosis; wherein said
method comprises administering to the individual a therapeutically
effective amount of an endokine-.alpha. polypeptide of claim
11.
20. A method of treating an individual having a disorder selected
from the group consisting of: AIDS, chronic lymphocyte disorder,
common variable immunodeficiency, a tumor, parasitic disease,
autoimmune diseases, lupus, arthritis, idiopathic thrombocytopenic
purpura, multiple sclerosis, chronic inflammation, acute
inflammation, acute allograft rejection, graft versus host disease,
transplant rejection, fetal resorption, fecal peritonitis, skin
allergies, bowel disease, a wound, sepsis, ALL, Hodgkins disease,
non-Hodgkins lymphoma, chronic lymphocyte leukemia, plasmacytomas,
multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases,
chronic myelogenous leukemia, chronic hypergammaglobulinemeia,
autoimmune hematological disorders, polychondritis, scleroderma,
Wegener granulomatosis, dermatomyositis, chronic active hepatitis,
myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic
sprue, autoimmune thyroiditis, idiopathic Addison's disease,
vitilogo, gluten-sensitive enteropathy, autoimmune neutropenias,
pemphigus vulgaris, Goodpasture's disease, bullous pemphigoid,
discoid lupus, dense deposit disease, endocrine opthalmopathy, IBD,
asthma, Graves disease, sarcoidosis, cirrhosis, juvenile diabetes,
insulin dependent diabetes mellitus, uveitis, autoimmune gastritis,
lymphopenias, polyarteritis nodosa, Sjogren's syndrome, Bechet's
disease, Hashimoto's disease, primary myxedema, polymyositis, mixed
connective tissue disease, keratoconjunctivitis sicca, vernal
keratoconjunctivitis, interstitial lung fibrosis,
glomerulonephritis, hepatitis, autoimmune hemolytic anemia, contact
sensitivity disease, monophasic EAE, SCIDS, Alzheimer's disease,
Parkinson's disease and primary lateral sclerosis; wherein said
method comprises administering to the individual a therapeutically
effective amount of an endokine-.alpha. polypeptide of claim
16.
21. A method of treating an individual having a disorder selected
from the group consisting of: autoimmune diseases, silicosis,
sarcoidosis, idiopathic pulmonary fibrosis, idiopathic
hyper-eosinophilic syndrome, endotoxic shock, atherosclerosis,
histamine-mediated allergic reactions, IgE-mediated allergic
reactions, chronic inflammation, acute inflammation, rheumatoid
arthritis, aplastic anemia, myelodysplastic syndrome, and a wound;
wherein said method comprises administering to the individual a
therapeutically effective amount of an endokine-.alpha. antagonist.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/427,956, filed Jun. 30, 2006, which is a divisional of U.S.
application Ser. No. 10/136,511, filed May 2, 2002 (now U.S. Pat.
No. 7,078,027, issued Jul. 18, 2006), which is a divisional of U.S.
application Ser. No. 09/513,584, filed Feb. 25, 2000 (now U.S. Pat.
No. 6,406,867, issued Jun. 18, 2002), which claims the benefit of
U.S. Provisional Applications Nos. 60/136,788, filed May 28, 1999,
and No. 60/122,099, filed Feb. 26, 1999; said U.S. application Ser.
No. 09/513,584 is also a continuation-in-part of U.S. application
Ser. No. 09/345,790, filed Jul. 1, 1999, (now U.S. Pat. No.
6,521,742, issued Feb. 18, 2003), which is a divisional of U.S.
application Ser. No. 08/912,227, filed Aug. 15, 1997, (now U.S.
Pat. No. 5,998,171, issued Dec. 7, 1999); said U.S. application
Ser. No. 08/912,227 claims the benefit of U.S. Provisional
Application No. 60/024,058, filed Aug. 16, 1996. Each of the
above-identified applications is incorporated by reference herein
in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns a novel member of the tumor
necrosis factor (TNF) family of cytokines. In particular, isolated
nucleic acid molecules are provided encoding the endokine alpha
protein. Endokine alpha polypeptides are also provided, as are
vectors, host cells and recombinant methods for producing the same.
Also provided are diagnostic and therapeutic methods concerning TNF
family-related disorders.
[0004] 2. Related Art
[0005] The cytokine known as tumor necrosis factor-.alpha.
(TNF.alpha.; also termed cachectin) is a protein 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)).
[0006] 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)).
[0007] 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)).
[0008] 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.
[0009] 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 (Kombluth, 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)).
[0010] 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.
[0011] 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).
[0012] 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.
[0013] 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)).
[0014] 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).
[0015] 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 different proteins. Gruss and
Dower, supra. Ligands for these receptors have been identified and
belong to at least two cytokine superfamilies. Gruss and Dower,
supra.
[0016] 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
TNF-like disorders.
SUMMARY OF THE INVENTION
[0017] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a cytokine that is
similar to TNF and is believed to have similar biological effects
and activities. This cytokine is named endokine alpha, and includes
endokine alpha polypeptides having at least a portion of the amino
acid sequence in FIG. 1 (SEQ ID NO:2) or an amino acid sequence
encoded by the cDNA clone deposited in a bacterial host as ATCC
Deposit Number 97640 on Jun. 27, 1996. The nucleotide sequence,
which was determined by sequencing the deposited endokine alpha
cDNA clone, contains an open reading frame encoding a polypeptide
of about 169 amino acid residues including an N-terminal
methionine, an intracellular domain of about 17 amino acid
residues, a transmembrane domain of about 26 amino acids, an
extracellular domain of about 126 amino acids, and a deduced
molecular weight for the complete protein of about 19 kDa.
[0018] 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 the endokine alpha polypeptide having
the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide
sequence encoding the endokine alpha polypeptide having the
complete amino acid sequence in SEQ ID NO:2 but minus the
N-terminal methionine residue; (c) a nucleotide sequence encoding
the endokine alpha polypeptide having the complete amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97640; and (d) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b) or (c) above.
[0019] Further embodiments of the invention include isolated
nucleic acid molecules that comprise or, alternatively, consist of,
a polynucleotide having a nucleotide sequence at least 80%, 85%,
90%, 92%, or 95% identical, and more preferably at least 96%, 97%,
98% or 99% identical, to any of the nucleotide sequences in (a),
(b), (c), or (d), above, or a polynucleotide which hybridizes under
stringent hybridization conditions to a polynucleotide in (a), (b),
(c), or (d), 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 endokine alpha
polypeptide having an amino acid sequence in (a), (b), (c), or (d),
above.
[0020] The invention is further directed to nucleic acid fragments
of the nucleic acid molecules described herein. Preferred nucleic
acid fragments include nucleic acid molecules which encode: a
polypeptide comprising the endokine alpha intracellular domain
(amino acid residues from about 1 to about 17 in FIG. 1 (SEQ ID
NO:2)); a polypeptide comprising the endokine alpha transmembrane
domain (amino acid residues from about 18 to about 43 in FIG. 1
(SEQ ID NO:2)); and a polypeptide comprising the endokine alpha
extracellular domain (amino acid residues from about 44 to about
169 in FIG. 1 (SEQ ID NO:2)).
[0021] 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 endokine alpha polypeptides or
peptides by recombinant techniques.
[0022] The invention further provides an isolated endokine alpha
polypeptide having an amino acid sequence selected from the group
consisting of: (a) the complete 169 amino acid sequence in SEQ ID
NO:2; (b) the complete 169 amino acid sequence in SEQ ID NO:2 but
minus the N-terminal methionine residue; (c) the complete amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97640; and (d) the amino acid sequence of an epitope-bearing
portion of any one of the polypeptides of (a), (b), or (c). The
polypeptides of the present invention also include polypeptides
having an amino acid sequence at least 80%, 85%, 90%, 92%, or 95%
identical, more preferably at least 96%, 97%, 98% or 99% identical
to those above.
[0023] Peptides or polypeptides having the amino acid sequence of
an epitope-bearing portion of a endokine 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.
[0024] In another embodiment, the invention provides an isolated
antibody that binds specifically to an endokine alpha polypeptide
having an amino acid sequence described in (a), (b), (c), or (d)
above.
[0025] Preferred polypeptide fragments according to the present
invention include a polypeptide comprising: the endokine alpha
intracellular domain, the endokine alpha transmembrane domain, and
the endokine alpha extracellular domain.
[0026] The invention further provides methods for isolating
antibodies that bind specifically to an endokine alpha polypeptide
having an amino acid sequence as described above. Such antibodies
may be useful diagnostically or therapeutically as antagonists in
the treatment of endokine alpha- and/or TNF-related disorders. The
invention also provides a diagnostic method for determining the
presence of a TNF-related disorder.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the nucleotide (SEQ ID NO: 1) and deduced amino
acid (SEQ ID NO:2) sequences of the endokine alpha protein. Amino
acids 1 to 17 represent the intracellular domain, amino acids 18 to
43 the transmembrane domain (the underlined sequence), and amino
acids 44 to 169 the extracellular domain (the remaining
sequence).
[0028] FIG. 2 shows the regions of similarity between the amino
acid sequences of the endokine alpha protein (SEQ ID NO:2), tissue
necrosis factor .alpha. (TNF-.alpha.) (SEQ ID NO:3), and TNF-.beta.
(SEQ ID NO:4). The J. Hein method was used with PAM 250 residue
weight table. Shading with solid black indicates residues that
match consensus exactly.
[0029] FIG. 3 provides an analysis of the endokine alpha amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, amino acid residues 44-54, 57-68,
69-78, 94-105, 108-132 and 148-158 in FIG. 1 correspond to the
shown highly antigenic regions of the endokine alpha protein.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding an endokine alpha
protein having an amino acid sequence shown in FIG. 1 (SEQ ID
NO:2), which was determined by sequencing a cloned cDNA. Endokine
alpha is a novel member of the tumor necrosis factor (TNF) ligand
family and shares sequence homology with human TNF.alpha. and
related TNF family members (FIG. 2). The nucleotide sequence shown
in FIG. 1 (SEQ ID NO: 1) was obtained by sequencing a cDNA clone,
which was deposited on Jun. 27, 1996, at the American Type Culture
Collection, Patent Depository, 10801 University Boulevard,
Manassas, Va. 20110-2209, and given accession number 97640. The
deposited clone is contained in the pBluescript SK(-) plasmid
(Stratagene, LaJolla, Calif.).
Nucleic Acid Molecules
[0031] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.99% 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 expected 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.
[0032] Unless otherwise indicated, each "nucleotide sequence" set
forth herein is presented as a sequence of deoxyribonucleotides
(abbreviated A, G, C and T). However, 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
deoxynucleotide (T) in the specified deoxynucleotide sequence in is
replaced by the ribonucleotide uridine (U). For instance, reference
to an RNA molecule having the sequence of FIG. 1 (SEQ ID NO: 1) set
forth using deoxyribonucleotide abbreviations is intended to
indicate an RNA molecule having a sequence in which each
deoxynucleotide A, G or C of SEQ ID NO: 1 has been replaced by the
corresponding ribonucleotide A, G or C, and each deoxynucleotide T
has been replaced by a ribonucleotide U.
[0033] Using the information provided herein, such as the
nucleotide sequence in FIG. 1, a nucleic acid molecule of the
present invention encoding an endokine alpha polypeptide can be
obtained using standard cloning and screening procedures, such as
those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in FIG. 1 (SEQ ID NO: 1) was discovered in a cDNA library derived
from human brain striatum. Expressed sequence tags corresponding to
a portion of the endokine alpha cDNA were also found in several
endothelial libraries and a fetal liver library.
[0034] The endokine alpha gene contains an open reading frame
encoding a protein of about 169 amino acid residues, an
intracellular domain of about 17 amino acids (amino acid residues
from about 1 to about 17 in FIG. 1 (SEQ ID NO:2)), a transmembrane
domain of about 26 amino acids (amino acid residues from about 18
to about 43 in FIG. 1 (SEQ ID NO:2)), an extracellular domain of
about 126 amino acids (amino acid residues from about 44 to about
169 in FIG. 1 (SEQ ID NO:2)); and a deduced molecular weight of
about 19 kDa. The endokine alpha protein shown in FIG. 1 (SEQ ID
NO: 2) is about 30% similar and about 22% identical to human
TNF-.alpha., which can be accessed on GenBank as Accession No.
U42764.
[0035] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above the actual
endokine alpha polypeptide encoded by the deposited cDNA comprises
about 169 amino acids, but can be anywhere in the range of about
154-184 amino acids. It will also be appreciated by reasonable
persons of skill in the art that, depending on the criteria used,
the exact `address` of the above-described endokine alpha protein
domains may differ. Thus, for example, the exact location of the
endokine alpha intracellular, transmembrane and extracellular
domains shown in FIG. 1 (SEQ ID NO:2) may vary slightly (e.g., the
exact address may differ by about 1 to about 5 residues compared to
that shown in FIG. 1) depending on the criteria used to define the
domain.
[0036] 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 can be the coding strand,
also known as the sense strand, or it can be the non-coding strand,
also referred to as the anti-sense strand.
[0037] 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.
[0038] However, a nucleic acid contained in a clone that is a
member of a library (e.g., a genomic or cDNA library) that has not
been isolated from other members of the library (e.g., in the form
of a homogeneous solution containing the clone and other members of
the library) or a chromosome isolated or removed from a cell or a
cell lysate (e.g., a "chromosome spread," as in a karyotype), is
not "isolated" for the purposes of the invention. As discussed
further herein, isolated nucleic acid molecules according to the
present invention may be produced naturally, recombinantly, or
synthetically.
[0039] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising the open reading frame (ORF) shown
in FIG. 1 (SEQ ID NO: 1) and further include nucleic acid molecules
substantially different than all or part of the ORF sequence shown
in FIG. 1 (SEQ ID NO: 1) but which, due to the degeneracy of the
genetic code, still encode the endokine alpha protein or a fragment
thereof. 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.
[0040] In another aspect, the invention provides isolated nucleic
acid molecules encoding the endokine alpha polypeptide having an
amino acid sequence encoded by the cDNA of the clone deposited as
ATCC Deposit No. 97640 on Jun. 27, 1996. The invention further
provides an isolated nucleic acid molecule having the nucleotide
sequence shown in FIG. 1 (SEQ ID NO: 1) or the nucleotide sequence
of the endokine 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 endokine alpha gene in human tissue,
for instance, by Northern blot analysis. As described in detail
below, detecting altered endokine alpha gene expression in certain
tissues or bodily fluids is indicative of certain disorders.
[0041] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in FIG. 1
(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, 100, 150, 200,
250, 300, 350, 400, 450, and 500 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 FIG. 1 (SEQ ID NO: 1). In this context, "about"
includes the particularly recited value and values larger or
smaller by several (5, 4, 3, 2 or 1) nucleotides. 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 FIG. 1
(SEQ ID NO: 1). Since the gene has been deposited and the
nucleotide sequence shown in FIG. 1 (SEQ ID NO 1) is provided,
generating such DNA fragments would be routine to the skilled
artisan. For example, restriction endonuclease cleavage or shearing
by sonication could easily be used to generate fragments of various
sizes. Alternatively, such fragments could be generated
synthetically.
[0042] In addition, the present inventors have also identified the
following related cDNA clone: HEMCG04R (SEQ ID NO: 11), which, by
BLAST analysis has 94% identity to nucleotides 26 to 482 of SEQ ID
NO: 1.
[0043] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding: a polypeptide comprising
or, alternatively, consisting of, the endokine alpha intracellular
domain (amino acid residues from about 1 to about 17 in FIG. 1 (SEQ
ID NO 2), or as encoded by the cDNA clone contained in ATCC Deposit
No. 97640); a polypeptide comprising or, alternatively, consisting
of, the endokine alpha transmembrane domain (amino acid residues
from about 18 to about 43 in FIG. 1 (SEQ ID NO 2), or as encoded by
the cDNA clone contained in ATCC Deposit No. 97640); and a
polypeptide comprising or, alternatively, consisting of, the
endokine alpha extracellular domain (amino acid residues from about
44 to about 169 in FIG. 1 (SEQ ID NO:2), or as encoded by the cDNA
clone contained in ATCC Deposit No. 97640).
[0044] Further preferred nucleic acid fragments of the present
invention include nucleic acid molecules encoding epitope-bearing
portions of the endokine alpha protein. In particular, such nucleic
acid fragments of the present invention include nucleic acid
molecules encoding a polypeptide comprising or, alternatively,
consisting of one, two, three or more of any of the following amino
acid sequences and polynucleotides encoding these polypeptides:
amino acid residues from about 44 to about 158 in FIG. 1 (SEQ ID
NO:2); amino acid residues from about 44 to about 54 in FIG. 1 (SEQ
ID NO:2); amino acid residues from about 57 to about 68 in FIG. 1
(SEQ ID NO:2); amino acid residues from about 69 to about 78 in
FIG. 1 (SEQ ID NO:2); amino acid residues from about 94 to about
105 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 108 to
about 132 in FIG. 1 (SEQ ID NO:2); and amino acid residues from
about 148 to about 158 in FIG. 1 (SEQ ID NO:2). The inventors have
determined that the above polypeptide fragments are antigenic
regions of the endokine alpha protein. Methods for determining
other such epitope-bearing portions of the endokine alpha protein
are described in detail below.
[0045] 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 complement of an endokine alpha
polynucleotide fragment described herein, or the cDNA clone
contained in ATCC Deposit 97640 made on Jun. 27, 1996. By
"stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH7.6), 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. By a polynucleotide which hybridizes to a "portion"
of a polynucleotide is intended a polynucleotide (either DNA or
RNA) hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0046] Of course, polynucleotides hybridizing to a larger portion
of the reference polynucleotide (e.g., the deposited cDNA clone),
for instance, a portion 50-500 nt in length, or even to the entire
length of the reference polynucleotide, are also useful as probes
according to the present invention, as are polynucleotides
corresponding to most, if not all, of the nucleotide sequence of
the deposited cDNA or the nucleotide sequence as shown in FIG. 1
(SEQ ID NO: 1). 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 FIG. 1 (SEQ ID NO: 1)). As indicated, such
portions are useful diagnostically either as a probe according to
conventional DNA hybridization techniques or as primers for
amplification of a target sequence by the polymerase chain reaction
(PCR), as described, for instance, in Sambrook, J. et al., eds.,
Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), the
entire disclosure of which is hereby incorporated herein by
reference.
[0047] Since an endokine alpha cDNA clone has been deposited and
its nucleotide sequence is provided in FIG. 1 (SEQ ID NO: 1),
generating polynucleotides which hybridize to a portion of the
endokine alpha cDNA molecule would be routine to the skilled
artisan. For example, restriction endonuclease cleavage or shearing
by sonication of the endokine alpha cDNA clone could easily be used
to generate DNA portions of various sizes which are polynucleotides
that hybridize to a portion of the endokine alpha cDNA molecule.
Alternatively, the hybridizing polynucleotides of the present
invention could be generated synthetically according to known
techniques.
[0048] Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3' terminal poly(A) tract of the endokine
alpha cDNA shown in FIG. 1 (SEQ ID NO: 1)), or to a complementary
stretch of T (or U) resides, would not be included in a
polynucleotide of the invention used to hybridize to a portion of a
nucleic acid of the invention, since such a polynucleotide would
hybridize to any nucleic acid molecule containing a poly(A) stretch
or the complement thereof (e.g., practically any double-stranded
cDNA clone).
[0049] As indicated, nucleic acid molecules of the present
invention that encode an endokine alpha protein may include, but
are not limited to, those encoding the amino acid sequence of the
polypeptide, by itself, the coding sequence for the polypeptide and
additional sequences, such as a pre-, or pro- or prepro-protein
sequence; the coding sequence of the polypeptide, with or without
the aforementioned additional coding sequences, together with
additional, non-coding sequences, including for example, but not
limited to, introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing--including splicing and
polyadenylation signals, e.g., ribosome binding and stability of
mRNA; an additional coding sequence which codes for additional
amino acids, such as those which provide additional
functionalities. Thus, for instance, the sequence encoding the
polypeptide can be fused to a marker sequence, such as a sequence
encoding a peptide which facilitates purification of the fused
polypeptide. In certain preferred embodiments of this aspect of the
invention, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (Qiagen, Inc.),
among others, many of which are publicly and/or 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 (HA) protein,
which has been described by Wilson et al., Cell 37:767 (1984).
Other such fusion proteins include the endokine alpha protein fused
to Fc at the N- or C-terminus.
[0050] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the endokine alpha protein.
Variants can 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.
Non-naturally occurring variants can be produced, e.g., using
art-known mutagenesis techniques.
[0051] Non-naturally occurring variants may be produced using
art-known mutagenesis techniques, which include, but are not
limited to oligonucleotide mediated mutagenesis, alanine scanning,
PCR mutagenesis, site directed mutagenesis (see e.g., Carter et
al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl.
Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells
et al., Gene 34:315 (1985)), restriction selection mutagenesis (see
e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415
(1986)).
[0052] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants can
be altered in coding or non-coding regions or both. Alterations in
the coding regions can 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
endokine alpha protein or portions thereof. Also especially
preferred in this regard are conservative substitutions. Most
highly preferred are nucleic acid molecules encoding the endokine
alpha protein having the amino acid sequence shown in FIG. 1 (SEQ
ID NO:2) or the endokine alpha amino acid sequence encoded by the
deposited cDNA clone.
[0053] Further embodiments of the invention include isolated
nucleic acid molecules comprising or, alternatively, consisting of,
a polynucleotide having a nucleotide sequence at least 80%, 85%,
90%, 92% or 95% identical, and more preferably at least 96%, 97%,
98%, or 99% identical to (a) a nucleotide sequence encoding the
polypeptide having the amino acid sequence in SEQ ID NO:2; (b) a
nucleotide sequence encoding the polypeptide having the amino acid
sequence in SEQ ID NO:2, but lacking the N-terminal methionine; (c)
a nucleotide sequence encoding the polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97640; or (d) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), or (c).
[0054] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding an endokine 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 endokine 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.
[0055] The reference (query) sequence may be the entire endokine
alpha encoding nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1)
or any endokine alpha polynucleotide fragment as described
herein.
[0056] As a practical matter, whether any particular nucleic acid
molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identical to, for instance, the nucleotide sequence shown in FIG. 1
or to the nucleotide sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as the
BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). BESTFIT uses the local
homology algorithm of Smith and Waterman, Adv. Appl. Math.
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.
[0057] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or
the length of the subject nucleotide sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence because of 5' or 3' deletions, not
because of internal deletions, a manual correction is made to the
results to take into consideration the fact that the FASTDB program
does not account for 5' and 3' truncations of the subject sequence
when calculating percent identity. For subject sequences truncated
at the 5' or 3' ends, relative to the query sequence, the percent
identity is corrected by calculating the number of bases of the
query sequence that are 5' and 3' of the subject sequence, which
are not matched/aligned, as a percent of the total bases of the
query sequence. A determination of whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this
embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score. For
example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the
5' end of the subject sequence and therefore, the FASTDB alignment
does not show a match/alignment of the first 10 bases at 5' end.
The 10 unpaired bases represent 10% of the sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in
the query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90 bases
were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100
base query sequence. This time the deletions are internal deletions
so that there are no bases on the 5' or 3' of the subject sequence
which are not matched/aligned with the query. In this case the
percent identity calculated by FASTDB is not manually corrected.
Once again, only bases 5' and 3' of the subject sequence which are
not matched/aligned with the query sequence are manually corrected
for. No other manual corrections are made for the purposes of this
embodiment.
[0058] The present application is directed to such nucleic acid
molecules which are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98%, or 99% identical to a nucleic acid sequence described above
irrespective of whether they encode a polypeptide having endokine
alpha protein activity. This is because, even where a particular
nucleic acid molecule does not encode a polypeptide having endokine
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 endokine alpha activity include, inter alia, (1)
isolating the endokine alpha gene or allelic variants thereof from
a cDNA library; (2) in situ hybridization (FISH) to metaphase
chromosomal spreads to provide precise chromosomal location of the
endokine alpha gene as described in Verma et al., Human
Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York
(1988); and (3) Northern Blot analysis for detecting endokine alpha
mRNA expression in specific tissues.
[0059] Preferred, however, are such nucleic acid molecules having
sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence described above which do, in
fact, encode a polypeptide having endokine alpha protein activity.
By "a polypeptide having endokine alpha activity" is intended
polypeptides exhibiting similar, but not necessarily identical,
activity as compared to the endokine alpha protein as measured in a
particular biological assay. Endokine alpha activity can be assayed
according to known methods. For example, a cytotoxicity assay or
cell proliferation assay can be used where endokine alpha
polypeptides are added to cells in culture and the effect of the
endokine on the cells is determined by measuring the decrease or
increase in cell numbers.
[0060] 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
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence described above will encode a polypeptide
"having endokine alpha protein activity." In fact, since degenerate
variants 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 endokine
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).
[0061] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
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. 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.
[0062] By "a polypeptide having endokine alpha functional activity"
is intended polypeptides exhibiting activity similar, but not
necessarily identical, to an activity of the endokine alpha
receptors of the present invention (either the full-length
polypeptide, or the splice variants), as measured, for example, in
a particular immunoassay or biological assay. For example, endokine
alpha activity can be measured by determining the ability of an
endokine alpha polypeptide to bind an endokine alpha ligand (e.g.,
TR11 (GITR, AITR)). Endokine alpha activity may also be measured by
determining the ability of a polypeptide, such as cognate ligand
which is free or expressed on a cell surface, to stimulate
proliferation, differentiation or activation, or to stimulate
TNF-.alpha. production, and/or to inhibit IL-12 production in cells
expressing the polypeptide, for example, B cells, T cells and
monocytes.
[0063] The present invention is further directed to fragments of
the isolated nucleic acid molecules (i.e. polynucleotides)
described herein. By a fragment of an isolated nucleic acid
molecule having, for example, the nucleotide sequence of the
deposited cDNA (clone 97640), a nucleotide sequence encoding the
polypeptide sequence encoded by the deposited cDNA, a nucleotide
sequence encoding the polypeptide sequence depicted in FIG. 1 (SEQ
ID NO:2), the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1),
or the complementary strand thereto, is intended fragments at least
15 nucleotides, and more preferably at least about 20 nucleotides,
still more preferably at least 30 nucleotides, and even more
preferably, at least about 40, 50, 100, 150, 200, 250, 300, 325,
350, 375, 400, 450, 500, 550, or 600 nucleotides in length. In this
context, "about" includes the particularly recited value and values
larger or smaller by several (5, 4, 3, 2 or 1) nucleotides. These
fragments have numerous uses which include, but are not limited to,
diagnostic probes and primers as discussed herein. Of course,
larger fragments, such as those of 501-1500 nucleotides in length
are also useful according to the present invention as are fragments
corresponding to most, if not all, of the nucleotide sequences of
the deposited cDNA (clone 97640) or as shown in FIG. 1 (SEQ ID NO:
1). By a fragment at least 20 nucleotides in length, for example,
is intended fragments which include 20 or more contiguous bases
from, for example, the nucleotide sequence of the deposited cDNA,
or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO: 1).
[0064] Representative examples of endokine alpha polynucleotide
fragments of the invention include, for example, fragments that
comprise, or alternatively, consist of, a sequence from about
nucleotide 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250,
251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to
550, 551 to 600, 600 to 650, 651 to 700, 701 to 750, 751 to 800,
801 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050, 1051
to 1100, 1101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 1300,
1301 to 1350, 1351 to 1400, 1401 to 1450, 1451 to 1500, 1501 to
1550, 1551 to 1600, 1601 to 1650, 1651 to 1700, 1701 to 1750, 1751
to 1800, and/or 1801 to 1840 of SEQ ID NO: 1, or the complementary
strand thereto, or the cDNA contained in the deposited clone. In
this context "about" includes the particularly recited ranges,
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both termini.
[0065] In specific embodiments, the polynucleotide fragments of the
invention comprise, or alternatively, consist of, a sequence from
nucleotide 961 to 1000, 1730 to 1770, 1770 to 1800, and/or 1800 to
1840, of SEQ ID NO: 1, or the complementary strand thereto.
Polynucleotides that hybridize to these polynucleotide fragments
are also encompassed by the invention.
[0066] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates an endokine alpha
functional activity. By a polypeptide demonstrating "functional
activity" is meant, a polypeptide capable of displaying one or more
known functional activities associated with a full-length endokine
alpha polypeptide. Such functional activities include, but are not
limited to, biological activity (e.g., stimulation of B cell
proliferation, differentiation or activation; stimulation of T cell
proliferation, differentiation or activation; stimulation of
TNF-.alpha.production in monocytes; and/or inhibition of IL-12
production in monocytes), antigenicity (ability to bind (or compete
with an endokine alpha polypeptide for binding) to an anti-endokine
alpha antibody), immunogenicity (ability to generate antibody which
binds to a endokine alpha polypeptide), ability to multimerize with
native endokine alpha and ability to bind to a receptor or ligand
for a endokine alpha polypeptide (e.g., TR11; see U.S. patent
application Ser. No. 09/176,200).
[0067] The functional activity of endokine alpha polypeptides, and
fragments, variants, derivatives, and analogs thereof, can be
assayed by various methods. For example, in one embodiment where
one is assaying for the ability to bind or compete with full-length
endokine alpha polypeptide for binding to anti-endokine alpha
antibody, various immunoassays known in the art can be used,
including, but not limited to, competitive and non-competitive
assay systems using techniques such as radioimmunoassays, ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold,
enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present
invention.
[0068] In another embodiment, where an endokine alpha ligand is
identified (e.g., TR11), or the ability of a polypeptide fragment,
variant or derivative of the invention to multimerize is being
evaluated, binding can be assayed, e.g., by means well-known in the
art, such as, for example, reducing and non-reducing gel
chromatography, protein affinity chromatography, and affinity
blotting. See generally, Phizicky, E., et al., Microbiol. Rev.
59:94-123 (1995). In another embodiment, physiological correlates
(signal transduction) of endokine alpha binding to its substrates
can be assayed.
[0069] In addition, assays described herein (see Examples 5-8) and
methods otherwise known in the art may routinely be applied to
measure the ability of endokine alpha polypeptides and fragments,
variants, derivatives and analogs thereof to elicit endokine alpha
related biological activity (e.g., stimulation of B cell
proliferation, differentiation or activation; stimulation of T cell
proliferation, differentiation or activation; stimulation of
TNF-.alpha. production in monocytes; and/or inhibition of IL-12
production in monocytes in vitro or in vivo). Other methods will be
known to the skilled artisan and are within the scope of the
invention.
[0070] Vectors and Host Cells
[0071] 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 endokine alpha polypeptides or portions thereof
by recombinant techniques.
[0072] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0073] 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.
[0074] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0075] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0076] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids. See, e.g.,
Ausubel, infra; Sambrook, infra.
[0077] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli: lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will include a translation initiating
AUG at the beginning and a termination codon appropriately
positioned at the end of the polypeptide to be translated.
[0078] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include bacterial cells, such as E. coli, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal
cells such as CHO, COS and Bowes melanoma cells; and plant cells.
Appropriate culture media and conditions for the above-described
host cells are known in the art.
[0079] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0080] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0081] 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).
[0082] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0083] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0084] 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. In a further
example, 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, as
indicated, a region(s) also may be added to the polypeptide to
facilitate purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art. A preferred fusion
protein comprises a heterologous region from immunoglobulin that is
useful to solubilize receptors. For example, EP A 0,464,533 (also,
Canadian counterpart 2,045,869) discloses fusion proteins
comprising various portions of constant region of immunoglobin
molecules together with another human protein or part thereof. In
many cases, the Fc part in the fusion protein is thoroughly
advantageous for use in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP A 0,232,262).
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, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(for example, hIL-5). See, D. Bennett et al., Journal of Molecular
Recognition 8:52-58 (1995) and K. Johanson et al., The Journal of
Biological Chemistry 270(16):9459-9471 (1995).
[0085] The endokine 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.
[0086] 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.
[0087] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., endokine alpha
coding sequence), and/or to include genetic material (e.g.,
heterologous polynucleotide sequences) that is operably associated
with endokine alpha polynucleotides of the invention, and which
activates, alters, and/or amplifies endogenous endokine alpha
polynucleotides. For example, techniques known in the art may be
used to operably associate heterologous control regions (e.g.,
promoter and/or enhancer) and endogenous endokine alpha
polynucleotide sequences via homologous recombination (see, e.g.,
U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication No. WO 96/29411, published Sep. 26, 1996; International
Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et
al., Nature 342:435-438 (1989), the disclosures of each of which
are incorporated by reference in their entireties).
Endokine Alpha Polypeptides and Peptides
[0088] The invention further provides an isolated endokine alpha
polypeptide having the amino acid sequence encoded by the deposited
cDNA, or the amino acid sequence in FIG. 1 (SEQ ID NO:2), or a
peptide or polypeptide comprising a portion of the above
polypeptides. The terms "peptide" and "oligopeptide" are considered
synonymous (as is commonly recognized) and each term can be used
interchangeably as the context requires to indicate a chain of at
least two amino acids coupled by peptidyl linkages. The word
"polypeptide" is used herein for chains containing more than ten
amino acid residues. All oligopeptide and polypeptide formulas or
sequences herein are written from left to right and in the
direction from amino terminus to carboxy terminus.
[0089] By "isolated" polypeptide or protein is intended a
polypeptide or protein removed from its native environment. For
example, recombinantly produced polypeptides and proteins expressed
in recombinant host cells are considered isolated for purposes of
the invention as are native or recombinant polypeptides and
proteins which have been substantially purified by any suitable
technique such as, for example, the one-step method described in
Smith and Johnson, Gene 67:31-40 (1988).
[0090] It will be recognized in the art that some amino acid
sequence of the endokine 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. In general, it is possible to replace residues
which form the tertiary structure, provided that residues
performing a similar function are used. In other instances, the
type of residue may be completely unimportant if the alteration
occurs at a non-critical region of the protein.
[0091] Thus, the invention further includes variations of the
endokine alpha polypeptide which show substantial endokine alpha
polypeptide activity or which include regions of endokine alpha
protein such as the protein fragments discussed below. Such mutants
include deletions, insertions, inversions, repeats, and type
substitutions (for example, substituting one hydrophilic residue
for another, but not strongly hydrophilic for strongly hydrophobic
as a rule). Small changes or such "neutral" amino acid
substitutions will generally have little effect on activity.
[0092] 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.
[0093] As indicated in detail above, further guidance concerning
which amino acid changes are likely to be phenotypically silent
(i.e., are not likely to have a significant deleterious effect on a
function) can be found in Bowie, J. U., et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990).
[0094] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0095] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the
endokine alpha protein. The prevention of aggregation is highly
desirable. Aggregation of proteins not only results in a loss of
activity but can also be problematic when preparing pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al.,
Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes
36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug
Carrier Systems 10:307-377 (1993)).
[0096] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993) describes certain mutations resulting in
selective binding of TNF-.alpha. to only one of the two known types
of TNF receptors. Thus, the endokine alpha of the present invention
may include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation.
[0097] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0098] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of substitutions
for any given endokine alpha polypeptide will not be more than 50,
40, 30, 20, 10, 5, or 3, depending on the objective.
[0099] Amino acids in the endokine 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.
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)).
[0100] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell. For example, a recombinantly produced version of the
endokine alpha polypeptide can be substantially purified by the
one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
[0101] The polypeptides of the present invention include the
polypeptides comprising or, alternatively, consisting of: (a) the
complete amino acid sequence as shown in FIG. 1 (SEQ ID NO:2); (b)
the complete amino acid sequence as shown in FIG. 1 (SEQ ID NO:2),
but minus the N-terminal methionine residue; (c) the amino acid
sequence of the endokine alpha polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
97640; and (d) the amino acid sequence of an epitope-bearing
portion of any one of the polypeptides of (a), (b), or (c), as well
as polypeptides which are at least 80%, 85%, 90%, 92% or 95%
identical, more preferably at least 96%, 97%, 98% or 99% identical
to a polypeptide described herein, and also include portions of
such polypeptides with at least 30 amino acids and more preferably
at least 50 amino acids.
[0102] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of an
endokine 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
sequence of the endokine 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.
[0103] As a practical matter, whether any particular polypeptide
comprises or, alternatively, consists of, a sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or
to the amino acid sequence encoded by deposited cDNA clone can be
determined conventionally using known computer programs such the
BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711. When using BESTFIT or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0104] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0105] The present inventors have discovered that the endokine
alpha protein is a 169 residue protein exhibiting three main
structural domains. The intracellular domain was identified within
residues from about 1 to about 17 in FIG. 1 (SEQ ID NO:2). The
transmembrane domain was identified within residues from about 18
to about 43 in FIG. 1 (SEQ ID NO:2). The extracellular domain was
identified within residues from about 44 to about 169 in FIG. 1
(SEQ ID NO:2). Thus, the invention further provides preferred
endokine alpha protein fragments comprising a polypeptide selected
from: the endokine alpha intracellular domain, the transmembrane
domain and the endokine alpha extracellular domain.
[0106] The extracellular domain of the endokine alpha protein can
be combined with parts of the constant domain of immunoglobulins
(IgG), resulting in chimeric polypeptides. These fusion proteins
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 the ligands than
the monomeric extracellular domains alone (Fountoulakis et al, J.
Biochem. 270:3958-3964 (1995)).
[0107] Polypeptide fragments of the present invention include
polypeptides comprising or alternatively, consisting of, an amino
acid sequence contained in SEQ ID NO:2, encoded by the cDNA
contained in the deposited clone, or encoded by nucleic acids which
hybridize (e.g., under stringent hybridization conditions) to the
nucleotide sequence contained in the deposited clone, or shown in
FIG. 1 (SEQ ID NO: 1) or the complementary strand thereto. Protein
fragments may be "free-standing," or comprised within a larger
polypeptide of which the fragment forms a part or region, most
preferably as a single continuous region. Representative examples
of polypeptide fragments of the invention, include, for example,
fragments that comprise or alternatively, consist of from about
amino acid residues: 1 to 50, 51 to 100, 101 to 150 and/or 151 to
169 of SEQ ID NO:2. In this context, "about" includes the
particularly recited ranges and ranges larger or smaller, by
several (5, 4, 3, 2, or 1) amino acids, at either terminus or both
termini. Moreover, polypeptide fragments can be at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 168
amino acids in length. Polynucleotides encoding these polypeptides
are also encompassed by the invention. Polynucleotides that
hybridize to the complement of these encoding polynucleotides are
also encompassed by the invention, as are the polypeptides encoded
by these hybridizing polynucleotides.
[0108] Among the especially preferred fragments of the invention
are fragments characterized by structural or functional attributes
of endokine alpha. Such fragments include amino acid residues that
comprise alpha-helix and alpha-helix-forming regions
("alpha-regions"), beta-sheet and beta-sheet-forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, surface forming regions, and high antigenic
index regions (i.e., containing four or more contiguous amino acids
having an antigenic index of greater than or equal to 1.5, as
identified using the default parameters of the Jameson-Wolf
program) of full-length endokine alpha (SEQ ID NO:2). Certain
preferred regions are those set out in FIG. 3 and include, but are
not limited to, regions of the aforementioned types identified by
analysis of the amino acid sequence depicted in FIG. 1 (SEQ ID
NO:2), such preferred regions include; Garnier-Robson predicted
alpha-regions, beta-regions, turn-regions, and coil-regions;
Chou-Fasman predicted alpha-regions, beta-regions, turn-regions,
and coil-regions; Kyte-Doolittle predicted hydrophilic and
hydrophobic regions; Eisenberg alpha and beta amphipathic regions;
Emini surface-forming regions; and Jameson-Wolf high antigenic
index regions, as predicted using the default parameters of these
computer programs. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0109] The data representing the structural or functional
attributes of endokine alpha set forth in FIG. 3 and/or Table 2, as
described above, was generated using the various modules and
algorithms of the DNA*STAR set on default parameters. In a
preferred embodiment, the data presented in columns VIII, IX, XIII,
and XIV of Table 2 can be used to determine regions of endokine
alpha which exhibit a high degree of potential for antigenicity.
Regions of high antigenicity are determined from the data presented
in columns VIII, IX, XIII, and/or IV by choosing values which
represent regions of the polypeptide which are likely to be exposed
on the surface of the polypeptide in an environment in which
antigen recognition may occur in the process of initiation of an
immune response.
[0110] Certain preferred regions in these regards are set out in
FIG. 3, but may, as shown in Table 2, be represented or identified
by using tabular representations of the data presented in FIG. 3.
The DNA*STAR computer algorithm used to generate FIG. 3 (set on the
original default parameters) was used to present the data in FIG. 3
in a tabular format (See Table 2). The tabular format of the data
in FIG. 3 may be used to easily determine specific boundaries of a
preferred region.
[0111] The above-mentioned preferred regions set out in FIG. 3 and
in Table 2 include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIG. 3. As set out in FIG. 3 and in Table 2,
such preferred regions include Garnier-Robson alpha-regions,
beta-regions, turn-regions, and coil-regions, Chou-Fasman
alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle
hydrophilic regions and hydrophobic regions, Eisenberg alpha- and
beta-amphipathic regions, Karplus-Schulz flexible regions, Emini
surface-forming regions and Jameson-Wolf regions of high antigenic
index.
[0112] Amino and Carboxy Terminal Deletions. As mentioned above,
even if deletion of one or more amino acids from the N-terminus of
a protein results in modification or loss of one or more biological
functions of the protein, other biological activities may still be
retained. Thus, the ability of shortened endokine alpha Madonnas to
induce and/or bind to antibodies which recognize the complete or
mature forms of the polypeptides generally will be retained when
less than the majority of the residues of the complete or mature
polypeptide are removed from the N-terminus. Whether a particular
polypeptide lacking N-terminal residues of a complete polypeptide
retains such immunologic activities can readily be determined by
routine methods described herein and otherwise known in the art. It
is not unlikely that an endokine alpha mutein with a large number
of deleted N-terminal amino acid residues may retain some
biological or immunogenic activities. In fact, peptides composed of
as few as six endokine alpha amino acid residues may often evoke an
immune response.
[0113] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the endokine alpha amino acid sequence shown in FIG. 1
(i.e., SEQ ID NO:2), up to the asparagine residue at position
number 164 and polynucleotides encoding such polypeptides. In
particular, the present invention provides polypeptides comprising
the amino acid sequence of residues n-169 of FIG. 1 (SEQ ID NO:2),
where n is an integer in the range of 2 to 164, and 165 is the
position of the first residue from the N-terminus of the complete
endokine alpha polypeptide believed to be required for at least
immunogenic activity of the endokine alpha polypeptide.
[0114] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of a member selected from the group
consisting of: residues P-2 to S-169; L-3 to S-169; S-4 to S-169;
H-5 to S-169; S-6 to S-169; R-7 to S-169; T-8 to S-169; Q-9 to
S-169; G-10 to S-169; A-11 to S-169; Q-12 to S-169; R-13 to S-169;
S-14 to S-169; S-15 to S-169; W-16 to S-169; K-17 to S-169; L-18 to
S-169; W-19 to S-169; L-20 to S-169; F-21 to S-169; C-22 to S-169;
S-23 to S-169; 1-24 to S-169; V-25 to S-169; M-26 to S-169; L-27 to
S-169; L-28 to S-169; F-29 to S-169; L-30 to S-169; C-31 to S-169;
S-32 to S-169; F-33 to S-169; S-34 to S-169; W-35 to S-169; L-36 to
S-169; 1-37 to S-169; F-38 to S-169; 1-39 to S-169; F-40 to S-169;
L-41 to S-169; Q-42 to S-169; L-43 to S-169; E-44 to S-169; T-45 to
S-169; A-46 to S-169; K-47 to S-169; E-48 to S-169; P-49 to S-169;
C-50 to S-169; M-51 to S-169; A-52 to S-169; K-53 to S-169; F-54 to
S-169; G-55 to S-169; P-56 to S-169; L-57 to S-169; P-58 to S-169;
S-59 to S-169; K-60 to S-169; W-61 to S-169; Q-62 to S-169; M-63 to
S-169; A-64 to S-169; S-65 to S-169; S-66 to S-169; E-67 to S-169;
P-68 to S-169; P-69 to S-169; C-70 to S-169; V-71 to S-169; N-72 to
S-169; K-73 to S-169; V-74 to S-169; S-75 to S-169; D-76 to S-169;
W-77 to S-169; K-78 to S-169; L-79 to S-169; E-80 to S-169; 1-81 to
S-169; L-82 to S-169; Q-83 to S-169; N-84 to S-169; G-85 to S-169;
L-86 to S-169; Y-87 to S-169; L-88 to S-169; 1-89 to S-169; Y-90 to
S-169; G-91 to S-169; Q-92 to S-169; V-93 to S-169; A-94 to S-169;
P-95 to S-169; N-96 to S-169; A-97 to S-169; N-98 to S-169; Y-99 to
S-169; N-100 to S-169; D-101 to S-169; V-102 to S-169; A-103 to
S-169; P-104 to S-169; F-105 to S-169; E-106 to S-169; V-107 to
S-169; R-108 to S-169; L-109 to S-169; Y-110 to S-169; K-111 to
S-169; N-112 to S-169; K-113 to S-169; D-114 to S-169; M-115 to
S-169; 1-116 to S-169; Q-117 to S-169; T-118 to S-169; L-119 to
S-169; T-120 to S-169; N-121 to S-169; K-122 to S-169; S-123 to
S-169; K-124 to S-169; I-125 to S-169; Q-126 to S-169; N-127 to
S-169; V-128 to S-169; G-129 to S-169; G-130 to S-169; T-131 to
S-169; Y-132 to S-169; E-133 to S-169; L-134 to S-169; H-135 to
S-169; V-136 to S-169; G-137 to S-169; D-138 to S-169; T-139 to
S-169; I-140 to S-169; D-141 to S-169; L-142 to S-169; I-143 to
S-169; F-144 to S-169; N-145 to S-169; S-146 to S-169; E-147 to
S-169; H-148 to S-169; Q-149 to S-169; V-150 to S-169; L-151 to
S-169; K-152 to S-169; N-153 to S-169; N-154 to S-169; T-155 to
S-169; Y-156 to S-169; W-157 to S-169; G-158 to S-169; 1-159 to
S-169; 1-160 to S-169; L-161 to S-169; L-162 to S-169; A-163 to
S-169; and N-164 to S-169 of the endokine alpha sequence shown in
FIG. 1. The present invention is also directed to nucleic acid
molecules comprising or, alternatively, consisting of, a
polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or 99% identical to the polynucleotide sequences encoding the
endokine alpha polypeptides described above. The present invention
also encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
[0115] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification or loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened endokine alpha mutein to induce and/or
bind to antibodies which recognize the complete or mature forms of
the polypeptide generally will be retained when less than the
majority of the residues of the complete or mature polypeptide are
removed from the C-terminus. Whether a particular polypeptide
lacking C-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that an endokine alpha mutein with a large number of deleted
C-terminal amino acid residues may retain some biological or
immunogenic activities. In fact, peptides composed of as few as six
endokine alpha amino acid residues may often evoke an immune
response.
[0116] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the endokine alpha
polypeptide shown in FIG. 1 (SEQ ID NO:2), up to the serine residue
at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides comprising the amino acid sequence of residues 1-m of
FIG. 1 (i.e., SEQ ID NO:2), where m is an integer in the range of 6
to 169, and 6 is the position of the first residue from the
C-terminus of the complete endokine alpha polypeptide believed to
be required for at least immunogenic activity of the endokine alpha
polypeptide.
[0117] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of a member selected from the group
consisting of: residues M-1 to I-168; M-1 to F-167; M-1 to Q-166;
M-1 to P-165; M-1 to N-164; M-1 to A-163; M-1 to L-162; M-1 to
L-161; M-1 to I-160; M-1 to I-159; M-1 to G-158; M-1 to W-157; M-1
to Y-156; M-1 to T-155; M-1 to N-154; M-1 to N-153; M-1 to K-152;
M-1 to L-151; M-1 to V-150; M-1 to Q-149; M-1 to H-148; M-1 to
E-147; M-1 to S-146; M-1 to N-145; M-1 to F-144; M-1 to I-143; M-1
to L-142; M-1 to D-141; M-1 to I-140; M-1 to T-139; M-1 to D-138;
M-1 to G-137; M-1 to V-136; M-1 to H-135; M-1 to L-134; M-1 to
E-133; M-1 to Y-132; M-1 to T-131; M-1 to G-130; M-1 to G-129; M-1
to V-128; M-1 to N-127; M-1 to Q-126; M-1 to I-125; M-1 to K-124;
M-1 to S-123; M-1 to K-122; M-1 to N-121; M-1 to T-120; M-1 to
L-119; M-1 to T-118; M-1 to Q-117; M-1 to I-116; M-1 to M-115; M-1
to D-114; M-1 to K-113; M-1 to N-112; M-1 to K-111; M-1 to Y-110;
M-1 to L-109; M-1 to R-108; M-1 to V-107; M-1 to E-106; M-1 to
F-105; M-1 to P-104; M-1 to A-103; M-1 to V-102; M-1 to D-101; M-1
to N-100; M-1 to Y-99; M-1 to N-98; M-1 to A-97; M-1 to N-96; M-1
to P-95; M-1 to A-94; M-1 to V-93; M-1 to Q-92; M-1 to G-91; M-1 to
Y-90; M-1 to I-89; M-1 to L-88; M-1 to Y-87; M-1 to L-86; M-1 to
G-85; M-1 to N-84; M-1 to Q-83; M-1 to L-82; M-1 to I-81; M-1 to
E-80; M-1 to L-79; M-1 to K-78; M-1 to W-77; M-1 to D-76; M-1 to
S-75; M-1 to V-74; M-1 to K-73; M-1 to N-72; M-1 to V-71; M-1 to
C-70; M-1 to P-69; M-1 to P-68; M-1 to E-67; M-1 to S-66; M-1 to
S-65; M-1 to A-64; M-1 to M-63; M-1 to Q-62; M-1 to W-61; M-1 to
K-60; M-1 to S-59; M-1 to P-58; M-1 to L-57; M-1 to P-56; M-1 to
G-55; M-1 to F-54; M-1 to K-53; M-1 to A-52; M-1 to M-51; M-1 to
C-50; M-1 to P-49; M-1 to E-48; M-1 to K-47; M-1 to A-46; M-1 to
T-45; M-1 to E-44; M-1 to L-43; M-1 to Q-42; M-1 to L-41; M-1 to
F-40; M-1 to I-39; M-1 to F-38; M-1 to I-37; M-1 to L-36; M-1 to
W-35; M-1 to S-34; M-1 to F-33; M-1 to S-32; M-1 to C-31; M-1 to
L-30; M-1 to F-29; M-1 to L-28; M-1 to L-27; M-1 to M-26; M-1 to
V-25; M-1 to I-24; M-1 to S-23; M-1 to C-22; M-1 to F-21; M-1 to
L-20; M-1 to W-19; M-1 to L-18; M-1 to K-17; M-1 to W-16; M-1 to
S-15; M-1 to S-14; M-1 to R-13; M-1 to Q-12; M-1 to A-11; M-1 to
G-10; M-1 to Q-9; M-1 to T-8; M-1 to R-7; and M-1 to S-6 of the
sequence of the endokine alpha sequence shown in FIG. 1. The
present invention is also directed to nucleic acid molecules
comprising or, alternatively, consisting of, a polynucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the polynucleotide sequences encoding the endokine
alpha polypeptides described above. The present invention also
encompasses the above polynucleotide sequences fused to a
heterologous polynucleotide sequence. Polypeptides encoded by these
polynucleotide sequences are also encompassed by the invention.
[0118] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
an endokine alpha polypeptide, which may be described generally as
having residues n-m of FIG. 1 (i.e., SEQ ID NO:2), where n and m
are integers as described above.
[0119] The endokine alpha polypeptides of the invention may be in
monomers or multimers (i.e., dimers, trimers, tetramers and higher
multimers). Accordingly, the present invention relates to monomers
and multimers of the endokine alpha polypeptides of the invention,
their preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the
polypeptides of the invention are monomers, dimers, trimers or
tetramers. In additional embodiments, the multimers of the
invention are at least dimers, at least trimers, or at least
tetramers.
[0120] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only endokine alpha polypeptides of the invention
(including endokine alpha fragments, variants, and fusion proteins,
as described herein). These homomers may contain endokine alpha
polypeptides having identical or different amino acid sequences. In
a specific embodiment, a homomer of the invention is a multimer
containing only endokine alpha polypeptides having an identical
amino acid sequence. In another specific embodiment, a homomer of
the invention is a multimer containing endokine alpha polypeptides
having different amino acid sequences. In specific embodiments, the
multimer of the invention is a homodimer (e.g., containing endokine
alpha polypeptides having identical or different amino acid
sequences) or a homotrimer (e.g., containing endokine alpha
polypeptides having identical or different amino acid sequences).
In additional embodiments, the homomeric multimer of the invention
is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
[0121] As used herein, the term heteromer refers to a multimer
containing heterologous polypeptides (i.e., polypeptides of a
different protein) in addition to the endokine alpha fragments and
endokine alpha polypeptides of the invention. In a specific
embodiment, the multimer of the invention is a heterodimer, a
heterotrimer, or a heterotetramer. In additional embodiments, the
heteromeric multimer of the invention is at least a heterodimer, at
least a heterotrimer, or at least a heterotetramer.
[0122] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the endokine alpha
polypeptides of the invention. Such covalent associations may
involve one or more amino acid residues contained in the
polypeptide sequence (e.g., that recited in SEQ ID NO:2, or
contained in the polypeptide encoded by the clone 97640). In one
instance, the covalent associations are cross-linking between
cysteine residues located within the polypeptide sequences which
interact in the native (i.e., naturally occurring) polypeptide. In
another instance, the covalent associations are the consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may involve one or more amino acid residues contained
in the heterologous polypeptide sequence in an endokine alpha
fusion protein. In one example, covalent associations are between
the heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in a endokine alpha-Fc fusion protein of the
invention (as described herein). In another specific example,
covalent associations of fusion proteins of the invention are
between heterologous polypeptide sequence from another TNF family
ligand/receptor member that is capable of forming covalently
associated multimers, such as for example, oseteoprotegerin (see,
e.g., International Publication No. WO 98/49305, the contents of
which are herein incorporated by reference in its entirety).
[0123] In another embodiment, two or more endokine alpha
polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising
multiple endokine alpha polypeptides separated by peptide linkers
may be produced using conventional recombinant DNA technology.
[0124] Another method for preparing multimer endokine alpha
polypeptides of the invention involves use of endokine alpha
polypeptides fused to a leucine zipper or isoleucine zipper
polypeptide sequence. Leucine zipper and isoleucine zipper domains
are polypeptides that promote multimerization of the proteins in
which they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., Science 240:1759,
(1988)), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
multimeric endokine alpha proteins are those described in PCT
application WO 94/10308, hereby incorporated by reference.
Recombinant fusion proteins comprising a soluble endokine alpha
polypeptide fused to a peptide that dimerizes or trimerizes in
solution are expressed in suitable host cells, and the resulting
soluble multimeric endokine alpha is recovered from the culture
supernatant using techniques known in the art.
[0125] Certain members of the TNF family of proteins are believed
to exist in trimeric form (Beutler and Huffel, Science 264:667,
1994; Banner et al., Cell 73:431, 1993). Thus, trimeric endokine
alpha may offer the advantage of enhanced biological activity.
Preferred leucine zipper moieties are those that preferentially
form trimers. One example is a leucine zipper derived from lung
surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric endokine alpha.
[0126] In another example, proteins of the invention are associated
by interactions between Flag.RTM. polypeptide sequence contained in
Flag.RTM.-endokine alpha fusion proteins of the invention. In a
further embodiment, associations proteins of the invention are
associated by interactions between a heterologous polypeptide
sequence contained in Flag.RTM.-endokine alpha fusion proteins of
the invention and anti-Flag.RTM. antibody.
[0127] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0128] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain and which can be incorporated by membrane
reconstitution techniques into liposomes (see, e.g., U.S. Pat. No.
5,478,925, which is herein incorporated by reference in its
entirety).
[0129] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller, M., et al., 1984, Nature
310:105-111). For example, a peptide corresponding to a fragment of
the endokine alpha polypeptides of the invention can be synthesized
by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino acids or chemical amino acid analogs can be
introduced as a substitution or addition into the endokine alpha
polynucleotide sequence. Non-classical amino acids include, but are
not limited to, to the D-isomers of the common amino acids,
2,4-diaminobutyric acid, .alpha.-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids,
and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0130] The invention encompasses endokine alpha polypeptides which
are differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including, but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0131] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends,
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0132] Also provided by the invention are chemically modified
derivatives of endokine alpha which may provide additional
advantages such as increased solubility, stability and circulating
time of the polypeptide, or decreased immunogenicity (see U.S. Pat.
No. 4,179,337). The chemical moieties for derivitization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0133] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0134] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0135] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective proteins chemically modified at the
N-terminus modification may be accomplished by reductive alkylation
which exploits differential reactivity of different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0136] The polypeptides of the present invention have uses which
include, but are not limited to, as sources for generating
antibodies that bind the polypeptides of the invention, and as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0137] Protein Modification
[0138] In addition, proteins of the invention can be chemically
synthesized using techniques known in the art (see, e.g.,
Creighton, Proteins: Structures and Molecular Principles, W.H.
Freeman & Co., N.Y. (1983), and Hunkapiller, M., et al., Nature
310:105-111 (1984)). For example, a peptide corresponding to a
fragment of the endokine-alpha polypeptides of the invention can be
synthesized by use of a peptide synthesizer. Furthermore, if
desired, nonclassical amino acids or chemical amino acid analogs
can be introduced as a substitution or addition into the
endokine-alpha polypeptide sequence. Non-classical amino acids
include, but are not limited to, to the D-isomers of the common
amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, alpha-Abu,
alpha-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, alpha-alanine, fluoro-amino acids, designer
amino acids such as alpha-methyl amino acids, Ca-methyl amino
acids, Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acids can be D (dextrorotary) or L
(levorotary).
[0139] Non-naturally occurring variants may be produced using
art-known mutagenesis techniques, which include, but are not
limited to oligonucleotide mediated mutagenesis, alanine scanning,
PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et
al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl.
Acids Res. 10:6487 (1982)), cassette mutagenesis (see, e.g., Wells
et al., Gene 34:315 (1985)), and restriction selection mutagenesis
(see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA
317:415 (1986)).
[0140] The invention additionally, encompasses endokine-alpha
polypeptides which are differentially modified during or after
translation, e.g., by glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including, but not limited to,
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH.sub.4, acetylation,
formylation, oxidation, reduction, metabolic synthesis in the
presence of tunicamycin, etc.
[0141] Additional post-translational modifications encompassed by
the invention include, for example, N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends,
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0142] Also provided by the invention are chemically modified
derivatives of endokine alpha which may provide additional
advantages such as increased solubility, stability and circulating
time of the polypeptide, or decreased immunogenicity (see U.S. Pat.
No. 4,179,337). The chemical moieties for derivitization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0143] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,
90,000, 95,000, or 100,000 kDa.
[0144] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al, Appl. Biochem.
Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al, Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
[0145] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on the functional or antigenic domains of the protein.
There are a number of attachment methods available to those skilled
in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues, glutamic acid residues
and the C-terminal amino acid residue. Sulfhydryl groups may also
be used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0146] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to a protein via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0147] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective proteins chemically modified at the
N-terminus may be accomplished by reductive alkylation which
exploits differential reactivity of different types of primary
amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0148] As indicated above, pegylation of the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol may be attached to the protein either directly
or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et
al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,
the disclosures of each of which are incorporated herein by
reference.
[0149] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monmethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[0150] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of
which is incorporated herein by reference. Pegylated protein
products produced using the reaction chemistries set out herein are
included within the scope of the invention.
[0151] The number of polyethylene glycol moieties attached to each
protein of the invention (i.e., the degree of substitution) may
also vary. For example, the pegylated proteins of the invention may
be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the
average degree of substitution within ranges such as 1-3, 2-4, 3-5,
4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule. Methods for determining the degree of
substitution are discussed, for example, in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0152] Antibodies and Epitopes
[0153] As described in detail below, the polypeptides of the
present invention can be used to raise polyclonal and monoclonal
antibodies, which are useful in diagnostic assays for detecting
endokine alpha protein expression as described below or as agonists
and antagonists capable of inhibiting endokine alpha protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" endokine alpha protein binding
proteins which are also candidate agonist and antagonist according
to the present invention. The yeast two hybrid system is described
in Fields and Song, Nature 340:245-246 (1989).
[0154] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. These immunogenic epitopes are believed to be confined
to a few loci on the molecule. On the other hand, a region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen, H. M. et al., Proc. Natl. Acad. Sci. USA
81:3998-4002 (1984).
[0155] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G. et al., Science 219:660-666 (1983). Peptides
capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Peptides that are extremely hydrophobic and those of six or fewer
residues generally are ineffective at inducing antibodies that bind
to the mimicked protein; longer, soluble peptides, especially those
containing proline residues, usually are effective. Sutcliffe et
al., supra, at 661. For instance, 18 of 30 peptides designed
according to these guidelines, containing 8-39 residues covering
75% of the sequence of the influenza virus hemagglutinin HA1
polypeptide chain, induced antibodies that reacted with the HA1
protein or intact virus; and 12/12 peptides from the MuLV
polymerase and 18/18 from the rabies glycoprotein induced
antibodies that precipitated the respective proteins.
[0156] 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. Thus, a high proportion of hybridomas obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with
the native protein. Sutcliffe et al., supra, at 663. The antibodies
raised by antigenic epitope-bearing peptides or polypeptides are
useful to detect the mimicked protein, and antibodies to different
peptides may be used for tracking the fate of various regions of a
protein precursor which undergoes posttranslational processing. The
peptides and anti-peptide antibodies may be used in a variety of
qualitative or quantitative assays for the mimicked protein, for
instance in competition assays since it has been shown that even
short peptides (e.g., about 9 amino acids) can bind and displace
the larger peptides in immunoprecipitation assays. See, for
instance, Wilson, I. A. et al., Cell 37:767-778 (1984) at 777. The
anti-peptide antibodies of the invention also are useful for
purification of the mimicked protein, for instance, by adsorption
chromatography using methods well known in the art.
[0157] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 15 to about 30 amino acids
contained within the amino acid sequence of a polypeptide of the
invention. However, peptides or polypeptides comprising a larger
portion of an amino acid sequence of a polypeptide of the
invention, containing about 30 to about 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are considered epitope-bearing
peptides or polypeptides of the invention and also are useful for
inducing antibodies that react with the mimicked protein.
Preferably, the amino acid sequence of the epitope-bearing peptide
is selected to provide substantial solubility in aqueous solvents
(i.e., the sequence includes relatively hydrophilic residues and
highly hydrophobic sequences are preferably avoided); and sequences
containing proline residues are particularly preferred.
[0158] Non-limiting examples of antigenic polypeptides that can be
used to generate endokine-specific polyclonal and monoclonal
antibodies include a polypeptide comprising or, alternatively,
consisting of one, two, three or more of any of the following amino
acid sequences and polynucleotides encoding these polypeptides:
amino acid residues from about 44 to about 158 in FIG. 1 (SEQ ID
NO:2); amino acid residues from about 44 to about 54 in FIG. 1 (SEQ
ID NO:2); amino acid residues from about 57 to about 68 in FIG. 1
(SEQ ID NO:2); amino acid residues from about 69 to about 78 in
FIG. 1 (SEQ ID NO:2); amino acid residues from about 94 to about
105 in FIG. 1 (SEQ ID NO:2); amino acid residues from about 108 to
about 132 in FIG. 1 (SEQ ID NO:2); and amino acid residues from
about 148 to about 158 in FIG. 1 (SEQ ID NO:2). As indicated above,
the inventors have determined that the above polypeptide fragments
are antigenic regions of the endokine alpha protein.
[0159] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger
polypeptide which acts as a carrier during recombinant production
and purification, as well as during immunization to produce
anti-peptide antibodies. Epitope-bearing peptides also may be
synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks. See, Houghten, R. A., Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Pat. No. 4,631,211 to
Houghten et al. (1986). In this procedure the individual resins for
the solid-phase synthesis of various peptides are contained in
separate solvent-permeable packets, enabling the optimal use of the
many identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously. Houghten et al, supra, at 5134.
[0160] 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).
Generally, animals may be immunized with free peptide; however,
anti-peptide antibody titer may be boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet
hemocyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde.
[0161] Animals such as rabbits, rats and mice are immunized with
either free or carrier-coupled peptides, for instance, by
intraperitoneal and/or intradermal injection of emulsions
containing about 100 .mu.g peptide or carrier protein and Freund's
adjuvant. Several booster injections may be needed, for instance,
at intervals of about two weeks, to provide a useful titer of
anti-peptide antibody which can be detected, for example, by ELISA
assay using free peptide adsorbed to a solid surface. The titer of
anti-peptide antibodies in serum from an immunized animal may be
increased by selection of anti-peptide antibodies, for instance, by
adsorption to the peptide on a solid support and elution of the
selected antibodies according to methods well known in the art.
[0162] 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. For instance, Geysen et al. (1984), supra,
discloses a procedure for rapid concurrent synthesis on solid
supports of hundreds of peptides of sufficient purity to react in
an enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art.
[0163] For instance, the immunologically important epitope in the
coat protein of foot-and-mouth disease virus was located by Geysen
et al. with a resolution of seven amino acids by synthesis of an
overlapping set of all 208 possible hexapeptides covering the
entire 213 amino acid sequence of the protein. Then, a complete
replacement set of peptides in which all 20 amino acids were
substituted in turn at every position within the epitope were
synthesized, and the particular amino acids conferring specificity
for the reaction with antibody were determined. Thus, peptide
analogs of the epitope-bearing peptides of the invention can be
made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen
(1987) further describes this method of identifying a peptide
bearing an immunogenic epitope of a desired protein.
[0164] 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 C.sub.1-C.sub.7-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.
[0165] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. Most preferably the antibodies
are human antigen binding antibody fragments of the present
invention which include, but are not limited to, Fab, Fab' and
F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a
V.sub.L or V.sub.H domain. The antibodies may be from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine, rabbit, goat, guinea pig, camel, horse, or
chicken.
[0166] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the invention
are any combinations of variable region(s) and hinge region, CH1,
CH2, and CH3 domains. The present invention further includes
monoclonal, polyclonal, chimeric, humanized, and human monoclonal
and polyclonal antibodies which specifically bind the polypeptides
of the present invention. The present invention further includes
antibodies which are anti-idiotypic to the antibodies of the
present invention.
[0167] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt, A. et al. (1991) J. Immunol. 147:60-69; U.S. Pat.
Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny, S. A. et al. (1992)J. Immunol. 148:1547-1553.
[0168] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which are recognized or specifically bound
by the antibody. The epitope(s) or polypeptide portion(s) may be
specified as described herein, e.g., by N-terminal and C-terminal
positions, by size in contiguous amino acid residues, or listed in
the Tables and Figures. Antibodies which specifically bind any
epitope or polypeptide of the present invention may also be
excluded. Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0169] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of the polypeptides
of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Further included in the present invention are antibodies which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the
present invention may also be described or specified in terms of
their binding affinity. Preferred binding affinities include those
with a dissociation constant or Kd less than 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-9M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0170] Antibodies of the present invention have uses that include,
but are not limited to, methods known in the art to purify, detect,
and target the polypeptides of the present invention including both
in vitro and in vivo diagnostic and therapeutic methods. For
example, the antibodies have use in immunoassays for qualitatively
and quantitatively measuring levels of the polypeptides of the
present invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference in the
entirety).
[0171] The antibodies of the present invention may be used either
alone or in combination with other compositions. The antibodies may
further be recombinantly fused to a heterologous polypeptide at the
N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions.
For example, antibodies of the present invention may be
recombinantly fused or conjugated to molecules useful as labels in
detection assays and effector molecules such as heterologous
polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO
91/14438; and WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396
387.
[0172] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. The term "monoclonal antibody" is
not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not to the method by which it is produced.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant and phage display technology.
[0173] Hybridoma techniques include those known in the art and
taught in Harlow et al., Antibodies: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,
in: Monoclonal Antibodies and T-cell Hybridomas, pp. 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties).
[0174] Fab and F(ab')2 fragments may be produced by proteolytic
cleavage, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab')2 fragments).
[0175] Alternatively, antibodies of the present invention can be
produced through the application of recombinant DNA technology and
phage display technology or through synthetic chemistry using
methods known in the art. For example, the antibodies of the
present invention can be prepared using various phage display
methods known in the art. In phage display methods, functional
antibody domains are displayed on the surface of a phage particle
which carries polynucleotide sequences encoding them. Phage with a
desired binding property are selected from a repertoire or
combinatorial antibody library (e.g. human or murine) by selecting
directly with antigen, typically antigen bound or captured to a
solid surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 with Fab, Fv or disulfide
stabilized Fv antibody domains recombinantly fused to either the
phage gene III or gene VIII protein. Examples of phage display
methods that can be used to make the antibodies of the present
invention include those disclosed in Brinkman U. et al. (1995) J.
Immunol. Methods 182:41-50; Ames, R. S. et al. (1995) J. Immunol.
Methods 184:177-186; Kettleborough, C. A. et al. (1994) Eur. J.
Immunol. 24:952-958; Persic, L. et al. (1997) Gene 187 9-18;
Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;
PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619;
WO 93/11236; WO 95/15982; and WO 95/20401; and U.S. Pat. Nos.
5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,
5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727
and 5,733,743 (said references incorporated by reference in their
entireties).
[0176] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in WO 92/22324;
Mullinax, R. L. et al., BioTechniques 12(6):864-869 (1992); and
Sawai, H. et al., AJRI 34:26-34 (1995); and Better, M. et al.,
Science 240:1041-1043 (1988) (said references incorporated by
reference in their entireties).
[0177] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu, L. et al., PNAS 90:7995-7999
(1993); and Skerra, A. et al., Science 240:1038-1040 (1988). For
some uses, including in vivo use of antibodies in humans and in
vitro detection assays, it may be preferable to use chimeric,
humanized, or human antibodies. Methods for producing chimeric
antibodies are known in the art. See e.g., Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S.
D. et al., J. Immunol. Methods 125:191-202 (1989); and U.S. Pat.
No. 5,807,715. Antibodies can be humanized using a variety of
techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S.
Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0
592 106; EP 0 519 596; Padlan E. A., Molecular Immunology
28(4/5):489-498 (1991); Studnicka G. M. et al., Protein Engineering
7(6):805-814 (1994); Roguska M. A. et al., PNAS 91:969-973 (1994)),
and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can
be made by a variety of methods known in the art including phage
display methods described above. See also U.S. Pat. Nos. 4,444,887,
4,716,111, 5,545,806, and 5,814,318; and international patent
application publication numbers WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741
(said references incorporated by reference in their
entireties).
[0178] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et
al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981;
Gillies, S. O. et al., PNAS 89:1428-1432 (1992); Fell, H. P. et
al., J. Immunol. 146:2446-2452 (1991) (said references incorporated
by reference in their entireties).
[0179] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the hinge region, CH1 domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to increase the in vivo half life of
the polypeptides or for use in immunoassays using methods known in
the art. The polypeptides may also be fused or conjugated to the
above antibody portions to form multimers. For example, Fc portions
fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher
multimeric forms can be made by fusing the polypeptides to portions
of IgA and IgM. Methods for fusing or conjugating the polypeptides
of the present invention to antibody portions are known in the art.
See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO
96/04388, WO 91/06570; Ashkenazi, A. et al., PNAS 88:10535-10539
(1991); Zheng, X. X. et al., J. Immunol. 154:5590-5600 (1994); and
Vil, H. et al., PNAS 89:11337-11341 (1992) (said references
incorporated by reference in their entireties).
[0180] The invention further relates to antibodies which act as
agonists or antagonists of the polypeptides of the present
invention. For example, the present invention includes antibodies
which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. Also include are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included are neutralizing antibodies
which bind the ligand and prevent binding of the ligand to the
receptor, as well as antibodies which bind the ligand, thereby
preventing receptor activation, but do not prevent the ligand from
binding the receptor. Further included are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. See e.g., WO
96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood
92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.
58(16):3668-3678 (1998); Harrop, J. A. et al., J. Immunol.
161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.
58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.
160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci. 111(Pt
2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard, J. et al., Cytokine 9(4):233-241
(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301
(1997); Taryman, R. E. et al., Neuron 14(4):755-762 (1995); Muller,
Y. A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al.,
Cytokine 8(1):14-20 (1996)(said references incorporated by
reference in their entireties).
[0181] The entire disclosure of each document cited in this section
on "Polypeptides and Peptides" is hereby incorporated herein by
reference.
Epitopes
[0182] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:2, or an epitope of the
polypeptide sequence encoded by a polynucleotide sequence contained
in deposited clone [Deposit information] or encoded by a
polynucleotide that hybridizes to the complement of the sequence of
SEQ ID NO:1 or contained in the clone deposited as ATCC Deposit
Number 97640 on Jun. 27, 1996 under stringent hybridization
conditions or lower stringency hybridization conditions as defined
supra. The present invention further encompasses polynucleotide
sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID
NO: 1), polynucleotide sequences of the complementary strand of a
polynucleotide sequence encoding an epitope of the invention, and
polynucleotide sequences which hybridize to the complementary
strand under stringent hybridization conditions or lower stringency
hybridization conditions defined supra.
[0183] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding, but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0184] Fragments that function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0185] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, and, most preferably, between about
15 to about 30 amino acids. Preferred polypeptides comprising
immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino
acid residues in length. Antigenic epitopes are useful, for
example, to raise antibodies, including monoclonal antibodies, that
specifically bind the epitope. Antigenic epitopes can be used as
the target molecules in immunoassays. (See, e.g., Wilson et al.,
Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666
(1983)).
[0186] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
e.g., Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,
Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen.
Virol. 66:2347-2354 (1985). A preferred immunogenic epitope
includes the secreted protein. The polypeptides comprising one or
more immunogenic epitopes may be presented for eliciting an
antibody response together with a carrier protein, such as an
albumin, to an animal system (such as, for example, rabbit or
mouse), or, if the polypeptide is of sufficient length (at least
about 25 amino acids), the polypeptide may be presented without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10
amino acids have been shown to be sufficient to raise antibodies
capable of binding to, at the very least, linear epitopes in a
denatured polypeptide (e.g., in Western blotting).
[0187] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.
66:2347-2354 (1985). If in vivo immunization is used, animals may
be immunized with free peptide; however, anti-peptide antibody
titer may be boosted by coupling the peptide to a macromolecular
carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
For instance, peptides containing cysteine residues may be coupled
to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as, for example,
rabbits, rats, and mice are immunized with either free or
carrier-coupled peptides, for instance, by intraperitoneal and/or
intradermal injection of emulsions containing about 100 micrograms
of peptide or carrier protein and Freund's adjuvant or any other
adjuvant known for stimulating an immune response. Several booster
injections may be needed, for instance, at intervals of about two
weeks, to provide a useful titer of anti-peptide antibody that can
be detected by, for example, ELISA assay using free peptide
adsorbed to a solid surface. The titer of anti-peptide antibodies
in serum from an immunized animal may be increased by selection of
anti-peptide antibodies, for instance, by adsorption to the peptide
on a solid support and elution of the selected antibodies according
to methods well known in the art.
[0188] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, and IgM), or portions thereof (CH1, CH2, CH3, or any
combination thereof and portions thereof) resulting in chimeric
polypeptides. Such fusion proteins may facilitate purification and
may increase half-life in vivo. This has been shown 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. See, e.g., EP
394,827; Traunecker et al., Nature 331:84-86 (1988). IgG fusion
proteins that have a disulfide-linked dimeric structure due to the
IgG portion desulfide bonds have also been found to be more
efficient in binding and neutralizing other molecules than
monomeric polypeptides or fragments thereof alone. See, e.g.,
Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). Nucleic
acids encoding the above epitopes can also be recombined with a
gene of interest as an epitope tag (e.g., the hemagglutinin ("HA")
tag or flag tag) to aid in detection and purification of the
expressed polypeptide. For example, a system described by Janknecht
et al. allows for the ready purification of non-denatured fusion
proteins expressed in human cell lines (Janknecht et al., Proc.
Natl. Acad. Sci. USA 88:8972-897 (1991)). In this system, the gene
of interest is subcloned into a vaccinia recombination plasmid such
that the open reading frame of the gene is translationally fused to
an amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix-binding domain for the fusion protein. Extracts
from cells infected with the recombinant vaccinia virus are loaded
onto Ni.sup.2+ nitriloacetic acid-agarose column and
histidine-tagged proteins can be selectively eluted with
imidazole-containing buffers.
[0189] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to generate variation in the polynucleotide sequence.
In another embodiment, polynucleotides of the invention, or the
encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. In another embodiment, one or
more components, motifs, sections, parts, domains, fragments, etc.,
of a polynucleotide coding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
Antibodies
[0190] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, preferably an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying
specific antibody-antigen binding). Antibodies of the invention
include, but are not limited to, polyclonal, monoclonal,
multispecific, human, humanized or chimeric antibodies, single
chain antibodies, Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above.
The term "antibody," as used herein, refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that immunospecifically binds an antigen. The immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule.
[0191] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, donkey, ship rabbit,
goat, guinea pig, camel, horse, or chicken. As used herein, "human"
antibodies include antibodies having the amino acid sequence of a
human immunoglobulin and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more
human immunoglobulin and that do not express endogenous
immunoglobulins, as described infra and, for example in, U.S. Pat.
No. 5,939,598 by Kucherlapati et al.
[0192] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; and WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; and 5,601,819; Kostelny et al., J. Immunol.
148:1547-1553 (1992).
[0193] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention that they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies that specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0194] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less than 85%, less than 80%, less than 75%, less than
70%, less than 65%, less than 60%, less than 55%, and less than 50%
identity (as calculated using methods known in the art and
described herein) to a polypeptide of the present invention are
also included in the present invention. Further included in the
present invention are antibodies that bind polypeptides encoded by
polynucleotides which hybridize to a polynucleotide of the present
invention under stringent hybridization conditions (as described
herein). Antibodies of the present invention may also be described
or specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2M,
10.sup.-2M, 5.times.10.sup.-3M, 10.sup.-3M, 5.times.10.sup.-4M,
10.sup.-4M, 5.times.10.sup.-5M, 10.sup.-5M, 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0195] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 90%, at least 80%, at
least 70%, at least 60%, or at least 50%.
[0196] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. The invention features both
receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor-specific antibodies which do not
prevent ligand binding, but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. For example,
receptor activation can be determined by detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the
receptor or its substrate by immunoprecipitation followed by
western blot analysis (for example, as described supra). In
specific embodiments, antibodies are provided that inhibit ligand
or receptor activity by at least 90%, at least 80%, at least 70%,
at least 60%, or at least 50% of the activity in absence of the
antibody.
[0197] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation. The antibodies may be
specified as agonists, antagonists or inverse agonists for
biological activities comprising the specific biological activities
of the peptides of the invention disclosed herein. The above
antibody agonists can be made using methods known in the art. See,
e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et
al., Blood 92(6):1981-1988 (1998); Chen, et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon, et al., J. Immunol. 160(7):3170-3179 (1998); Prat et
al., J. Cell. Sci. 111(Pt 2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine
9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):
11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);
Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference
herein in their entireties).
[0198] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0199] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalent and non-covalent
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs, or
toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; and
WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
[0200] The antibodies of the invention include derivatives that are
modified, e.g., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to, specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0201] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include, but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0202] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0203] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well-known in the art
and are discussed in detail in Example 3. Briefly, mice can be
immunized with a polypeptide of the invention or a cell expressing
such peptide. Once an immune response is detected, e.g., antibodies
specific for the antigen are detected in the mouse serum, the mouse
spleen is harvested and splenocytes isolated. The splenocytes are
then fused by well-known techniques to any suitable myeloma cells,
for example cells from cell line SP20 available from the ATCC.
Hybridomas are selected and cloned by limited dilution. The
hybridoma clones are then assayed by methods known in the art for
cells that secrete antibodies capable of binding a polypeptide of
the invention. Ascites fluid, which generally contains high levels
of antibodies, can be generated by immunizing mice with positive
hybridoma clones.
[0204] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0205] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0206] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0207] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0208] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol.
Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397, which are incorporated herein by reference in their
entireties. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and framework regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties.)
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al., Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS
91:969-973 (1994)), and chain shuffling (U.S. Pat. No.
5,565,332).
[0209] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0210] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(Int. Rev. Immunol. 13:65-93 (1995)). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.
Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0211] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0212] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444 (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
Polynucleotides Encoding Antibodies
[0213] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:2.
[0214] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligation of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0215] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be obtained from a
suitable source (e.g., an antibody cDNA library, or a cDNA library
generated therefrom, or nucleic acid, preferably poly A+ RNA,
isolated therefrom, or any tissue or cells expressing the antibody,
such as hybridoma cells selected to express an antibody of the
invention) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR may
then be cloned into replicable cloning vectors using any method
well known in the art.
[0216] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (See, for example,
the techniques described in Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current
Protocols in Molecular Biology, John Wiley & Sons, NY (1998),
which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0217] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0218] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al, Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine m-Ab and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0219] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988,
Science 242:423-42; Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al, Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
Methods of Producing Antibodies
[0220] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0221] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, e.g., a heavy or light
chain of an antibody of the invention, requires construction of an
expression vector containing a polynucleotide that encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody, or portion thereof (preferably
containing the heavy or light chain variable domain), of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
a protein by expressing a polynucleotide containing an antibody
encoding nucleotide sequence are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0222] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, operably linked to a heterologous promoter. In preferred
embodiments for the expression of double-chained antibodies,
vectors encoding both the heavy and light chains may be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
[0223] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0224] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to a matrix glutathione-agarose beads followed by elution
in the presence of free glutathione. The pGEX vectors are designed
to include thrombin or factor Xa protease cleavage sites so that
the cloned target gene product can be released from the GST
moiety.
[0225] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0226] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0227] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0228] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0229] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB
TECH 11(5):155-215 (May 1993)); and hygro, which confers resistance
to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly known in the art of recombinant DNA technology which can
be used are described in Ausubel et al., eds., Current Protocols in
Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and 13, Dracopoli et al., eds, Current
Protocols in Human Genetics, John Wiley & Sons, NY (1994);
Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are
incorporated by reference herein in their entireties.
[0230] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, "The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells," in DNA Cloning,
Vol. 3. (Academic Press, New York, 1987)). When a marker in the
vector system expressing antibody is amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody gene, production of the antibody
will also increase (Crouse et al., Mol. Cell. Biol. 3:257
(1983)).
[0231] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, Nature
322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0232] Once an antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
Antibody Conjugates
[0233] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20 or 50 amino acids of the polypeptide) of
the present invention to generate fusion proteins. The fusion does
not necessarily need to be direct, but may occur through linker
sequences. The antibodies may be specific for antigens other than
polypeptides (or portion thereof, preferably at least 10, 20 or 50
amino acids of the polypeptide) of the present invention. For
example, antibodies may be used to target the polypeptides of the
present invention to particular cell types, either in vitro or in
vivo, by fusing or conjugating the polypeptides of the present
invention to antibodies specific for particular cell surface
receptors. Antibodies fused or conjugated to the polypeptides of
the present invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452 (1991), which are incorporated by
reference in their entireties.
[0234] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341 (1992) (said references incorporated by
reference in their entireties).
[0235] As discussed, supra, the polypeptides of the present
invention may be fused or conjugated to the above antibody portions
to increase the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to facilitate purification. One
reported example describes 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 394,827; Traunecker et al., Nature 331:84-86
(1988)). The polypeptides of the present invention fused or
conjugated to an antibody having disulfide-linked dimeric
structures (due to the IgG) may also be more efficient in binding
and neutralizing other molecules, than the monomeric secreted
protein or protein fragment alone. (Fountoulakis et al., J.
Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a
fusion protein is beneficial in therapy and diagnosis, and thus can
result in, for example, improved pharmacokinetic properties. (EP A
232,262). Alternatively, deleting the Fc part after the fusion
protein has been expressed, detected, and purified, would be
desired. For example, the Fc portion may hinder therapy and
diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have 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);
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0236] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitates their purification. In preferred embodiments, 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. Other
peptide tags useful for purification include, but are not limited
to, the "HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))
and the "flag" tag.
[0237] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal
ions which can be conjugated to antibodies for use as diagnostics
according to the present invention. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc.
[0238] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion. A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0239] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, a thrombotic agent or an anti-angiogenic agent, e.g.,
angiostatin or endostatin; or, biological response modifiers such
as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0240] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0241] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. eds., pp.
243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et
al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. eds., pp. 475-506 (1985); "Analysis,
Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies
For Cancer Detection And Therapy, Baldwin et al. eds., pp. 303-16
(Academic Press 1985), and Thorpe et al., "The Preparation And
Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.
62:119-58 (1982).
[0242] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0243] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
Assays for Antibody Binding
[0244] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
(1994), which is incorporated by reference herein in its entirety).
Exemplary immunoassays are described briefly below (but are not
intended by way of limitation).
[0245] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel (et al., eds, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
(19914) at 10.16.1.
[0246] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel, et al., eds, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
(1994) at 10.8.1.
[0247] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel, et al., eds, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York
(1994) at 11.2.1.
[0248] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest is conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
[0249] Endokine Alpha Related Disorder Diagnosis
[0250] Endokine alpha is a new member of the TNF family of
cytokines. For endokine alpha related disorders, it is believed
that substantially altered (increased or decreased) levels of
endokine alpha gene expression can be detected in 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" endokine alpha gene expression level, that
is, the endokine 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 an endokine
alpha-related disorder, which involves measuring the expression
level of the gene encoding the endokine alpha protein in tissue or
other cells or body fluid from an individual and comparing the
measured gene expression level with a standard endokine alpha gene
expression level, whereby an increase or decrease in the gene
expression level compared to the standard is indicative of an
endokine alpha related disorder.
[0251] By individual is intended mammalian individuals, preferably
humans. By "measuring the expression level of the gene encoding the
endokine alpha protein" is intended qualitatively or quantitatively
measuring or estimating the level of the endokine alpha protein or
the level of the mRNA encoding the endokine 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 endokine alpha protein level or mRNA
level in a second biological sample). Preferably, the endokine
alpha protein level or mRNA level in the first biological sample is
measured or estimated and compared to a standard endokine 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 involving endokine alpha. As
will be appreciated in the art, once a standard endokine alpha
protein level or mRNA level is known, it can be used repeatedly as
a standard for comparison.
[0252] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains endokine alpha protein or mRNA. As
indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain
secreted mature endokine alpha protein, or tissue sources found to
express endokine alpha. 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.
[0253] The present invention is useful for diagnosis of various
endokine alpha-related disorders in mammals, preferably humans, as
similar to TNF-like disorders known in the art or as presented
herein. These include disorders associated with immunomodulation
and inflammation, cell proliferation, angiogenesis, tumor
metastases, apoptosis, sepsis and endotoxemia.
[0254] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the
single-step-guanidinium-thiocyanate-phenol-chloroform method
described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159
(1987). Levels of mRNA encoding an endokine alpha polypeptide 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).
[0255] Northern blot analysis can be performed as described in
Harada et al., Cell 63:303-312 (1990). Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. Endokine alpha protein
cDNA labeled according to any appropriate method (such as the
.sup.32P-multiprimed DNA labeling system (Amersham)) is used as
probe. After hybridization overnight, the filter is washed and
exposed to x-ray film. cDNA for use as probe according to the
present invention is described in the sections above and will
preferably at least 15 bp in length.
[0256] S1 mapping can be performed as described in Fujita et al.,
Cell 49:357-367 (1987). To prepare probe DNA for use in S1 mapping,
the sense strand of above-described cDNA is used as a template to
synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding the endokine alpha protein). Northern
blot analysis can be performed as described above.
[0257] Preferably, levels of mRNA encoding the endokine alpha
protein are assayed using the RT-PCR method described in Makino et
al., Technique 2:295-301 (1990). By this method, the
radioactivities of the "amplicons" in the polyacrylamide gel bands
are linearly related to the initial concentration of the target
mRNA. Briefly, this method involves adding total RNA isolated from
a biological sample in a reaction mixture containing a RT primer
and appropriate buffer. After incubating for primer annealing, the
mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase
inhibitor and reverse transcriptase. After incubation to achieve
reverse transcription of the RNA, the RT products are then subject
to PCR using labeled primers. Alternatively, rather than labeling
the primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding the endokine alpha protein) is quantified using an imaging
analyzer. RT and PCR reaction ingredients and conditions, reagent
and gel concentrations, and labeling methods are well known in the
art. Variations on the RT-PCR method will be apparent to the
skilled artisan.
[0258] Any set of oligonucleotide primers which will amplify
reverse transcribed target mRNA can be used and can be designed as
described in the sections above.
[0259] Assaying endokine alpha protein levels in a biological
sample can occur using any art-known method. Preferred for assaying
endokine alpha protein levels in a biological sample are
antibody-based techniques. For example, endokine alpha protein
expression in tissues can be studied with classical
immunohistological methods. In these, the specific recognition is
provided by the primary antibody (polyclonal or monoclonal), but
the secondary detection system can utilize fluorescent, enzyme, or
other conjugated secondary antibodies. As a result, an
immunohistological staining of tissue section for pathological
examination is obtained. Tissues can also be extracted, e.g., with
urea and neutral detergent, for the liberation of endokine alpha
protein for Western-blot or dot/slot assay (Jalkanen, M., et al.,
J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.
Biol. 105:3087-3096 (1987)). In this technique, which is based on
the use of cationic solid phases, quantitation of endokine alpha
protein can be accomplished using isolated endokine alpha protein
as a standard. This technique can also be applied to body fluids.
With these samples, a molar concentration of endokine alpha protein
will aid to set standard values of endokine alpha protein content
for different body fluids, like serum, plasma, urine, synovial
fluid, spinal fluid, etc. The normal appearance of endokine alpha
protein amounts can then be set using values from healthy
individuals, which can be compared to those obtained from a test
subject.
[0260] Other antibody-based methods useful for detecting endokine
alpha protein levels include immunoassays, such as the enzyme
linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
For example, endokine alpha protein-specific monoclonal antibodies
can be used both as an immunoadsorbent and as an enzyme-labeled
probe to detect and quantify the endokine alpha protein. The amount
of endokine alpha protein present in the sample can be calculated
by reference to the amount present in a standard preparation using
a linear regression computer algorithm. Such an ELISA for detecting
a tumor antigen is described in Tacobelli et al., Breast Cancer
Research and Treatment 11:19-30 (1988). In another ELISA assay, two
distinct specific monoclonal antibodies can be used to detect
endokine alpha protein in a body fluid. In this assay, one of the
antibodies is used as the immunoadsorbent and the other as the
enzyme-labeled probe.
[0261] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting endokine alpha protein with immobilized antibody and,
without washing, contacting the mixture with the labeled antibody.
The "two-step" assay involves washing before contacting the mixture
with the labeled antibody. Other conventional methods may also be
employed as suitable. It is usually desirable to immobilize one
component of the assay system on a support, thereby allowing other
components of the system to be brought into contact with the
component and readily removed from the sample.
[0262] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labeled antibody/substrate reaction. Besides enzymes, other
suitable labels include 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.
[0263] In addition to assaying endokine alpha protein levels in a
biological sample obtained from an individual, endokine alpha
protein can also be detected in vivo by imaging. Antibody labels or
markers for in vivo imaging of endokine 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.
[0264] A endokine alpha protein-specific antibody or antibody
portion which has been labeled with an appropriate detectable
imaging moiety, such as a radioisotope (for example, .sup.131I,
.sup.111In, .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 a 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 moieties 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 portion will then preferentially accumulate at
the location of cells which contain endokine alpha protein. In vivo
tumor imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Portions" (Chapter 13 in Tumor Imaging: The Radiochemical Detection
of Cancer, Burchiel, S. W. and Rhodes, B. A. eds., Masson
Publishing Inc. (1982)).
[0265] Endokine alpha-protein specific antibodies for use in the
present invention can be raised against the intact endokine alpha
protein or an antigenic polypeptide portion thereof, which may
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.
[0266] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody portions (such as, for example, Fab and F(ab').sub.2
portions) which are capable of specifically binding to endokine
alpha protein. Fab and F(ab').sub.2 portions lack the Fc portion 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 portions are
preferred.
[0267] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
endokine alpha protein or an antigenic portion thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies.
[0268] In a preferred method, a preparation of endokine alpha
protein is prepared and purified as described above 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.
[0269] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or endokine alpha protein
binding portions thereof). Such monoclonal antibodies can be
prepared using hybridoma technology (see, e.g., Colligan, Current
Protocols in Immunology, Wiley Interscience, New York (1990-1996);
Harlow & Lane, Antibodies: A Laboratory Manual, Chs. 6-9, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1988); Ausubel,
infra, at Chapter 11, these references entirely incorporated herein
by reference).
[0270] In general, such procedures involve immunizing an animal
(preferably a mouse) with an endokine alpha polypeptide antigen or
with an endokine alpha polypeptide-expressing cell. Suitable cells
can be recognized by their capacity to bind anti-endokine alpha
protein antibody. Such cells may be cultured in any suitable tissue
culture medium (e.g., Earle's modified Eagle's medium supplemented
with 10% fetal bovine serum (inactivated at about 56.degree. C.),
supplemented with about 10 .mu.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 (e.g.,
parent myeloma cell line (SP.sub.2O), available from the American
Type Culture Collection (ATCC) (Manassas, Va., USA)). 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); Harlow & Lane,
infra, Chapter 7. The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding the endokine alpha antigen.
[0271] Alternatively, additional antibodies capable of binding to
the endokine 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 therefore it is possible to obtain an antibody which
binds to a second antibody. In accordance with this method,
endokine 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 endokine alpha protein-specific antibody can be
blocked by the endokine alpha protein antigen. Such antibodies
comprise anti-idiotypic antibodies to the endokine alpha
protein-specific antibody and can be used to immunize an animal to
induce formation of further endokine alpha protein-specific
antibodies.
[0272] It will be appreciated that Fab and F(ab').sub.2 and other
portions of the antibodies of the present invention may be used
according to the methods disclosed herein. Such portions are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab portions) or pepsin (to produce F(ab').sub.2
portions). Alternatively, endokine alpha protein-binding portions
can be produced through the application of recombinant DNA
technology or through synthetic chemistry.
[0273] Where in vivo imaging is used to detect enhanced levels of
endokine 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).
[0274] Further suitable labels for the endokine alpha
protein-specific antibodies of the present invention are provided
below. Examples of suitable enzyme labels include malate
dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, and acetylcholine esterase.
[0275] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In and .sup.99mTc are preferred isotopes
where in vivo imaging is used since they avoid the problem of
dehalogenation of the .sup.125I or .sup.131I-labeled monoclonal
antibody by the liver. In addition, these radionucleotides have a
more favorable gamma emission energy for imaging (Perkins et al.,
Eur. J. Nucl. Med. 10:296-301 (1985); Carasquillo et al., J. Nucl.
Med. 28:281-287 (1987)). For example, .sup.111In coupled to
monoclonal antibodies with 1-(p-isothiocyanatobenzyl)-DPTA has
shown little uptake in non-tumorous tissues, particularly the
liver, and therefore enhances specificity of tumor localization
(Esteban et al., J. Nucl. Med. 28:861-870 (1987)).
[0276] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0277] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0278] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin.
[0279] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0280] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and Fe.
[0281] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (Clin. Chim. Acta 70:1-31
(1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40 (1977)).
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which
methods are incorporated by reference herein.
[0282] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG 1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. Most preferably the antibodies
are human antigen binding antibody fragments of the present
invention include, but are not limited to, Fab, Fab' and F(ab')2,
Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a VL or
VH domain. The antibodies may be from any animal origin including
birds and mammals. Preferably, the antibodies are human, murine,
rabbit, goat, guinea pig, camel, horse, or chicken.
[0283] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the invention
are any combinations of variable region(s) and hinge region, CH1,
CH2, and CH3 domains. The present invention further includes
chimeric, humanized, and human monoclonal and polyclonal antibodies
which specifically bind the polypeptides of the present invention.
The present invention further includes antibodies which are
anti-idiotypic to the antibodies of the present invention.
[0284] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt, A. et al., J. Immunol. 147:60-69 (1991); U.S. Pat.
Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny, S. A. et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified
in terms of the epitope(s) or portion(s) of a polypeptide of the
present invention which are recognized or specifically bound by the
antibody. The epitope(s) or polypeptide portion(s) may be specified
as described herein, e.g., by N-terminal and C-terminal positions,
by size in contiguous amino acid residues, or listed in the Tables
and Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0285] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of the polypeptides
of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Further included in the present invention are antibodies which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the
present invention may also be described or specified in terms of
their binding affinity. Preferred binding affinities include those
with a dissociation constant or Kd less than 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0286] Antibodies of the present invention have uses that include,
but are not limited to, methods known in the art to purify, detect,
and target the polypeptides of the present invention including both
in vitro and in vivo diagnostic and therapeutic methods. For
example, the antibodies have use in immunoassays for qualitatively
and quantitatively measuring levels of the polypeptides of the
present invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference in the
entirety).
[0287] The antibodies of the present invention may be used either
alone or in combination with other compositions. The antibodies may
further be recombinantly fused to a heterologous polypeptide at the
N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions.
For example, antibodies of the present invention may be
recombinantly fused or conjugated to molecules useful as labels in
detection assays and effector molecules such as heterologous
polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO
91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396
387.
[0288] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. Monoclonal antibodies can be
prepared using a wide of techniques known in the art including the
use of hybridoma and recombinant technology. See, e.g., Harlow et
al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal
Antibodies and T-cell Hybridomas, pp. 563-681 (Elsevier, N.Y.,
1981) (said references incorporated by reference in their
entireties).
[0289] Fab and F(ab')2 fragments may be produced by proteolytic
cleavage, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab')2 fragments).
[0290] Alternatively, antibodies of the present invention can be
produced through the application of recombinant DNA technology or
through synthetic chemistry using methods known in the art. For
example, the antibodies of the present invention can be prepared
using various phage display methods known in the art. In phage
display methods, functional antibody domains are displayed on the
surface of a phage particle which carries polynucleotide sequences
encoding them. Phage with a desired binding property are selected
from a repertoire or combinatorial antibody library (e.g. human or
murine) by selecting directly with antigen, typically antigen bound
or captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 with Fab, Fv
or disulfide stabilized Fv antibody domains recombinantly fused to
either the phage gene III or gene VIII protein. Examples of phage
display methods that can be used to make the antibodies of the
present invention include those disclosed in Brinkman U. et al., J.
Immunol. Methods 182:41-50 (1995); Ames, R. S. et al., J. Immunol.
Methods 184:177-186 (1995); Kettleborough, C. A. et al, Eur. J.
Immunol. 24:952-958 (1994); Persic, L. et al., Gene 187:9-18
(1997); Burton, D. R. et al., Advances in Immunology 57:191-280
(1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.
5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,
5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727
and 5,733,743 (said references incorporated by reference in their
entireties).
[0291] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in WO 92/22324;
Mullinax, R. L. et al., BioTechniques 12(6):864-869 (1992); and
Sawai, H. et al., AJRI 34:26-34 (1995); and Better, M. et al.,
Science 240:1041-1043 (1988) (said references incorporated by
reference in their entireties).
[0292] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al (1991) Methods in
Enzymology 203:46-88; Shu, L. et al., PNAS 90:7995-7999 (1993); and
Skerra, A. et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies, S. D. et al., J.
Immunol. Methods 125:191-202 (1989); and U.S. Pat. No. 5,807,715.
Antibodies can be humanized using a variety of techniques including
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101;
and 5,585,089), veneering or resurfacing (EP 0592 106; EP 0519596;
Padlan E. A., Molecular Immunology 28(4/5):489-498 (1991);
Studnicka G. M. et al., Protein Engineering 7(6):805-814 (1994);
Roguska M. A. et al, PNAS 91:969-973) (1994), and chain shuffling
(U.S. Pat. No. 5,565,332). Human antibodies can be made by a
variety of methods known in the art including phage display methods
described above. See also, U.S. Pat. Nos. 4,444,887, 4,716,111,
5,545,806, and 5,814,318; and WO 98/46645 (said references
incorporated by reference in their entireties).
[0293] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et
al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981;
Gillies, S. O. et al, PNAS 89:1428-1432 (1992); Fell, H. P. et al.,
J. Immunol. 146:2446-2452 (1991) (said references incorporated by
reference in their entireties).
[0294] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the hinge region, CH1 domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to increase the in vivo half life of
the polypeptides or for use in immunoassays using methods known in
the art. The polypeptides may also be fused or conjugated to the
above antibody portions to form multimers. For example, Fc portions
fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher
multimeric forms can be made by fusing the polypeptides to portions
of IgA and IgM. Methods for fusing or conjugating the polypeptides
of the present invention to antibody portions are known in the art.
See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO
96/04388, WO 91/06570; Ashkenazi, A. et al., PNAS 88:10535-10539
(1991); Zheng, X. X. et al., J. Immunol. 154:5590-5600 (1995); and
Vil, H. et al., PNAS 89:11337-11341 (1992) (said references
incorporated by reference in their entireties).
[0295] The invention further relates to antibodies which act as
agonists or antagonists of the polypeptides of the present
invention. For example, the present invention includes antibodies
which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding, but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. Also include are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included are neutralizing antibodies
which bind the ligand and prevent binding of the ligand to the
receptor, as well as antibodies which bind the ligand, thereby
preventing receptor activation, but do not prevent the ligand from
binding the receptor. Further included are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. see e.g., WO
96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood
92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.
58(16):3668-3678 (1998); Harrop, J. A. et al., J. Immunol.
161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.
58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.
160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci. 111(Pt
2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard, J. et al., Cytokinde 9(4):233-241
(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301
(1997); Taryman, R. E. et al. Neuron 14(4):755-762 (1995); Muller,
Y. A. et al., Structure 6(9): 1153-1167 (1998); Bartunek, P. et
al., Cytokine 8(1):14-20 (1996) (said references incorporated by
reference in their entireties).
Transgenic Animals
[0296] The polypeptides of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[0297] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, Mol. Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated
by reference herein in its entirety. Further, the contents of each
of the documents recited in this paragraph is herein incorporated
by reference in its entirety.
[0298] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)), each of which is herein incorporated by
reference in its entirety).
[0299] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric animals. The transgene may be integrated as a
single transgene or as multiple copies such as in concatamers,
e.g., head-to-head tandems or head-to-tail tandems. The transgene
may also be selectively introduced into and activated in a
particular cell type by following, for example, the teaching of
Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236
(1992)). The regulatory sequences required for such a cell-type
specific activation will depend upon the particular cell type of
interest, and will be apparent to those of skill in the art. When
it is desired that the polynucleotide transgene be integrated into
the chromosomal site of the endogenous gene, gene targeting is
preferred. Briefly, when such a technique is to be utilized,
vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory
sequences required for such a cell-type specific inactivation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art. The contents of each of the
documents recited in this paragraph is herein incorporated by
reference in its entirety.
[0300] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[0301] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[0302] Transgenic and "knock-out" animals of the invention have
uses which include, but are not limited to, animal model systems
useful in elaborating the biological function of endokine alpha
polypeptides, studying conditions and/or disorders associated with
aberrant endokine alpha expression, and in screening for compounds
effective in ameliorating such conditions and/or disorders.
[0303] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells, etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and
implanted in the body, e.g., genetically engineered fibroblasts can
be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each
of which is incorporated by reference herein in its entirety. See
also U.S. Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative
Selection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi,
et al., Cells and Non-Human Organisms Containing Predetermined
Genomic Modifications and Positive-Negative Selection Methods and
Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder, et al.,
Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner,
et al., Genetic Transformation of Zygotes); each of which is hereby
incorporated by reference in its entirety).
[0304] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[0305] Antagonists
[0306] In specific embodiments, antagonists according to the
present invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:1, or the complementary strand thereof,
and/or to nucleotide sequences contained in the deposited clone
97640. In one embodiment, antisense sequence is generated
internally by the organism, in another embodiment, the antisense
sequence is separately administered (see, for example, O'Connor,
J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).
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 251:1300 (1991). The methods are based on
binding of a polynucleotide to a complementary DNA or RNA.
[0307] For example, the 5' coding portion of a polynucleotide that
encodes the mature 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 the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[0308] In one embodiment, the endokine alpha antisense nucleic acid
of the invention is produced intracellularly by transcription from
an exogenous sequence. For example, a vector or a portion thereof,
is transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the
endokine alpha antisense nucleic acid. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others know in the art,
used for replication and expression in vertebrate cells. Expression
of the sequence encoding endokine alpha, or fragments thereof, can
be by any promoter known in the art to act in vertebrate,
preferably human cells. Such promoters can be inducible or
constitutive. Such promoters include, but are not limited to, the
SV40 early promoter region (Bemoist and Chambon, Nature 29:304-310
(1981), the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the
herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci.
U.S.A. 78:1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)),
etc.
[0309] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of an endokine alpha gene. However, absolute complementarity,
although preferred, is not required. A sequence "complementary to
at least a portion of an RNA," referred to herein, means a sequence
having sufficient complementarity to be able to hybridize with the
RNA, forming a stable duplex; in the case of double stranded
endokine alpha antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base
mismatches with an endokine alpha RNA it may contain and still form
a stable duplex (or triplex as the case may be). One skilled in the
art can ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0310] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
1994, Nature 372:333-335. Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of the
nucleotide sequence shown in FIG. 1 could be used in an antisense
approach to inhibit translation of endogenous endokine alpha mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of endokine alpha mRNA, antisense nucleic
acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at
least 50 nucleotides.
[0311] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 (1987);
PCT Publication No. WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(see, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or
intercalating agents. (see, e.g., Zon, Pharm. Res. 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0312] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
a-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0313] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0314] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0315] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids
Res. 15:6625-6641 (1987)). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., Nucl. Acids Res.
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,
FEBS Lett. 215:327-330 (1987)).
[0316] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.,
Nucl. Acids Res. 16:3209 (1988), methylphosphonate oligonucleotides
can be prepared by use of controlled pore glass polymer supports
(Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451 (1988)),
etc.
[0317] While antisense nucleotides complementary to the endokine
alpha coding region sequence could be used, those complementary to
the transcribed untranslated region are most preferred.
[0318] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (see, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al,
Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy endokine
alpha mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking
regions that form complementary base pairs with the target mRNA.
The sole requirement is that the target mRNA have the following
sequence of two bases: 5'-UG-3'. The construction and production of
hammerhead ribozymes is well known in the art and is described more
fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are
numerous potential hammerhead ribozyme cleavage sites within the
nucleotide sequence of endokine alpha (FIG. 1). Preferably, the
ribozyme is engineered so that the cleavage recognition site is
located near the 5' end of the endokine alpha mRNA; i.e., to
increase efficiency and minimize the intracellular accumulation of
non-functional mRNA transcripts.
[0319] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express endokine alpha in vivo. DNA constructs encoding the
ribozyme may be introduced into the cell in the same manner as
described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct
"encoding" the ribozyme under the control of a strong constitutive
promoter, such as, for example, pol III or pol II promoter, so that
transfected cells will produce sufficient quantities of the
ribozyme to destroy endogenous endokine alpha messages and inhibit
translation. Since ribozymes unlike antisense molecules, are
catalytic, a lower intracellular concentration is required for
efficiency.
[0320] Endogenous gene expression can also be reduced by
inactivating or "knocking out" the endokine alpha gene and/or its
promoter using targeted homologous recombination. (e.g., see
Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi,
Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989);
each of which is incorporated by reference herein in its entirety).
For example, a mutant, non-functional polynucleotide of the
invention (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous polynucleotide sequence (either the
coding regions or regulatory regions of the gene) can be used, with
or without a selectable marker and/or a negative selectable marker,
to transfect cells that express polypeptides of the invention in
vivo. In another embodiment, techniques known in the art are used
to generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art. The
contents of each of the documents recited in this paragraph is
herein incorporated by reference in its entirety.
[0321] In other embodiments, antagonists according to the present
invention include soluble forms of endokine alpha (e.g., fragments
of the endokine alpha polypeptide shown in FIG. 1 that include the
ligand binding domain from the extracellular region of the full
length receptor). Such soluble forms of endokine alpha, which may
be naturally occurring or synthetic, antagonize endokine alpha
mediated signaling by competing with the cell surface bound forms
of the receptor for binding to TNF-family ligands. Antagonists of
the present invention also include antibodies specific for
TNF-family ligands and endokine alpha-Fc fusion proteins.
[0322] By a "TNF-family ligand" is intended naturally occurring,
recombinant, and synthetic ligands that are capable of binding to a
member of the TNF receptor family and inducing and/or blocking the
ligand/receptor signaling pathway. Members of the TNF ligand family
include, but are not limited to, TNF-a, lymphotoxin-a (LT-a, also
known as TNF-b), LT-b (found in complex heterotrimer LT-a2-b),
FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor
(NGF).
[0323] TNF-.alpha. has been shown to protect mice from infection
with herpes simplex virus type 1 (HSV-1). Rossol-Voth et al., J.
Gen. Virol. 72:143-147 (1991). The mechanism of the protective
effect of TNF-.alpha. is unknown but appears to involve neither
interferons nor NK cell killing. One member of the TNFR family has
been shown to mediate HSV-1 entry into cells. Montgomery et al.,
Eur. Cytokine Newt. 7:159 (1996). Further, antibodies specific for
the extracellular domain of this TNFR block HSV-1 entry into cells.
Thus, endokine alpha antagonists of the present invention include
both endokine alpha amino acid sequences and antibodies capable of
preventing TNFR mediated viral entry into cells. Such sequences and
antibodies can function by either competing with cell surface
localized TNFR for binding to virus or by directly blocking binding
of virus to cell surface receptors.
[0324] Antibodies according to the present invention may be
prepared by any of a variety of standard methods using endokine
alpha receptor immunogens of the present invention. Such endokine
alpha receptor immunogens include the endokine alpha receptor
protein shown in FIG. 1 (SEQ ID NO:2) (which may or may not include
a leader sequence) and polypeptide fragments of the receptor
comprising the ligand binding, extracellular, transmembrane, the
intracellular domains of the endokine alpha receptors, or any
combination thereof.
[0325] Polyclonal and monoclonal antibody agonists or antagonists
according to the present invention can be raised according to the
methods disclosed herein and/or known in the art, such as, for
example, those methods described in Tartaglia and Goeddel, J. Biol.
Chem. 267(7):4304-4307 (1992)); Tartaglia et al., Cell 73:213-216
(1993)), and PCT Application WO 94/09137 (the contents of each of
these three applications are herein incorporated by reference in
their entireties), and are preferably specific to polypeptides of
the invention having the amino acid sequence of SEQ ID NO:2.
[0326] As one of skill in the art will appreciate, endokine alpha
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with heterologous
polypeptide sequences. For example, the polypeptides of the present
invention may be fused with the constant domain of immunoglobulins
(IgA, IgE, IgG, and IgM) or portions thereof (CH1, CH2, CH3, and
any combination thereof, including both entire domains and portions
thereof), resulting in chimeric polypeptides. These fusion proteins
facilitate purification and show an increased half-life.
[0327] The techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of
endokine alpha thereby effectively generating agonists and
antagonists of endokine alpha. See generally, U.S. Pat. Nos.
5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and
Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O.,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and
Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents
and publications are hereby incorporated by reference). In one
embodiment, alteration of endokine alpha polynucleotides and
corresponding polypeptides may be achieved by DNA shuffling. DNA
shuffling involves the assembly of two or more DNA segments into a
desired endokine alpha molecule by homologous, or site-specific,
recombination. In another embodiment, endokine alpha
polynucleotides and corresponding polypeptides may be altered by
being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of endokine alpha may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules. In
preferred embodiments, the heterologous molecules are, for example,
TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta),
LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL,
CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International
Publication No. WO 96/14328), AIM-I (International Publication No.
WO 97/33899), AIM-II (International Publication No. WO 97/34911),
APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha
(International Publication Nos. WO 98/07880 and WO 98/18921), OPG,
OX40, nerve growth factor (NGF), and soluble forms of Fas, CD30,
CD27, CD40 and 4-IBB, DR3 (International Publication No. WO
97/33904), DR4 (International Publication No. WO 98/32856), TR5
(International Publication No. WO 98/30693), TR6 (International
Publication No. WO 98/30694), TR7 (International Publication No. WO
98/41629), TRANK, TR9 (International Publication No. WO 98/56892),
TR10 (International Publication No. WO 98/54202), 312C2
(International Publication No. WO 98/06842), TR12, and TNF-R1,
TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1/DR4/APO-2, TRAIL-R2/DR5,
DcR1/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, and
v-FLIP.
[0328] In further preferred embodiments, the heterologous molecules
are any member of the TNF family.
[0329] Chromosome Assays
[0330] 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.
[0331] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of an endokine 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. Typically, in
accordance with routine procedures for chromosome mapping, some
trial and error may be necessary to identify a genomic probe that
gives a good in situ hybridization signal.
[0332] In some cases, in addition, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified portion.
[0333] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of portions from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0334] 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).
[0335] 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).
[0336] 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.
[0337] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0338] Endokine Alpha Uses
[0339] The Tumor Necrosis Factor (TNF) family ligands are known to
be among the most pleiotropic cytokines, inducing a large number of
cellular responses, including cytotoxicity, anti-viral activity,
immunoregulatory activities, and the transcriptional regulation of
several genes (Goeddel, D. V. et al., "Tumor Necrosis Factors: Gene
Structure and Biological Activities," Symp. Quant. Biol. 51:597-609
(1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev.
Biochem. 57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988);
Fiers, W., FEBS Lett. 285:199-224 (1991)). The TNF-family ligands
induce such various cellular responses by binding to TNF-family
receptors.
[0340] Endokine alpha polynucleotides, polypeptides, agonists or
antagonists of the invention may be used in developing treatments
for any disorder mediated (directly or indirectly) by defective, or
insufficient amounts of endokine alpha. Endokine alpha
polypeptides, agonists or antagonists may be administered to a
patient (e.g., mammal, preferably human) afflicted with such a
disorder. Alternatively, a gene therapy approach may be applied to
treat such disorders. Disclosure herein of endokine alpha
nucleotide sequences permits the detection of defective endokine
alpha genes, and the replacement thereof with normal endokine
alpha-encoding genes. Defective genes may be detected in in vitro
diagnostic assays, and by comparison of the endokine alpha
nucleotide sequence disclosed herein with that of a endokine alpha
gene derived from a patient suspected of harboring a defect in this
gene.
[0341] In another embodiment, the polypeptides of the present
invention are used as a research tool for studying the biological
effects that result from inhibiting TR11/endokine alpha
interactions on different cell types. endokine alpha polypeptides
also may be employed in in vitro assays for detecting TR11 or
endokine alpha or the interactions thereof.
[0342] As indicated above, 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)). 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)). A number of studies
have suggested that TNF is an important mediator of the cachexia in
cancer, infectious pathology, and in other catabolic states.
[0343] Thus, the endokine alpha protein of the present invention
can be used for tumor targeting, preferably, after conjugation with
radioisotypes or cytostatic drugs (Gruss and Dower, Blood
85(12):3378-3404 (1995)). Endokine alpha can be used in patients
with melanoma and sarcoma for tumor regression and extension of
patient life span through a local injection or used in isolated
limb perfusion (Aggarwal and Natarajan, Eur. Cytokine Netw.
7(2):92-124 (1996)).
[0344] The endokine alpha of the present invention can also have a
therapeutic role in specific situations, for example, activity
against viral, bacterial, yeast, fungal, and other infections
(including toxoplasma gondii, schistosoma mansoni, listeria
monocytogens and BCG). These effects of endokine alpha can be
indirect and thus preferably, mediated through activation of
macrophages, eosinophils, fibroblasts, or neutrophils.
[0345] TNF is also 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 (Kombluth, S. K. et al.,
J. Immunol. 137:2585-2591 (1986)) and other cytokines. 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)).
[0346] Neutralizing antisera or mAbs to TNF have been shown in
mammals other than man to abrogate adverse phaysiological 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)). 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).
[0347] As endokine alpha is believed to exhibit many of the
biological effects of TNF, the present invention is further
directed to antibody-based therapies which involve administering an
anti-endokine alpha antibody to a mammalian, preferably human,
patient for treating one or more of the above-described disorders.
Methods for producing anti-endokine alpha polyclonal and monoclonal
antibodies are described in detail above. Such antibodies may be
provided in pharmaceutically acceptable compositions as known in
the art or as described herein.
[0348] Polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, are useful in the diagnosis
and treatment or prevention of a wide range of diseases and/or
conditions. Such diseases and conditions include, but are not
limited to, cancer (e.g., immune cell related cancers, breast
cancer, prostate cancer, ovarian cancer, follicular lymphoma,
gliobalstoma, cancer associated with mutation or alteration of p53,
brain tumor, bladder cancer, uterocervical cancer, colon cancer,
colorectal cancer, non-small cell carcinoma of the lung, small cell
carcinoma of the lung, stomach cancer, etc.), lymphoproliferative
disorders (e.g., lymphadenopathy and lymphomas (e.g., EBVinduced
lymphoproliferations and Hodgkin's disease), microbial (e.g.,
viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2
infection, herpesvirus infection (including, but not limited to,
HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection,
poxvirus infection, human papilloma virus infection, hepatitis
infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori
infection, invasive Staphylococcia, etc.), parasitic infection,
nephritis, bone disease (e.g., osteoporosis), atherosclerosis,
pain, cardiovascular disorders (e.g., neovascularization,
hypovascularization or reduced circulation (e.g., ischemic disease
(e.g., myocardial infarction, stroke, etc.)), AIDS, allergy,
inflammation, neurodegenerative disease (e.g., Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, pigmentary
retinitis, cerebellar degeneration, etc.), graft rejection (acute
and chronic), graft vs. host disease, diseases due to
osteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissue
destruction in rheumatism, liver disease (e.g., acute and chronic
hepatitis, liver injury, and cirrhosis), autoimmune disease (e.g.,
multiple sclerosis, myasthenia gravis, rheumatoid arthritis,
systemic lupus erythematosus, immune complex glomerulonephritis,
autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's
disease, Hashimoto's thyroiditis, inflammatory autoimmune diseases,
etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes,
diabetic complications (e.g., diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy), influenza, asthma, psoriasis,
glomerulonephritis, septic shock, and ulcerative colitis.
[0349] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in promoting
angiogenesis, wound healing (e.g., wounds, burns, and bone
fractures), and regulating bone formation and treating
osteoporosis.
[0350] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are also useful as an adjuvant
to enhance immune responsiveness to specific antigen and/or
anti-viral immune responses.
[0351] More generally, polynucleotides and/or polypeptides of the
invention and/or agonists and/or antagonists thereof are useful in
regulating (i.e., elevating or reducing) immune response. For
example, polynucleotides and/or polypeptides of the invention may
be useful in preparation or recovery from surgery, trauma,
radiation therapy, chemotherapy, and transplantation, or may be
used to boost immune response and/or recovery in the elderly and
immunocompromised individuals. Alternatively, polynucleotides
and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful as immunosuppressive agents, for
example in the treatment or prevention of autoimmune disorders or
in the prevention of transplant rejection. In specific embodiments,
polynucleotides and/or polypeptides of the invention are used to
treat or prevent chronic inflammatory, allergic or autoimmune
conditions, such as those described herein or are otherwise known
in the art.
[0352] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding endokine
alpha locally or systemically in the body or by direct cytotoxicity
of the antibody, e.g., as mediated by complement (CDC) or by
effector cells (ADCC). Some of these approaches are described in
more detail below. Armed with the teachings provided herein, one of
ordinary skill in the art will know how to use the antibodies of
the present invention for diagnostic, monitoring or therapeutic
purposes without undue experimentation.
[0353] The pharmaceutical compositions of the present invention may
be administered by any means that achieve their intended purpose.
Amounts and regimens for the administration of antibodies, their
fragments or derivatives can be determined readily by those with
ordinary skill in the clinical art of treating TNF-related
disease.
[0354] For example, administration may be by parenteral,
subcutaneous, intravenous, intramuscular, intraperitoneal,
transdermal, or buccal routes. Alternatively, or concurrently,
administration may be by the oral route. The dosage administered
will be dependent upon the age, health, and weight of the
recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired.
[0355] Compositions within the scope of this invention include all
compositions wherein the antibody, fragment or derivative is
contained in an amount effective to achieve its intended purpose.
While individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the art.
The effective dose is a function of the individual chimeric or
monoclonal antibody, the presence and nature of a conjugated
therapeutic agent (see below), the patient and his clinical status,
and can vary from about 10 .mu.g/kg body weight to about 5000 mg/kg
body weight. The preferred dosages comprise 0.1 to 500 mg/kg body
wt.
[0356] In addition to the pharmacologically active compounds, the
new pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Preferably,
the preparations contain from about 0.01 to 99 percent, preferably
from about 20 to 75 percent of active compound(s), together with
the excipient.
[0357] Similarly, preparations of an endokine alpha antibody or
fragment of the present invention for parenteral administration,
such as in detectably labeled form for imaging or in a free or
conjugated form for therapy, include sterile aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oil
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media, parenteral vehicles including sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's,
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, such as those based on Ringer's dextrose, and the
like. Preservatives and other additives may also be present, such
as, for example, antimicrobials, anti-oxidants, chelating agents,
and inert gases and the like. See, generally, Remington's
Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton, Pa.,
1980.
[0358] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating a subject having or
developing endokine alpha related disorders as described herein.
Such treatment comprises parenterally administering single or
multiple doses of the antibody, a fragment or derivative, or a
conjugate thereof.
[0359] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hemopoietic growth factors,
etc., which serve to increase the number or activity of effector
cells which interact with the antibodies.
[0360] Since circulating concentrations of endokine alpha (like
TNF) tend to be extremely low, in the range of about 10 pg/ml in
non-septic individuals, and reaching about 50 pg/ml in septic
patients and above 100 pg/ml in the sepsis syndrome for TNF
(Hammerle, A. F. et al., 1989, supra) or may be only be detectable
at sites of endokine alpha-related disorders, it is preferred to
use high affinity and/or potent in vivo endokine alpha-inhibiting
and/or neutralizing antibodies, fragments or regions thereof, for
both endokine alpha immunoassays and therapy of endokine related
disorders. Such antibodies, fragments, or regions, will preferably
have an affinity for human endokine alpha, expressed as Ka, of at
least 10.sup.8 M.sup.-1, more preferably, at least 10.sup.9
M.sup.-1, such as 5.times.10.sup.8 M.sup.-1,
8.times.10.sup.8M.sup.-1, 2.times.10.sup.9 M.sup.-1,
4.times.10.sup.9 M.sup.-1, 6.times.10.sup.9 M.sup.-1,
8.times.10.sup.9 M.sup.-1.
[0361] Preferred for human therapeutic use are high affinity murine
and murine/human or human/human chimeric antibodies, and fragments,
regions and derivatives having potent in vivo endokine-inhibiting
and/or neutralizing activity, according to the present invention,
e.g., that block endokine-induced IL-1, IL-6 or TNF secretion,
procoagulant activity, expression of cell adhesion molecules such
as ELAM-1 and ICAM-1 and mitogenic activity, in vivo, in situ, and
in vitro.
[0362] Additional preferred embodiments of the invention include,
but are not limited to, the use of endokine-.alpha. polypeptides
and functional agonists in the following applications:
[0363] A vaccine adjuvant that enhances immune responsiveness to
specific antigen.
[0364] An adjuvant to enhance tumor-specific immune responses.
[0365] An adjuvant to enhance anti-viral immune responses.
[0366] As a stimulator of B cell responsiveness to pathogens.
[0367] As a activator of T cells.
[0368] As an agent that elevates the immune status of a individual
prior to their receipt of immunosuppressive therapies.
[0369] As an agent to accelerate recovery of immunocompromised
individuals; As an agent to boost immunoresponsiveness among aged
populations; As an immune system enhancer following bone marrow
transplant.
[0370] As an agent to direct an individuals immune system towards
development of a humoral response (i.e. TH2) and/or a TH1 cellular
response.
[0371] As a means to induce tumor proliferation and thus make it
more susceptible to anti-neoplastic agents. For example multiple
myeloma is a slowly dividing disease and is thus refractory to
virtually all anti-neoplastic regimens. If these cells were forced
to proliferate more rapidly their susceptibility profile would
likely change.
[0372] As a B cell and other ligand expressing cell (e.g.,
endothelial cells) specific binding protein to which specific
activators or inhibitors of cell growth may be attached. The result
would be to focus the activity of such activators or inhibitors
onto normal, diseased, or neoplastic B cell populations.
[0373] As a means of detecting B-lineage cells and/or ligand
expressing cells (e.g., endothelial cells) by virtue of its
specificity. This application may require labeling the protein with
biotin or other agents to afford a means of detection.
[0374] As a stimulator of B cell production in pathologies such as
AIDS, chronic lymphocyte disorder and/or Common Variable
Immunodificiency;
[0375] As part of a B cell selection device the function of which
is to isolate B cells as well as other ligand expressing cells
(e.g., endothelial cells) from a heterogenous mixture of cell
types. Endokine alpha could be coupled to a solid support to which
B cells would then specifically bind. Unbound cells would be washed
out and the bound cells subsequently eluted. This technique would
allow purging of tumor cells from, for example, bone marrow or
peripheral blood prior to transplant.
[0376] As a therapy for generation and/or regeneration of lymphoid
tissues following surgery, trauma or genetic defect.
[0377] As a gene-based therapy for genetically inherited disorders
resulting in immuno-incompetence such as observed among SCID
patients.
[0378] As an antigen for the generation of antibodies to inhibit or
enhance endokine alpha mediated responses.
[0379] As a means of activating monocytes/macrophages to defend
against parasitic diseases that effect monocytes such as
Leshmania.
[0380] As pretreatment of bone marrow samples prior to transplant.
Such treatment would increase B cell representation and thus
accelerate recovery.
[0381] As a means of regulating secreted cytokines that are
elicited by endokine alpha.
[0382] Inhibition of unwanted TH1 responses. There is strong
evidence to demonstrate that IL-12 is an important cytokine in
directing TH1 differentiation. Endokine alpha-induced inhibition of
IL-12 production might be helpful in controlling TH 1-associated
conditions, such as autoimmune diseases, inflammation, acute
allograft rejection, fetal reabsorption.
[0383] Reduction of inflammatory response. It was shown that
induction of IL-10 and MCP-1 ameliorate experimental fecal
peritonitis.
[0384] Enhanced resistance to pathogens. Products of the oxidative
burst, such as H.sub.2O.sub.2, are used by monocytes for the
killing of phagocytosed pathogens or for the extracellular
destruction of cells.
[0385] All of the above described applications as they may apply to
veterinary medicine.
[0386] Antagonists of endokine alpha include binding and/or
inhibitory antibodies, antisense nucleic acids, ribozymes or
soluble forms of the endokine alpha receptor(s). These would be
expected to reverse many of the activities of the ligand described
above as well as find clinical or practical application as:
[0387] A means of blocking various aspects of immune responses to
foreign agents or self. Examples include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and pathogens.
Although our current data speaks directly to the potential role of
endokine alpha in B cell, T cell and monocyte related pathologies,
it remains possible that other cell types may gain expression or
responsiveness to endokine alpha. Thus, endokine alpha may, like
CD40 and its ligand, be regulated by the status of the immune
system and the microenvironment in which the cell is located.
[0388] A therapy for preventing the B and/or T cell proliferation
and Ig secretion associated with autoimmune diseases such as
idiopathic thrombocytopenic purpura, systemic lupus erythramatosus
and MS.
[0389] An inhibitor of B and or T cell migration in endothelial
cells, This activity disrupts tissue architecture or cognate
responses and is useful, for example, in disrupting immune
responses, and blocking sepsis.
[0390] An inhibitor of graft versus host disease or transplant
rejection.
[0391] A therapy for B cell and/or T cell malignancies such as ALL,
Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte
leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and
EBV-transformed diseases.
[0392] A therapy for chronic hypergammaglobulinemeia evident in
such diseases as monoclonalgammopathy of undetermined significance
(MGUS), Waldenstrom's disease, and related idiopathic
monoclonalgammopathies.
[0393] A means of decreasing the involvement of B cells and Ig
associated with Chronic Myelogenous Leukemia.
[0394] An immunosuppressive agent(s).
[0395] An inhibitor of signaling pathways involving ERK1, COX2 and
Cyclin D2 which have been associated with endokine alpha induced B
cell activation.
[0396] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described above.
[0397] The antagonists may be employed for instance to inhibit
endokine alpha 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.
[0398] 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 endokine alpha polypeptides of the present
invention. The antagonists may also be employed for treating
atherosclerosis, by preventing monocyte infiltration in the artery
wall.
[0399] 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.
[0400] 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
endokine alpha. 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.
[0401] Antibodies against endokine alpha may be employed to bind to
and endokine alpha activity to treat ARDS, by preventing
infiltration of neutrophils into the lung after injury. The
antagonists and antagonists of the instant may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described hereinafter.
[0402] Agonists and antagonists of the invention also have uses in
stimulating wound and tissue repair, stimulating angiogenesis,
stimulating the repair of vascular or lymphatic diseases or
disorders. Additionally, agonists and antagonists of the invention
may be used to stimulate the regeneration of mucosal surfaces.
[0403] The compositions of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the compositions of
the invention, include but not limited to, other members of the TNF
family, chemotherapeutic agents, antibiotics, steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic
agents, cytokines and/or growth factors. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[0404] In one embodiment, the compositions of the invention are
administered in combination with other members of the TNF family.
TNF, TNF-related or TNF-like molecules that may be administered
with the compositions of the invention include, but are not limited
to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), TR6 (International
Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921, OX40, and nerve growth
factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB,
TR2 (International Publication No. WO 96/34095), DR3 (International
Publication No. WO 97/33904), DR4 (International Publication No. WO
98/32856), TR5 (International Publication No. WO 98/30693), TR6
(International Publication No. WO 98/30694), TR7 (International
Publication No. WO 98/41629), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[0405] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, cyclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents that act by suppressing the function of
responding T cells.
[0406] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, tetracycline,
metronidazole, amoxicillin, beta-lactamases, aminoglycosides,
macrolides, quinolones, fluoroquinolones, cephalosporins,
erythromycin, ciprofloxacin, and streptomycin.
[0407] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0408] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0409] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and
TNF-alpha.
[0410] In an additional embodiment, the compositions of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the compositions
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (P1GF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (P1GF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B186), as
disclosed in International Publication Number WO 96/26736; Vascular
Endothelial Growth Factor-D (VEGF-D), as disclosed in International
Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D), as disclosed in International Publication Number
WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as
disclosed in German Patent Number DE19639601. The above mentioned
references are incorporated herein by reference.
[0411] In an additional embodiment, the compositions of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0412] In additional embodiments, the compositions of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
[0413] Endokine alpha compositions of the invention are also
suitably administered by sustained-release systems. Suitable
examples of sustained-release compositions include suitable
polymeric materials (such as, for example, semi-permeable polymer
matrices in the form of shaped articles, e.g., films, or
microcapsules), suitable hydrophobic materials (for example, as an
emulsion in an acceptable oil) or ion exchange resins, and
sparingly soluble derivatives (such as, for example, a sparingly
soluble salt).
[0414] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, and 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).
[0415] Sustained-release compositions also include liposomally
entrapped compositions of the invention (see generally, Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing Endokine alpha polypeptide may be 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
Endokine alpha polypeptide therapy.
[0416] In another embodiment sustained release compositions of the
invention include crystal formulations known in the art.
[0417] In yet an additional embodiment, the compositions of the
invention are delivered by way of a pump (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574
(1989)). Other controlled release systems are discussed in the
review by Langer (Science 249:1527-1533 (1990)).
[0418] The compositions of the invention may be administered alone
or in combination with other adjuvants. Adjuvants that may be
administered with the compositions of the invention include, but
are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE
(Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a
specific embodiment, compositions of the invention are administered
in combination with alum. In another specific embodiment,
compositions of the invention are administered in combination with
QS-21. Further adjuvants that may be administered with the
compositions of the invention include, but are not limited to,
monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,
CRL1005, aluminum salts, MF-59, and Virosomal adjuvant technology.
Vaccines that may be administered with the compositions of the
invention include, but are not limited to, vaccines directed toward
protection against MMR (measles, mumps, rubella), polio, varicella,
tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae
B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,
cholera, yellow fever, Japanese encephalitis, poliomyelitis,
rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23.TM..
Combinations may be administered either concomitantly, e.g., as an
admixture, separately, but simultaneously or concurrently; or
sequentially. This includes presentations in which the combined
agents are administered together as a therapeutic mixture, and also
procedures in which the combined agents are administered
separately, but simultaneously, e.g., as through separate
intravenous lines into the same individual. Administration "in
combination" further includes the separate administration of one of
the compounds or agents given first, followed by the second.
[0419] In another specific embodiment, compositions of the
invention are used in combination with PNEUMOVAX-23.TM. to treat,
prevent, and/or diagnose infection and/or any disease, disorder,
and/or condition associated therewith. In one embodiment,
compositions of the invention are used in combination with
PNEUMOVAX-23.TM. to treat, prevent, and/or diagnose any Gram
positive bacterial infection and/or any disease, disorder, and/or
condition associated therewith. In another embodiment, compositions
of the invention are used in combination with PNEUMOVAX-23.TM. to
treat, prevent, and/or diagnose infection and/or any disease,
disorder, and/or condition associated with one or more members of
the genus Enterococcus and/or the genus Streptococcus. In another
embodiment, compositions of the invention are used in any
combination with PNEUMOVAX-23.TM. to treat, prevent, and/or
diagnose infection and/or any disease, disorder, and/or condition
associated with one or more members of the Group B streptococci. In
another embodiment, compositions of the invention are used in
combination with PNEUMOVAX-23.TM. to treat, prevent, and/or
diagnose infection and/or any disease, disorder, and/or condition
associated with Streptococcus pneumoniae.
[0420] The compositions of the invention may be administered alone
or in combination with other therapeutic agents, including, but not
limited to, chemotherapeutic agents, antibiotics, antivirals,
steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic agents and cytokines. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately, but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately, but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[0421] In one embodiment, the compositions of the invention are
administered in combination with other members of the TNF family.
TNF, TNF-related or TNF-like molecules that may be administered
with the compositions of the invention include, but are not limited
to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), AIM-II (International
Publication No. WO 97/34911 and WO 98/18921), APRIL (J. Exp. Med.
188(6):1185-1190), endokine-alpha (International Publication No. WO
98/07880), TR6 (International Publication No. WO 98/30694), OPG,
OX40, and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TR6 (International Publication No. WO
98/30694), TR7 (International Publication No. WO 98/41629), TRANK,
TR9 (International Publication No. WO 98/56892), TR10
(International Publication No. WO 98/54202), 312C2 (International
Publication No. WO 98/06842), and TR12.
[0422] In a preferred embodiment, the compositions of the invention
are administered alone or in combination with CD40 ligand (CD40L),
a soluble form of CD40L (e.g., AVREND), biologically active
fragments, variants, or derivatives of CD40L, anti-CD40L antibodies
(e.g., agonistic or antagonistic antibodies), and/or anti-CD40
antibodies (e.g., agonistic or antagonistic antibodies).
[0423] In certain embodiments, compositions of the invention are
administered in combination with antiretroviral agents, nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors. Nucleoside
reverse transcriptase inhibitors that may be administered in
combination with the compositions of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the
compositions of the invention, include, but are not limited to,
VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM. (delavirdine), and
SUSTIVA.TM. (efavirenz). Protease inhibitors that may be
administered in combination with the compositions of the invention,
include, but are not limited to, CRIXIVAN.TM. (indinavir),
NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and VIRACEPT.TM.
(nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors may be used in
any combination with compositions of the invention to treat,
prevent, and/or diagnose AIDS and/or to treat, prevent, and/or
diagnose HIV infection.
[0424] In other embodiments, compositions of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the compositions of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RTFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat, prevent, and/or
diagnose an opportunistic Pneumocystis carinii pneumonia infection.
In another specific embodiment, compositions of the invention are
used in any combination with ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., and/or ETHAMBUTOL.TM. to prophylactically treat,
prevent, and/or diagnose an opportunistic Mycobacterium avium
complex infection. In another specific embodiment, compositions of
the invention are used in any combination with RIFABUTIN.TM.,
CLARITHROMYCIN.TM., and/or AZITHROMYCIN.TM. to prophylactically
treat, prevent, and/or diagnose an opportunistic Mycobacterium
tuberculosis infection. In another specific embodiment,
compositions of the invention are used in any combination with
GANCICLOVIR.TM., FOSCARNET.TM., and/or CIDOFOVIR.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
cytomegalovirus infection. In another specific embodiment,
compositions of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
fungal infection. In another specific embodiment, compositions of
the invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic herpes simplex virus type I and/or type TI
infection. In another specific embodiment, compositions of the
invention are used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic Toxoplasma gondii infection. In another specific
embodiment, compositions of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
bacterial infection.
[0425] In a further embodiment, the compositions of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the compositions of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[0426] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole, penicillins, quinolones, rifampin, streptomycin,
sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
[0427] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs cyclophosphamide, cyclophosphamide IV,
methylprednisolone, prednisolone, azathioprine, FK-506,
15-deoxyspergualin, and other immunosuppressive agents that act by
suppressing the function of responding T cells.
[0428] In specific embodiments, compositions of the invention are
administered in combination with immunosuppressants.
Immunosuppressants preparations that may be administered with the
compositions of the invention include, but are not limited to,
ORTHOCLONE.TM. (OKT3), SANDIMMUNE.TM. NEORAL.TM./SANGDYA.TM.
(cyclosporin), PROGRAF.TM. (tacrolimus), CELLCEPT.TM.
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE.TM.
(sirolimus). In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow
transplantation.
[0429] In a preferred embodiment, the compositions of the invention
are administered in combination with steroid therapy. Steroids that
may be administered in combination with the compositions of the
invention, include, but are not limited to, oral corticosteroids,
prednisone, and methylprednisolone (e.g., IV methylprednisolone).
In a specific embodiment, compositions of the invention are
administered in combination with prednisone. In a further specific
embodiment, the compositions of the invention are administered in
combination with prednisone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and prednisone are those described
herein, and include, but are not limited to, azathioprine,
cylophosphamide, and cyclophosphamide IV. In a another specific
embodiment, compositions of the invention are administered in
combination with methylprednisolone. In a further specific
embodiment, the compositions of the invention are administered in
combination with methylprednisolone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and methylprednisolone are those
described herein, and include, but are not limited to,
azathioprine, cylophosphamide, and cyclophosphamide IV.
[0430] In a preferred embodiment, the compositions of the invention
are administered in combination with an antimalarial. Antimalarials
that may be administered with the compositions of the invention
include, but are not limited to, hydroxychloroquine, chloroquine,
and/or quinacrine.
[0431] In a preferred embodiment, the compositions of the invention
are administered in combination with an NSAID.
[0432] In a nonexclusive embodiment, the compositions of the
invention are administered in combination with one, two, three,
four, five, ten, or more of the following drugs: NRD-101 (Hoechst
Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium
(Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed
disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto),
eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech
Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis),
JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong
Wha), darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1
(synergen; Amgen), IPR-6001 (Institute for Pharmaceutical
Research), trocade (Hoffman-La Roche), EF-5 (Scotia
Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149
(Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck),
ST-1482 (Sigma-Tau), and butixocort propionate (WarnerLambert).
[0433] In a preferred embodiment, the compositions of the invention
are administered in combination with one, two, three, four, five or
more of the following drugs: methotrexate, sulfasalazine, sodium
aurothiomalate, auranofin, cyclosporine, penicillamine,
azathioprine, an antimalarial drug (e.g., as described herein),
cyclophosphamide, chlorambucil, gold, ENBREL.TM. (Etanercept),
anti-TNF antibody, and prednisolone.
[0434] In a more preferred embodiment, the compositions of the
invention are administered in combination with an antimalarial,
methotrexate, anti-TNF antibody, ENBREL.TM. and/or suflasalazine.
In one embodiment, the compositions of the invention are
administered in combination with methotrexate. In another
embodiment, the compositions of the invention are administered in
combination with anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
methotrexate and anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
suflasalazine. In another embodiment, the compositions of the
invention are administered in combination with methotrexate,
anti-TNF antibody, and suflasalazine. In another embodiment, the
compositions of the invention are administered in combination
ENBREL.TM.. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM. and
methotrexate. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM.,
methotrexate and suflasalazine. In another embodiment, the
compositions of the invention are administered in combination with
ENBREL.TM., methotrexate and suflasalazine. In other embodiments,
one or more antimalarials is combined with one of the above-recited
combinations. In a specific embodiment, the compositions of the
invention are administered in combination with an antimalarial
(e.g., hydroxychloroquine), ENBREL.TM., methotrexate and
suflasalazine. In another specific embodiment, the compositions of
the invention are administered in combination with an antimalarial
(e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and
methotrexate.
[0435] In an additional embodiment, compositions of the invention
are administered alone or in combination with one or more
intravenous immune globulin preparations. Intravenous immune
globulin preparations that may be administered with the
compositions of the invention include, but are not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM., and
GAMIMUNE.TM.. In a specific embodiment, compositions of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0436] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0437] In another embodiment, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0438] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or any combination of the
components of CHOP. In another embodiment, compositions of the
invention are administered in combination with Rituximab. In a
further embodiment, compositions of the invention are administered
with Rituxmab and CHOP, or Rituxmab and any combination of the
components of CHOP.
[0439] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5,
IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha,
IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another
embodiment, compositions of the invention may be administered with
any interleukin, including, but not limited to, IL-1 alpha,
IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, 1 L-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, IL-21, and IL-22. In preferred embodiments, the compositions
of the invention are administered in combination with IL4 and
IL10.
[0440] In an additional embodiment, the compositions of the
invention are administered with a chemokine. In another embodiment,
the compositions of the invention are administered with chemokine
beta-8, chemokine beta-1, and/or macrophage inflammatory protein-4.
In a preferred embodiment, the compositions of the invention are
administered with chemokine beta-8.
[0441] In an additional embodiment, the compositions of the
invention are administered in combination with an IL-4 antagonist.
IL-4 antagonists that may be administered with the compositions of
the invention include, but are not limited to: soluble IL-4
receptor polypeptides, multimeric forms of soluble IL-4 receptor
polypeptides; anti-IL-4 receptor antibodies that bind the IL-4
receptor without transducing the biological signal elicited by
IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more
IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do
not transduce the biological signal elicited by IL-4. Preferably,
the antibodies employed according to this method are monoclonal
antibodies (including antibody fragments, such as, for example,
those described herein).
[0442] In an additional embodiment, the compositions of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the compositions of the invention include, but are not limited to,
LEUKINE.TM. (SARGRAMOSTIM.TM.) and NEUPOGEN.TM.
(FILGRASTIM.TM.).
[0443] In an additional embodiment, the compositions of the
invention are administered in combination with fibroblast growth
factors. Fibroblast growth factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0444] Additionally, the compositions of the invention may be
administered alone or in combination with other therapeutic
regimens, including, but not limited to, radiation therapy. Such
combinatorial therapy may be administered sequentially and/or
concomitantly.
Therapeutic Uses
[0445] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the described disorders.
Therapeutic compounds of the invention include, but are not limited
to, antibodies of the invention (including fragments, analogs and
derivatives thereof as described herein) and nucleic acids encoding
antibodies of the invention (including fragments, analogs and
derivatives thereof as described herein). The antibodies of the
invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein such as, for example autoimmune
diseases, disorders, or conditions associated with such diseases or
disorders (including, but not limited to, autoimmune hemolytic
anemia, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis, relapsing polychondritis, rheumatic heart disease,
glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis,
Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,
Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,
Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin
dependent diabetes mellitis, and autoimmune inflammatory eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis), systemic lupus erhythematosus, Goodpasture's
syndrome, Pemphigus, Receptor autoimmunities such as, for example,
Graves' Disease, Myasthenia Gravis, and insulin resistance,
autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,
rheumatoid arthritis, schleroderma with anti-collagen antibodies,
mixed connective tissue disease, polymyositis/dermatomyositis,
pernicious anemia, idiopathic Addison's disease, infertility,
glomerulonephritis such as primary glomerulonephritis and IgA
nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes
millitus, and adrenergic drug resistance (including adrenergic drug
resistance with asthma or cystic fibrosis), chronic active
hepatitis, primary biliary cirrhosis, other endocrine gland
failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome,
urticaria, atopic dermatitis, asthma, inflammatory myopathies, and
other inflammatory, granulamatous, degenerative, and atrophic
disorders, and immunodeficiencies or conditions associated with
such diseases or disorders, including, but not limited to, graft
versus host disease and graft rejection.
[0446] In a specific embodiment, antibodies of the invention are be
used to treat, inhibit, prognose, diagnose or prevent rheumatoid
arthritis.
[0447] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent systemic
lupus erythematosis.
[0448] The treatment and/or prevention of diseases and disorders
associated with aberrant expression and/or activity of a
polypeptide of the invention includes, but is not limited to,
alleviating symptoms associated with those diseases and disorders.
Antibodies of the invention may be provided in pharmaceutically
acceptable compositions as known in the art or as described
herein.
[0449] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0450] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0451] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0452] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides, including fragments thereof. Preferred binding
affinities include those with a dissociation constant or Kd less
than 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, and 10.sup.-15 M.
[0453] In one embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing Endokine alpha
polypeptides or anti-Endokine alpha antibodies associated with
heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs) to targeted cells, expressing the membrane-bound form of
Endokine alpha on their surface, or alternatively, an Endokine
alpha receptor (e.g., TR11) on their surface. Endokine alpha
polypeptides or anti-Endokine alpha antibodies of the invention may
be associated with heterologous polypeptides, heterologous nucleic
acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic
and/or covalent interactions.
[0454] In one embodiment, the invention provides a method for the
specific delivery of compositions of the invention to cells by
administering polypeptides of the invention (e.g., Endokine alpha
or anti-Endokine alpha antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the
invention provides a method for delivering a therapeutic protein
into the targeted cell. In another example, the invention provides
a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA
that can integrate into the cell's genome or replicate episomally
and that can be transcribed) into the targeted cell.
[0455] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention (e.g.,
Endokine alpha polypeptides or anti-Endokine alpha antibodies) in
association with toxins or cytotoxic prodrugs.
[0456] In a specific embodiment, the invention provides a method
for the specific destruction of cells expressing TR11 on their
surface (e.g., activated T cells, and/or T cell and/or B cell
related leukemias or lymphomas) by administering Endokine alpha
polypeptides in association with toxins or cytotoxic prodrugs.
[0457] In another specific embodiment, the invention provides a
method for the specific destruction of cells expressing the
membrane-bound form of Endokine alpha on their surface (e.g.,
endothelial cells) by administering anti-Endokine alpha antibodies
in association with toxins or cytotoxic prodrugs.
[0458] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, cytotoxins
(cytotoxic agents), or any molecules or enzymes not normally
present in or on the surface of a cell that under defined
conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.103Pd, .sup.133Xe,
.sup.131I, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd, .sup.169Yb, .sup.51Cr,
.sup.54Mn, .sup.75Se, .sup.113Sn, .sup.90Yttrium, .sup.117Tin,
.sup.186Rhenium, .sup.166Holmium, and .sup.188Rhenium; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin.
[0459] Techniques known in the art may be applied to label proteins
(including antibodies) of the invention. Such techniques include,
but are not limited to, the use of bifunctional conjugating agents
(see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604;
5,274,119; 4,994,560; and 5,808,003; the contents of each of which
are hereby incorporated by reference in its entirety). A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0460] By "cytotoxic prodrug" is meant a non-toxic compound that is
converted by an enzyme, normally present in the cell, into a
cytotoxic compound. Cytotoxic prodrugs that may be used according
to the methods of the invention include, but are not limited to,
glutamyl derivatives of benzoic acid mustard alkylating agent,
phosphate derivatives of etoposide or mitomycin C, cytosine
arabinoside, daunorubisin, and phenoxyacetamide derivatives of
doxorubicin.
[0461] The compositions of the invention may be administered to an
animal (including, but not limited to, those listed above, and also
including transgenic animals) incapable of producing functional
endogenous antibody molecules or having an otherwise compromised
endogenous immune system, but which is capable of producing human
immunoglobulin molecules by means of a reconstituted or partially
reconstituted immune system from another animal (see, e.g.,
published PCT Application Nos. WO 98/24893, WO 96/34096, WO
96/33735, and WO 91/10741). Compositions of the invention include,
but are not limited to, endokine-alpha polypeptides and
polynucleotides and agonists and antagonists thereof, antibodies,
anti-antibodies, etc.
[0462] The compositions described herein may be used as a vaccine
adjuvant that enhances immune responsiveness to specific antigen.
In a specific embodiment, the vaccine adjuvant is an endokine alpha
polypeptide described herein. In another specific embodiment, the
vaccine adjuvant is an endokine alpha polynucleotide described
herein (i.e., the endokine alpha polynucleotide is a genetic
vaccine adjuvant). As discussed herein, endokine alpha
polynucleotides may be administered using techniques known in the
art, including but not limited to, liposomal delivery, recombinant
vector delivery, injection of naked DNA, and gene gun delivery.
[0463] The compositions described herein may also be an adjuvant
used to enhance tumor-specific immune responses.
[0464] Anti-viral immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include, but are not
limited to, virus and virus associated diseases or symptoms
described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: AIDS, meningitis,
Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific
embodiment, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: HIV/AIDS,
Respiratory syncytial virus, Dengue, Rotavirus, Japanese B
encephalitis, Influenza A and B, Parainfluenza, Measles,
Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,
Herpes simplex, and yellow fever. In another specific embodiment,
the compositions of the invention are used as an adjuvant to
enhance an immune response to the HIV gp120 antigen.
[0465] Anti-bacterial or anti-fungal immune responses that may be
enhanced using the compositions of the invention as an adjuvant,
include bacteria or fungus and bacteria or fungus associated
diseases or symptoms described herein or otherwise known in the
art. In specific embodiments, the compositions of the invention are
used as an adjuvant to enhance an immune response to a bacteria or
fungus, disease, or symptom selected from the group consisting of:
tetanus, Diphtheria, botulism, and meningitis type B. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: Vibrio
cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella
paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium
(malaria).
[0466] Anti-parasitic immune responses that may be enhanced using
the compositions of the invention as an adjuvant, include parasite
and parasite associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to Plasmodium (malaria).
[0467] The compositions of the invention may be used as a
stimulator of B or T cell responsiveness to pathogens.
[0468] The compositions of the invention may be used as an agent
that elevates the immune status of an individual prior to their
receipt of immunosuppressive therapies; as an agent to induce
higher affinity antibodies; as an agent to increase serum
immunoglobulin concentrations; as an agent to accelerate recovery
of immunocompromised individuals; as an agent to boost
immunoresponsiveness among aged populations; and as an immune
system enhancer prior to, during, or after bone marrow transplant
and/or other transplants (e.g., allogeneic or xenogeneic organ
transplantation).
[0469] With respect to transplantation, compositions of the
invention may be administered prior to, concomitant with, and/or
after transplantation. In a specific embodiment, compositions of
the invention are administered after transplantation, prior to the
beginning of recovery of T-cell populations. In another specific
embodiment, compositions of the invention are first administered
after transplantation after the beginning of recovery of T cell
populations, but prior to full recovery of B cell populations.
[0470] The compositions of the invention may be used as an agent to
boost immunoresponsiveness among B cell and/or T cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy. B cell
immunodeficiencies that may be ameliorated or treated by
administering the endokine alpha polypeptides or polynucleotides of
the invention, or agonists thereof, include, but are not limited
to, severe combined immunodeficiency (SCID)-X linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),
X-linked agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class TI deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency. T cell
immunodeficiencies that may be ameliorated or treated by
administering the endokine alpha polypeptides or polynucleotides of
the invention, or agonists thereof, include, but are not limited
to, DiGeorge anomaly, thymic hypoplasia, chronic mucocutaneous
candidiasis, natural killer cell deficiency, idiopathic CD4+
T-lymphocytopenia, and immunodeficiency with predominant T-cell
defect, graft versus host disease, graft rejections and
inflammation associated with an immuno-deficiency.
[0471] Additional conditions resulting in an acquired loss of B or
T cell function that may be ameliorated or treated by administering
the endokine alpha polypeptides or polynucleotides of the
invention, or agonists thereof, include, but are not limited to,
HIV Infection, AIDS, bone marrow transplant, and B cell chronic
lymphocytic leukemia (CLL).
[0472] Compositions of the invention may also be used as an agent
to boost immunoresponsiveness among individuals having a temporary
immune deficiency. Conditions resulting in a temporary immune
deficiency that may be ameliorated or treated by administering the
endokine alpha polypeptides or polynucleotides of the invention, or
agonists thereof, include, but are not limited to, recovery from
viral infections (e.g., influenza), conditions associated with
malnutrition, recovery from infectious mononucleosis, or conditions
associated with stress, recovery from measles, recovery from blood
transfusion, and recovery from surgery.
[0473] In a preferred embodiment, endokine alpha polynucleotides,
polypeptides, and/or agonists and/or antagonists thereof are used
to treat, prevent, and/or diagnose diseases or disorders affecting
or conditions associated with any one or more of the various mucous
membranes of the body. Such diseases or disorders include, but are
not limited to, for example, mucositis, mucoclasis, mucocolitis,
mucocutaneous leishmaniasis (such as, for example, American
leishmaniasis, leishmaniasis americana, nasopharyngeal
leishmaniasis, and New World leishmaniasis), mucocutaneous lymph
node syndrome (for example, Kawasaki disease), mucoenteritis,
mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial
dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid
degeneration, myxomatous degeneration, myxomatosis, mucoid medial
degeneration (for example, cystic medial necrosis), mucolipidosis
(including, for example, mucolipidosis I, mucolipidosis II,
mucolipidosis III, and mucolipidosis IV), mucolysis disorders,
mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis
(such as, for example, type I mucopolysaccharidosis (i.e., Hurler's
syndrome), type IS mucopolysaccharidosis (i.e., Scheie's syndrome
or type V mucopolysaccharidosis), type II mucopolysaccharidosis
(i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e.,
Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e.,
Morquio's syndrome), type VI mucopolysaccharidosis (i.e.,
Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e.,
mucopolysaccharidosis due to beta-glucuronidase deficiency), and
mucosulfatidosis), mucopolysacchariduria, mucopurulent
conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal
disease (i.e., bovine virus diarrhea), mucous colitis (such as, for
example, mucocolitis and myxomembranous colitis), and
mucoviscidosis (such as, for example, cystic fibrosis, cystic
fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic
disease of the pancreas, mucoviscidosis, and viscidosis). In a
highly preferred embodiment, endokine alpha polynucleotides,
polypeptides, and/or agonists and/or antagonists thereof are used
to treat, prevent, and/or diagnose mucositis, especially as
associated with chemotherapy.
[0474] Endokine alpha polynucleotides or polypeptides of the
invention, or agonists or antagonists thereof, may be used to
diagnose, prognose, treat or prevent one or more of the following
diseases or disorders, or conditions associated therewith: primary
immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki
syndrome, bone marrow transplant (e.g., recent bone marrow
transplant in adults or children), chronic B-cell lymphocytic
leukemia, HIV infection (e.g., adult or pediatric HIV infection),
chronic inflammatory demyelinating polyneuropathy, and
post-transfusion purpura.
[0475] Additionally, Endokine alpha polynucleotides or polypeptides
of the invention, or agonists or antagonists thereof, may be used
to diagnose, prognose, treat or prevent one or more of the
following diseases, disorders, or conditions associated therewith,
Guillain-Barre syndrome, anemia (e.g., anemia associated with
parvovirus B 19), patients with stable multiple myeloma who are at
high risk for infection (e.g., recurrent infection), autoimmune
hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia),
thrombocytopenia (e.g., neonatal thrombocytopenia), and
immune-mediated neutropenia, transplantation (e.g., cytamegalovirus
(CMV)-negative recipients of CMV-positive organs),
hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with
risk factor for infection or morbidity), epilepsy (e.g.,
intractable epilepsy), systemic vasculitic syndromes, myasthenia
gravis (e.g., decompensation in myasthenia gravis),
dermatomyositis, and polymyositis.
[0476] Endokine alpha polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, may be used
to treat, prevent, and/or diagnose various immune system-related
disorders and/or conditions associated with these disorders, in
mammals, preferably humans. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune
cells. This inappropriate recognition results in an immune response
leading to the destruction of the host tissue. Therefore, the
administration of endokine alpha polynucleotides or polypeptides of
the invention and/or agonists and/or antagonists thereof that can
inhibit an immune response, particularly the proliferation of B
cells and/or the production of immunoglobulins, may be an effective
therapy in treating and/or preventing autoimmune disorders. Thus,
in preferred embodiments, endokine alpha antagonists of the
invention (e.g., polypeptide fragments of endokine alpha and
anti-endokine alpha antibodies) are used to treat, prevent, and/or
diagnose an autoimmune disorder.
[0477] Autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, and/or diagnosed with the
endokine alpha polynucleotides, polypeptides, and/or antagonists of
the invention (e.g., anti-endokine alpha antibodies), include, but
are not limited to, autoimmune hemolytic anemia, autoimmune
neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g.,
IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes
mellitis, and autoimmune inflammatory eye disease.
[0478] Additional autoimmune disorders (that are highly probable)
that may be treated, prevented, and/or diagnosed with the
compositions of the invention include, but are not limited to,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis) (often characterized, e.g., by cell-mediated and
humoral thyroid cytotoxicity), systemic lupus erhythematosus (often
characterized, e.g., by circulating and locally generated immune
complexes), Goodpasture's syndrome (often characterized, e.g., by
anti-basement membrane antibodies), Pemphigus (often characterized,
e.g., by epidermal acantholytic antibodies), Receptor
autoimmunities such as, for example, Graves' Disease (often
characterized, e.g., by TSH receptor antibodies), Myasthenia Gravis
(often characterized, e.g., by acetylcholine receptor antibodies),
and insulin resistance (often characterized, e.g., by insulin
receptor antibodies), autoimmune hemolytic anemia (often
characterized, e.g., by phagocytosis of antibody-sensitized RBCs),
and autoimmune thrombocytopenic purpura (often characterized, e.g.,
by phagocytosis of antibody-sensitized platelets.
[0479] Additional autoimmune disorders that may be treated,
prevented, and/or diagnosed with the compositions of the invention
include, but are not limited to, rheumatoid arthritis (often
characterized, e.g., by immune complexes in joints), schleroderma
with anti-collagen antibodies (often characterized, e.g., by
nucleolar and other nuclear antibodies), mixed connective tissue
disease (often characterized, e.g., by antibodies to extractable
nuclear antigens (e.g., ribonucleoprotein)),
polymyositis/dermatomyositis (often characterized, e.g., by
nonhistone ANA), pernicious anemia (often characterized, e.g., by
antiparietal cell, microsomes, and intrinsic factor antibodies),
idiopathic Addison's disease (often characterized, e.g., by humoral
and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g., by antispermatozoal antibodies),
glomerulonephritis (often characterized, e.g., by glomerular
basement membrane antibodies or immune complexes) such as primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid (often
characterized, e.g., by IgG and complement in basement membrane),
Sjogren's syndrome (often characterized, e.g., by multiple tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes
millitus (often characterized, e.g., by cell-mediated and humoral
islet cell antibodies), and adrenergic drug resistance (including
adrenergic drug resistance with asthma or cystic fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
[0480] Additional autoimmune disorders (that are possible) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention include, but are not limited to, chronic active
hepatitis (often characterized, e.g., by smooth muscle antibodies),
primary biliary cirrhosis (often characterized, e.g., by
mitchondrial antibodies), other endocrine gland failure (often
characterized, e.g., by specific tissue antibodies in some cases),
vitiligo (often characterized, e.g., by melanocyte antibodies),
vasculitis (often characterized, e.g., by Ig and complement in
vessel walls and/or low serum complement), post-MI (often
characterized, e.g., by myocardial antibodies), cardiotomy syndrome
(often characterized, e.g., by myocardial antibodies), urticaria
(often characterized, e.g., by IgG and IgM antibodies to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM
antibodies to IgE), asthma (often characterized, e.g., by IgG and
IgM antibodies to IgE), inflammatory myopathies, and many other
inflammatory, granulamatous, degenerative, and atrophic
disorders.
[0481] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, and/or diagnosed
using anti-endokine alpha antibodies.
[0482] In a specific preferred embodiment, rheumatoid arthritis is
treated, prevented, and/or diagnosed using anti-endokine alpha
antibodies and/or other antagonist of the invention.
[0483] In a specific preferred embodiment, lupus is treated,
prevented, and/or diagnosed using anti-endokine alpha antibodies
and/or other antagonist of the invention.
[0484] In a specific preferred embodiment, nephritis associated
with lupus is treated, prevented, and/or diagnosed using
anti-endokine alpha antibodies and/or other antagonist of the
invention.
[0485] In a specific embodiment, endokine alpha polynucleotides or
polypeptides, or antagonists thereof (e.g., anti-endokine alpha
antibodies) are used to treat or prevent systemic lupus
erythematosus and/or diseases, disorders or conditions associated
therewith. Lupus-associated diseases, disorders, or conditions that
may be treated or prevented with endokine alpha polynucleotides or
polypeptides, or antagonists of the invention, include, but are not
limited to, hematologic disorders (e.g., hemolytic anemia,
leukopenia, lymphopenia, and thrombocytopenia), immunologic
disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies),
rashes, photosensitivity, oral ulcers, arthritis, fever, fatigue,
weight loss, serositis (e.g., pleuritus (pleuricy)), renal
disorders (e.g., nephritis), neurological disorders (e.g.,
seizures, peripheral neuropathy, CNS related disorders),
gastroinstestinal disorders, Raynaud phenomenon, and pericarditis.
In a preferred embodiment, the endokine alpha polynucleotides or
polypeptides, or antagonists thereof (e.g., anti-endokine alpha
antibodies) are used to treat or prevent renal disorders associated
with systemic lupus erythematosus. In a most preferred embodiment,
Endokine alpha polynucleotides or polypeptides, or antagonists
thereof (e.g., anti-endokine alpha antibodies) are used to treat or
prevent nephritis associated with systemic lupus erythematosus.
[0486] In certain embodiments, soluble endokine alpha polypeptides
of the invention, or agonists thereof, are administered, to treat,
prevent, prognose and/or diagnose an immunodeficiency (e.g., severe
combined immunodeficiency (SCID)-X linked, SCID-autosomal,
adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency, DiGeorge
anomaly, thymic hypoplasia, chronic mucocutaneous candidiasis,
natural killer cell deficiency, idiopathic CD4+ T-lymphocytopenia,
and immunodeficiency with predominant T-cell defect or conditions
associated with an immunodeficiency.
[0487] In a specific embodiment, endokine alpha polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose common
variable immunodeficiency.
[0488] In a specific embodiment, endokine alpha polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose X-linked
agammaglobulinemia.
[0489] In another specific embodiment, endokine alpha polypeptides
or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose severe
combined immunodeficiency (SCID).
[0490] In another specific embodiment, endokine alpha polypeptides
or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose
Wiskott-Aldrich syndrome.
[0491] In another specific embodiment, endokine alpha polypeptides
or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose X-linked
Ig deficiency with hyper IgM.
[0492] In another specific embodiment, endokine alpha polypeptides
or polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, prognose and/or diagnose DiGeorge
anomaly.
[0493] In another specific embodiment, the combination of IL-10 and
endokine alpha polypeptides or polynucleotides can advantageously
be used in the suppression of pathology, associated with T cell
responses, in particular, autoimmune diseases, graft-versus-host
disease (GVHD) and tissue graft rejection. The invention can be
used to suppress cell-mediated reactions such as allograft
rejection and GVHD. Moreover, considering the diverse biological
activities of IL-10, the concurrent use of IL-10 and endokine alpha
polypeptides or polynucleotides may support GVL
(graft-versus-leukemia) in allogeneic bone marrow transplants. In
another specific embodiment, the compositions of the invention may
be used to prevent the rejection or prolong the survival of
allogeneic transplants of skin, bone, neuronal tissue, synovium,
heart, kidney, pancreas, bone marrow, small intestine, lung,
combined heart-lung, corneal tissue, liver, etc. The transplanted
tissue itself is typically human in origin, but may also be from
another species such as a rhesus monkey, baboon or pig. As used
herein, the term "tissue" includes individual cells, such as blood
cells, including progenitors and precursors thereof, and pancreatic
cells, as well as solid organs and the like. The term solid organ
means a heart, skin, a liver, a lung, a cornea, a kidney, a
pancreas, an intestine, endocrine glands, a bladder, skeletal
muscles, etc. In another specific embodiment, the compositions of
the invention may be used to treat a large category of diseases,
prior to and/or after onset thereof, such diseases including, but
not being limited to, autoimmune diseases, including, but not
limited to, inflammatory conditions with an etiology including an
autoimmune component such as arthritis (for example rheumatoid
arthritis, arthritis chronica progrediente and arthritis deformans)
and rheumatic diseases.
[0494] Autoimmune diseases may be divided into two general types,
namely systemic autoimmune diseases (exemplified by arthritis,
lupus and scleroderma), and organ specific (exemplified by multiple
sclerosis, diabetes and atherosclerosis, in which latter case the
vasculature is regarded as a specific organ). Specific autoimmune
diseases for which the compositions of the invention may be
employed include, but, are not limited to, autoimmune hematological
disorders (including, e.g., hemolytic anemia, aplastic anemia,
pernicious anemia, pure red cell anemia and idiopathic
thrombocytopenia), systemic lupus erythematosus, polychondritis,
scleroderma, Wegener granulomatosis, dermatomyositis, chronic
active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson
syndrome, idiopathic sprue, autoimmune inflammatory bowel disease
(including, e.g., ulcerative colitis and Crohn's disease),
autoimmune thyroiditis, idiopathic Addison's disease, vitilogo,
gluten-sensitive enteropathy, autoimmune neutropenias, pemphigus
vulgaris, Goodpasture's disease, bullous pemphigoid, discoid lupus,
dense deposit disease, endocrine opthalmopathy, IBD, asthma, Graves
disease, sarcoidosis, multiple sclerosis, cirrhosis including
primary biliary cirrhosis, juvenile diabetes (diabetes mellitus
type 1), insulin dependent diabetes mellitus (i.e., IDDM, or
autoimmune diabetes), uveitis (anterior and posterior), autoimmune
gastritis, lymphopenias, polyarteritis nodosa, Sjogren's syndrome,
Bechet's disease, Hashimoto's disease, primary myxedema,
polymyositis, mixed connective tissue disease, keratoconjunctivitis
sicca and vernal keratoconjunctivitis, interstitial lung fibrosis,
psoriatic arthritis, glomerulonephritis (with and without nephrotic
syndrome, e.g., including idiopathic nephrotic syndrome or minimal
change nephropathy), juvenile dermatomyositis, hepatitis including
chronic active hepatitis, organ rejection, and other afflictions.
Other diseases and conditions include, but are not limited to,
autoimmune thyroiditis, autoimmune hemolytic anemia, and contact
sensitivity disease, which may, for example, be caused by plant
matter, such as poison ivy. Other diseases and conditions include,
but are not limited to, suppressing chronic and acute monophasic
EAE, HIV-related conditions, AIDS, SCIDS, adjuvant arthritis, a
lymphatic malignancy or immune disorder, and neurodegenerative
diseases such as amyotrophic lateral sclerosis, Alzheimer's
disease, Parkinson's disease and primary lateral sclerosis.
Further, the inventive compositions can be used to promote wound
healing and to treat infectious diseases.
[0495] The diseases set forth above, as referred to herein, include
those exhibited by animal models for such diseases, such as, for
example non-obese diabetic (NOD) mice for IDDM and experimental
autoimmune encephalomyelitis (EAE) mice for multiple sclerosis.
Other conditions include immune system-related miscarriage and
inflammatory disorders. The discoveries of the present invention
may also be applied to treat autoimmune diseases which manifest as
infertility, including endometriosis.
[0496] Further, it is becoming increasingly apparent that many
vascular disorders, including atherosclerotic forms of such
disorders, have an autoimmune component, and a number of patients
with vascular disease have circulating auto antibodies. In general,
the compositions of the invention are useful in immunomodulation,
especially immunosuppression, and in the treatment of leukemias
characterized by over-proliferation of T-lymphocytes, including
virally-induced leukemias, e.g., HTLV-1-induced leukemia. An
improvement or amelioration in immune function can be assessed by
observation of partial or total restoration of the ability to mount
an immune response. In the case of autoimmune disease, an
improvement or amelioration can best be assessed by a significant
reduction or disappearance of a clinical symptom associated with
inflammation caused by the autoimmune disease, for example, joint
pain or swelling or stiffness in rheumatoid arthritis; number of
major attacks in chronic-relapsing multiple sclerosis;
stabilization or improvement of motor function in chronic
progressive multiple sclerosis; intestinal inflammation in the case
of Chron's disease; and serological measurements (such as antibody
to double-stranded DNA, complement components and circulating
immune complexes), and number and severity of skin flare-ups or
myalgras, arthralgia, leukopenia, or thrombocytopenia for systemic
lupus erythematosus. The symptoms which can be used to monitor
efficacy of a regimen in autoimmune disease are generally
well-known in the art. The compositions of the invention can be
applied to induce T cell tolerance to a variety of antigens. For
example, T cell tolerance can be induced to a soluble antigen
(e.g., a soluble protein). T cells can be tolerized to antigens
involved in autoimmune diseases or disorders associated with
abnormal immune responses. For example, in one embodiment, the
antigen is an autoantigen. In another embodiment, the antigen is an
allergan. Alternatively, T cells can be tolerized to antigens
expressed on foreign cells. Accordingly, in yet other embodiments,
the antigen is an alloantigen or xenoantigen. Induction of T cell
tolerance to alloantigens and xenoantigens is of particular use in
transplantation, for example to inhibit rejection by a transplant
recipient of a donor graft, e.g., a tissue or organ graft or bone
marrow transplant. Additionally, tolerization of donor T cells
within a bone marrow graft is useful for inhibiting graft versus
host disease.
Gene Therapy
[0497] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, byway of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0498] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0499] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993);
TIBTECH 11(5):155-215 (May 1993)). Methods commonly known in the
art of recombinant DNA technology which can be used are described
in Ausubel et al., eds., Current Protocols in Molecular Biology,
John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0500] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody nucleic acids (Koller and Smithies,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al.,
Nature 342:435-438 (1989)). In specific embodiments, the expressed
antibody molecule is a single chain antibody; alternatively, the
nucleic acid sequences include sequences encoding both the heavy
and light chains, or fragments thereof, of the antibody.
[0501] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0502] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where they are expressed to produce
the encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering them
so that they become intracellular, e.g., by infection using
defective or attenuated retrovirals or other viral vectors (see
U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or
by use of microparticle bombardment (e.g., a gene gun; Biolistic,
Dupont), or coating with lipids or cell-surface receptors or
transfecting agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec.
23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis
et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.); and WO
93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989)).
[0503] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an antibody of the invention are used. For
example, a retroviral vector can be used (see Miller et al., Meth.
Enzymol. 217:581-599 (1993)). These retroviral vectors have been to
delete retroviral sequences that are not necessary for packaging of
the viral genome and integration into host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[0504] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0505] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0506] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0507] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including, but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92 (1985)) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0508] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0509] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0510] In a preferred embodiment, the cells used for gene therapy
is autologous to the patient.
[0511] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598, dated Apr. 28, 1994; Stemple and
Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio.
21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771
(1986)).
[0512] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[0513] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
[0514] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0515] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0516] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
or compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0517] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0518] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler, eds., Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327).
[0519] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise, eds., CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball, eds., Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0520] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0521] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0522] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0523] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0524] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0525] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0526] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0527] 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.
Optionally 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.
Diagnosis and Imaging
[0528] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or disorders associated with the aberrant expression and/or
activity of a polypeptide of the invention. The invention provides
for the detection of aberrant expression of a polypeptide of
interest, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0529] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0530] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see 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 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;
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99Tc); luminescent labels, such
as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0531] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of the interest in an animal, preferably a mammal and
most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0532] 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 99mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific 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)).
[0533] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0534] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0535] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0536] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
Kits
[0537] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0538] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0539] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0540] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0541] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or calorimetric substrate (Sigma, St.
Louis, Mo.).
[0542] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0543] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0544] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Expression and Purification of Endokine Alpha in E. coli
[0545] The DNA sequence encoding the endokine alpha protein in the
deposited cDNA clone is amplified using PCR oligonucleotide primers
specific to the amino terminal sequences of the endokine alpha
protein. Additional nucleotides containing restriction sites to
facilitate cloning are added to the 5' and 3' sequences,
respectively.
[0546] The 5' oligonucleotide primer has the sequence GCG CCA TGG
CTA AGT TTG GAC CAT (SEQ ID NO:5) containing the underlined Nco I
restriction site.
[0547] The 3' primer has the sequence GCG AAG CTT TCA AGT CTC TAG
GAG ATG (SEQ ID NO:6) containing the underlined HindIII restriction
site.
[0548] The restriction sites are convenient to restriction enzyme
sites in the bacterial expression vector pQE60, which is used for
bacterial expression in M15/rep4 host cells in these examples.
(Qiagen, Inc., Chatsworth, Calif., 91311). pQE60 encodes ampicillin
antibiotic resistance ("Amp.sup.r") and contains a bacterial origin
of replication ("ori"), an IPTG inducible promoter, a ribosome
binding site ("RBS"), a 6-His tag and restriction enzyme sites.
[0549] The amplified endokine alpha protein DNA and the vector
pQE60 both are digested with NcoI and HindIII and the digested DNAs
are then ligated together. Insertion of the endokine alpha protein
DNA into the restricted pQE60 vector places the endokine alpha
protein coding region downstream of and operably linked to the
vector's IPTG-inducible promoter and in-frame with an initiating
ATG appropriately positioned for translation of endokine alpha
protein.
[0550] The ligation mixture is transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kan.sup.r"), is used in carrying out the illustrative
example described here. This strain, which is only one of many that
are suitable for expressing endokine alpha protein, is available
commercially from Qiagen.
[0551] 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.
[0552] 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).
[0553] The O/N culture is used to inoculate a large culture, at a
dilution of approximately 1:100 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-thiogalactopyranoside ("IPTG") is then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation and disrupted, by standard
methods. Inclusion bodies are purified from the disrupted cells
using routine collection techniques, and protein is solubilized
from the inclusion bodies into 8M urea. The 8M urea solution
containing the solubilized protein is passed over a PD-10 column in
2.times. phosphate-buffered saline ("PBS"), thereby removing the
urea, exchanging the buffer and refolding the protein. The protein
is purified by a further step of chromatography to remove
endotoxin. Then, it is sterile filtered. The sterile filtered
protein preparation is stored in 2.times.PBS.
Example 2
Cloning and Expression of Endokine Alpha in a Baculovirus
Expression System
[0554] The cDNA sequence encoding the endokine alpha protein in the
deposited clone is amplified using PCR oligonucleotide primers
corresponding to 5' and 3' regions of the gene.
[0555] The 5' primer has the sequence GC GGA TCC CGA GAC TGC TAA
GGA GCC (SEQ ID NO:7) containing the underlined BamHI restriction
enzyme site and containing nucleotides encoding a portion of the
endokine alpha protein in FIG. 1.
[0556] The 3' primer has the sequence GC GGA TCC CTA GGA GAT GAA
TTG GGG ATT TG (SEQ ID NO:8) containing the underlined BamHI
restriction site and containing a sequence complementary to that
encoding a portion of the endokine alpha protein in FIG. 1.
[0557] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc.,
LaJolla, Calif.). The fragment then is digested with BamHI and
again is purified on a 1% agarose gel. This fragment is designated
herein F2.
[0558] The vector pA2-GP is used to express the endokine alpha
protein in the baculovirus expression system, using standard
methods, as described in Summers et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This
expression vector contains the strong polyhedrin promoter of the
Autographa californica nuclear polyhedrosis virus (AcMNPV) followed
by convenient restriction sites. The signal peptide of AcMNPV gp67,
including the N-terminal methionine, is located just upstream of a
BamHI site. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For an easy
selection of recombinant virus, the beta-galactosidase gene from E.
coli is inserted in the same orientation as the polyhedrin promoter
and is followed by the polyadenylation signal of the polyhedrin
gene. The polyhedrin sequences are flanked at both sides by viral
sequences for cell-mediated homologous recombination with wild-type
viral DNA to generate viable virus that express the cloned
polynucleotide.
[0559] Many other baculovirus vectors could be used in place of
pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of
skill readily will appreciate, that construction provides
appropriately located signals for transcription, translation,
trafficking and the like, such as an in-frame AUG and a signal
peptide, as required. Such vectors are described in Luckow et al.,
Virology 170: 31-39, among others.
[0560] The plasmid is digested with the restriction enzyme BamHI
and then is 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 "V2".
[0561] Fragment F2 and the dephosphorylated plasmid V2 are ligated
together with T4 DNA ligase. E. coli HB101 cells are transformed
with ligation mix and spread on culture plates. Bacteria are
identified that contain the plasmid with the human endokine alpha
gene by digesting DNA from individual colonies using BamHI and then
analyzing the digestion product by gel electrophoresis. The
sequence of the cloned fragment is confirmed by DNA sequencing.
[0562] 5 .mu.g of the plasmid 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). 1 .mu.g of BaculoGold.TM. virus DNA
and 5 .mu.g of the plasmid 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 rocked back and forth to mix the newly
added solution. The plate is then incubated for 5 hours at
27.degree. C. After 5 hours the transfection solution is removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. The plate is put back into an
incubator and cultivation is continued at 27.degree. C. for four
days.
[0563] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with "Blue Gal" (Life Technologies Inc.,
Gaithersburg) 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).
[0564] Four days after serial dilution, the virus is added to the
cells. After appropriate incubation, blue stained plaques are
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses is then resuspended in an Eppendorf tube
containing 200 .mu.l of Grace's medium. The agar is removed by a
brief centrifugation and the supernatant 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. A clone
containing properly inserted endokine alpha is identified by DNA
analysis including restriction mapping and sequencing of this
plasmid.
[0565] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus at a multiplicity of infection ("MOI") of about 2
(about 1 to about 3). Six hours later the medium is removed and is
replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Gaithersburg). 42 hours
later, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then they are harvested by
centrifugation, lysed and the labeled proteins are visualized by
SDS-PAGE and autoradiography.
Example 3
Cloning and Expression in CHO Cells
[0566] The vector pC4 is used for the expression of endokine alpha
protein. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC
Accession No. 37146). The 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., J. Biol. Chem. 253:1357-1370 (1978),
Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143
(1990), Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68)
(1991). Cells grown in increasing concentrations of MTX develop
resistance to the drug by overproducing the target enzyme, DHFR, as
a result of amplification of the DHFR gene. If a second gene is
linked to the DHFR gene, it is usually co-amplified and
over-expressed. It is 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.
[0567] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen et al., Molec. Cell. Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early
gene of human cytomegalovirus (CMV) (Boshart et al., Cell
41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow integration of
the genes. 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 endokine alpha in a regulated way in mammalian cells (Gossen,
M., & Bujard, H., Proc. Natl. Acad. Sci. USA 89: 5547-5551
(1992)). 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.
[0568] The plasmid pC4 is digested with the restriction enzymes
BamHI and Asp718I and then dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0569] The DNA sequence encoding the complete endokine alpha
protein including its leader sequence is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of
the gene. The 5' primer has the sequence 5' GCG GGA TCC GCC ATC ATG
CCT TTA AGC CAT TC 3' (SEQ ID NO:9) containing the underlined BamHI
restriction enzyme site followed by an efficient signal for
initiation of translation in eukaryotes, as described by Kozak, M.,
J. Mol. Biol. 196:947-950 (1987), and 17 bases of the coding
sequence of endokine alpha shown in FIG. 1 (SEQ ID NO: 1). The 3'
primer has the sequence 5' GC GGA TCC CTA GGA GAT GAA TTG GGG ATT
TG 3' (SEQ ID NO: 10) containing the underlined Asp718I restriction
site followed by nucleotides complementary to the non-translated
region of the endokine alpha gene shown in FIG. 1 (SEQ ID NO:
1).
[0570] The amplified fragment is digested with the endonucleases
BamHI and Asp718I 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.
[0571] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. 5 .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using
lipofectin (Felgner et al., supra). The plasmid pSV2neo 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 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 reverse phase HPLC analysis.
Example 4
Tissue Distribution of Endokine Alpha Expression
[0572] Northern blot analysis was carried out to examine the levels
of expression of the gene encoding the endokine alpha protein in
human tissues, using methods described by, among others, Sambrook
et al., supra. A cDNA probe containing the entire nucleotide
sequence of the endokine alpha protein of the present invention
(SEQ ID NO: 1) was labeled with .sup.32P using the Rediprime.TM.
DNA labeling system (Amersham Life Science), according to
manufacturer's instructions. After labelling, the probe was
purified using a CHROMA SPIN-100.TM. column (Clontech Laboratories,
Inc.), according to manufacturer's protocol number PT1200-1. The
purified labelled probe was then used to examine various human
tissues for the expression of the gene encoding the endokine alpha
protein.
[0573] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) were obtained
from Clontech and were examined with labelled probe using
ExpressHyb.TM. Hybridization Solution (Clontech) according to
manufacturer's protocol number PT 1190-1. Following hybridization
and washing, the blots were mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
[0574] Expression of the gene encoding an endokine alpha protein of
the present invention was detected in human brain striatum and
pancreas tissue.
Example 5
Identification of a Novel Activation-Inducible Protein of the TNF
Receptor Superfamily and its Ligand
Background
[0575] Members of the TNFR superfamily share similar multiple
cysteine-rich pseudorepeats of the extracellular domain, each
containing 30-45 amino acids with six cysteines (Smith, C. A., et
al, Cell 76:959-962 (1994)). Except for the death domain-containing
family which includes TNFR1 (Schall, T. J., et al, Cell 61:361-370
(1990)), Fas (Trauth, B. C., et al, Science 245:301-305 (1989),
Yonehara, S., et al, J. Exp. Med. 169:1747-1756 (1989), and Oehm,
A., et al, J. Biol. Chem. 267:10709-10715 (1992)), DR3 (Chinnaiyan,
A. M., et al, Science 274:990-992 (1996), Kitson, J., et al, Nature
384:372-375 (1996), Bodmer, J.-L., et al, Immunity 6:79-88 (1997),
and Screaton, G. R., et al, Proc. Natl. Acad. Sci. USA 94:4615-4619
(1997)), DR4 (Wiley, S. R., et al, Immunity 3:673-682 (1995),
Pitti, R. M., et al, J. Biol. Chem. 271:12687-2690 (1996), and Pan,
G., et al, Science 276:111-113 (1997)), DR5 (Walczak, H., et al,
EMBO J. 16:5386-5397 (1997), MacFarlane, M., et al, J. Biol. Chem.
272:25417-25420 (1997), Schneider, P., et al, Immunity 7:831-836
(1997), Chaudhary, P. M., et al, Immunity 7:821-830 (1997), and
Sheridan, J. P., et al, Science 277:818-821 (1997)), and decoy
TRAIL receptors (Marsters, S. A., et al, Cur. Biol. 7:1003-1006
(1997), Pan, G., et al, Science 277:815-815 (1997), Degli-Esposti,
M. A., et al, J. Exp. Med. 186:1165-1170 (1997), and Degli-Esposti,
M. A., et al, Immunity 7:813-820 (1997)), no remarkable similarity
is found within the intracellular domain of these molecules.
However, there is a striking homology in the cytoplasmic domains of
murine and human 4-1BB, CD27, and murine GITR within TNFR
superfamily members (Kwon, B. S., et al, Proc. Natl. Acad. Sci. USA
86:1963-1967 (1989), Camerimi, D., et al, J. Immunol. 147:3165-3169
(1991), and Nocentini, G., et al, Proc. Natl. Acad. Sci. USA
94:6216-6221 (1997)). Acidic amino acids are especially highly
conserved in the cytoplasmic domain of this subfamily. Like other
TNFR superfamily members (Smith, C. A., et al, Cell 76:959-962
(1994)), this subfamily is implicated in diverse biological
functions. First of all, 4-1BB and CD27 molecules provide strong
costimulatory signals for T cell proliferation when ligated with
their respective ligands or with agonistic antibodies (Smith, C.
A., et al, Cell 76:959-962 (1994), and Pollok, K. E., et al, J.
Immunol. 150:771-781 (1993)). In addition to functioning as an
accessory molecule, CD27 induces apoptosis, which is mediated by a
death domain-containing molecule called Siva (Prasad, K. V. S., et
al, Proc. Natl. Acad. Sci. USA 94:6346-6351 (1997)). Recently
identified murine GITR is shown to inhibit TCR-induced apoptosis
(Nocentini, G., et al, Proc. Natl. Acad. Sci. USA 94:6216-6221
(1997)).
[0576] Although the immunological functions of subfamily members
have been relatively well defined, insights into their signal
transduction pathway have only recently been revealed (Arch, R. H.,
et al, Mol. Cell. Biol. 18:558-565 (1998), Jang, I. K., et al,
Biochem. Biophys. Res. Com. 242:613-620 (1998), Saoulli, K., et al,
J. Exp. Med. 187:1849-1862 (1998), and Akiba, H., et al, J. Biol.
Chem. 273:13353-13358 (1998)). Two groups (Arch, R. H., et al, Mol.
Cell. Biol. 18:558-565 (1998), and Jang, I. K., et al, Biochem.
Biophys. Res. Come 242:613-620 (1998)) have provided data
indicating that association of 4-1BB with TRAF2 molecules initiates
a signal cascade leading to activation of NF-.kappa.B. In the CD27
signaling pathway, both TRAF2 and TRAF5 mediate NF-.kappa.B and
SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase)
activation and NIK (NF-.kappa.B-inducing kinase) is a common
downstream kinase of TRAF2 and TRAF5 (Akiba, H., et al, J. Biol.
Chem. 273:13353-13358 (1998)).
[0577] Because the number of TNFR members is rapidly expanding, it
was expected that even more numbers of the superfamily would exist.
By a PCR-based strategy with murine GITR sequence and searching an
EST (expressed sequence tag) database, a new member of the TNFR was
discovered and named TR11. The following provides a
characterization of the receptor TR11 and its ligand, endokine
alpha.
Experimental Procedures
[0578] cDNA cloning. A database containing more than two million
ESTs obtained from over 750 different cDNA libraries was generated
by Human Genome Sciences, Inc., using high throughput automated DNA
sequence analysis of randomly selected human cDNA clones. A
specific homology and motif search using the known amino acid
sequence and motif of TNFR members against this database revealed
several ESTs with a translated sequence 35-55% homologous to that
of the TNFR family. Several clones were identified from cDNA
libraries of PHA-activated T cells, T helper cells, leukocytes, a
healing abdomen wound, primary dendritic cells and adipose tissue.
A full-length TR-11 cDNA clone encoding an intact N-terminal signal
peptide was obtained from a human activated T-cell library and
selected for further investigation (see, U.S. patent application
Ser. No. 09/176,200 filed Oct. 21, 1998). The complete cDNA
sequence of both strands of this clone was determined, and its
homology to TNFR members was confirmed. The same gene was also
identified by a PCR-based strategy with murine GITR sequence.
Similarly, endokine-.alpha. (TNF ligand 6) was identified through a
systematic comparison of sequence homology with TNF ligand family
members. Partial endokine-.alpha. sequences which were 25%
homologous to that of TNF ligand family members were identified
from endothelial, HUVEC (human umbilical vein endothelial cell),
brain, and fetal liver cDNA libraries. A full-length cDNA clone was
obtained from a human brain cDNA library.
[0579] Expression vectors. Full-length and HA (hemaglutinin A
epitope)-tagged TR-11 encoding the putative entire TR-11 protein
(amino acids 26-234) were amplified by PCR using sense
(5'CTAGCTAGCTAGVVVAGCGCCCC ACCGGGGGTCCC-3', and
5'-CTAGCTAGCTAGCTATCCATAT
GATGTTCCAGATTATGCTCAGCGCCCCACCGGGGGTCCC-3', respectively) and
anti-sense (5-AAGGAAAAAAGCGGGCCGCTCACACCCACAGGTCTCCCAG-3') primers,
cut with Nhe I/Not I, and fused in frame downstream of a CD5 leader
sequence (Jang, I. K., et al, Biochem. Biophys. Res. Com.
242:613-620 (1998)) into the pcDNA3.1 (pcDNA3.1/CD5L-TR-11) and
pcDNA3 (pcDNA3/CD5L-TR-11), respectively. Full-length
endokine-.alpha. was amplified by PCR (sense,
5-AGACCCAAGCTTTTGAAAATGAT ATGAGACGC-3'; anti-sense,
5'-AGACGGGATCCTCCTCCTATAGTAAGAAGGC-3'), cut with Hind III/BamHI,
and inserted into pcDNA3.1 (pcDNA3.1/endokine-.alpha.) and pCEP4
(Invitrogen, Carlsbad, Calif.; pCEP4/endokine-.alpha.). pRK5-based
expression vectors encoding Flag-tagged full-length TRAF1, TRAF2,
TRAF3, TRAF5, TRAF6, NIK, dominant negative TRAF2 (dnTRAF2), or
dnNIK have been described (Jang, I. K., et al, Biochem. Biophys.
Res. Com. 242:613-620 (1998), Rothe, M., et al, Science
269:1421-1427 (1995), Hu, H. M., et al, J. Biol. Chem.
269:30069-30072 (1994), Nakano, H., et al, J. Biol. Chem.
271:14661-14664 (1996), Takeuchi, M., et al, J. Biol. Chem.
271:19935-19942 (1996), Cao, Z., et al, Nature 383:443-446 (1996),
and Song, H. Y., et al, Proc. Natl. Acad. Sci. USA 94:9792-9796
(1997)). The NF-.kappa.B-dependent E-selectin-luciferase reporter
gene (pELAM-Luc) and pRSV-.beta.-galactosidase (pRSV-.beta.-gal)
plasmids were also described elsewhere (Rothe, M., et al, Science
269:1421-1427 (1995), and Schindler, U., et al, Mol. Cell. Biol.
14:5820-9796 (1994)).
[0580] Northern blot and RT (reverse transcriptase)-PCR analysis.
For Northern blot analysis, cDNA probes were labeled with .sup.32P
using the Rediprime DNA labeling system (Amersham Life Science,
Arlington Height, Ill.), according to the manufacturer's
instructions. Unincorporated nucleotide was removed from the
labeled probe using CHROMA SPIN-100 (Clonetech, Palo Alto, Calif.).
Two human multiple tissue poly(A) RNA blots containing
approximately 2 .mu.g of poly(A) RNA per lane from various human
tissues were purchased from Clontech. In addition, two cell line
blots containing 20 mg total RNA from different cell lines were
used. Northern blotting was performed with the Expressed
Hybridization Solution (Clonetech) according to the manufacturer's
manual. For RT-PCR analysis, total RNA was isolated from human PBMC
after stimulation with dexamethasone, PMA/ionomycin, or
anti-CD3/CD28 mAbs, and from unstimulated or LPS-stimulated HUVEC
cells. RT-PCR was performed under standard conditions.
[0581] Interaction of TR-11 with TRAFs. pcDNA3/CD5L-TR-11-HA
plasmid (5 .mu.g/110 cm-plate) was co-transfected into HEK293 EBNA
cells (2.times.10.sup.6 cells/plate) by the standard calcium
phosphate precipitation method with pRK/TRAF1, 2, 3, 5, or 6-Flag
vector (5 .mu.g/plate). Twenty four-hours after transfection, cells
were lysed with 1 ml of lysis buffer (50 mM HEPES [pH7.4], 250 mM
NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 10% glycerol, and protease
inhibitors). For immunoprecipitation, lysates were incubated with
anti-Flag M2 (Eastman Kodak, Rochester, N.Y.) or control murine
IgG1 mAb at 4.degree. C. for 1 h, followed by incubation with 201
of a 1:1 slurry of protein G-Sepharose (PharMingen, San Diego,
Calif.) for another hour. Precipitates were thoroughly washed with
lysis buffer, then fractionated on a 10% SDS-polyacrylamide gel
before transfer to PVDF membrane (Millipore, Bedfore, Mass.).
Western blot analysis was performed with anti-HA mAb coupled with
horseradish peroxidase (Boehringer Mannheim, Indianapolis, Ind.)
and visualized using the enhanced chemiluminescence Western
blotting detection system (Amersham).
[0582] Analysis of NF-.kappa.B by reporter assay. Approximately
0.5.times.10.sup.6 HEK293 EBNA cells/well were seeded on 6-well
plates. After 24 h, cells were transfected by the standard calcium
phosphate precipitation method using various combinations of
pcDNA3.1/CD5L-TR-11 plus pRK5 plasmids encoding TRAFs, dnTRAF2,
NIK, or dnNIK. The total amount of plasmid was adjusted to 2.0
.mu.g by adding empty vector. Twenty-four hours after transfection,
cells were lysed in 200 .mu.l reporter lysis buffer (Promega,
Madison, Wis.). Luciferase activity was measured using 20 .mu.l
cell extract. 5 .mu.l cell extract was used to assay
.beta.-galactosidase activity as an internal control, and
luminescence values were normalized by individual
.beta.-galactosidase activity.
[0583] Recombinant protein production and purification. TR-11-Fc
fusion protein was used for ligand screening and cell-binding
experiments. A fragment encoding the predicted extracellular domain
of TR-11 (amino acids 26-139) was amplified using a sense primer
flanked by an Nhe I site
(5'-AGACCCAAGCTTGTGGGCTCTTGAAACCCGGCATG-3') and an antisense primer
flanked by a Bgl II site (5'-GAAAGATCTGGGCTCTGCCGG
CGGGGACCCTGGGAC-3'). The amplified fragment was cut with Nhe I/Bgl
II and cloned into mammalian vector pCEP4, in frame with CD5L at
the 5' end and with the Fc portion of human IgG1 at the 3' end
(pCEP4/CD5L-TR-11-Fc). pCEP4/CD5L-TR-11-Fc was transfected into
HEK293 EBNA cells. TR-11-Fc fusion protein was purified from
pCEP4/CD5L-TR-11-Fc-transfected HEK293 EBNA cell supernatants using
protein G column. To generate a Flag-tagged soluble form of
endokine-.alpha. protein (amino acids 39-169), the flag-tagged
endokine-.alpha. expression vector
(pCEP4/CD5L-endokine-.alpha.-Flag) was constructed by PCR
amplification of endokine-.alpha. coding sequences using sense
(5'-CTAGCTAGCCCAGCGCCCCGACTACAAGGACGACGATGACAAGGAGACTGCTAAGGAG
CCC-3') and antisense (5'-CCGCTCGAGCTATAG TAAGAAGGCTCC-3') primers,
digesting the product with Nhe I/Xho I and cloning into pCEP4, in
frame with the CD5L sequence. The construct was expressed in HEK293
EBNA cells. Transfected cell supernatants containing secreted
endokine-.alpha.-Flag were harvested and used for binding assays.
For some experiments, endokine-.alpha.-Flag protein was purified
from harvested supernatants, using anti-Flag gel (Sigma, St. Louis.
Mo.) according to the manufacturer's instructions.
[0584] Binding assay. Protein binding assays were done essentially
as described (Pan, G., et al, Science 276:111-113 (1997)). For
cell-binding assays, HEK293 EBNA cells were transfected using
pcDNA3.1/CD5L-TR-11 or pcDNA3.1, as described above. Forty-eight
hours after transfection, cells were harvested and incubated
consecutively with endokine-.alpha.-Flag-containing supernatant,
anti-Flag antibody, and FITC-conjugated anti-mouse IgG antibody
(Southern Biotechnology, Birmingham, Ala.). Flow cytometry analysis
was performed using the Becton Dickinson FACScan (San Jose,
Calif.). Jurkat T cells were stably transfected by electroporation
using linearized pcDNA3.1/CD5L-TR-11, and selected in the presence
of Zeocin (Invitrogen). A binding assay for this cell line was
performed as described above. To test the ability of TR-11-Fc
fusion protein to bind membrane-bound endokine-.alpha.,
pCEP4/endokine-.alpha. was stably transfected into HEK293 EBNA
cells. After selection in the presence of hygromycin,
endokine-.alpha.-expressing cells were harvested and incubated with
TR-11-Fc protein, followed by FITC-conjugated anti-human IgG1
antibody (Southern Biotechnology). The Becton Dickinson FACScan was
used for flow cytometry analysis.
Results and Discussion
[0585] TR-11 was identified by searching an EST database and by a
PCR-based strategy with murine GITR sequence. A full-length cDNA of
a clone from a human activated T-cell cDNA library, which is
tentatively named TR-11 (for activation-inducible TNFR family
member), encodes a 234 amino acid type I transmembrane protein with
a calculated MW of 25 kDa. The receptor has a signal peptide (the
first 25 amino acids) and a single transmembrane region (amino
acids 140-158). When compared with the extracellular domain of
other TNFR family members, TR-11 displays three cysteine-rich
pseudorepeats corresponding to the second, third, and fourth TNFR
motif, respectively. The first cysteine pseudorepeat contains eight
cysteine residues and lacks C4. Therefore, it is unlikely that the
canonical pattern of C1-C2, C3-C5, and C4-C6 disulfide bridges
exist in this motif. The second pseudorepeat shows some features of
the third TNFR motif, but it is atypical in that C5 is not present
even though it contains 7 cysteine residues. The third pseudorepeat
shows extensive homologies with the fourth pseudorepeat of 4-1BB.
The cytoplasmic domain contains acidic amino acids which are highly
conserved in the cytoplasmic domains of 4-1BB, CD27, and GITR.
Overall, TR-11 exhibits a high homology (55% identity) to murine
GITR, but there is a mismatch in the first cysteine-rich
pseudorepeat between GITR and TR-11, because the first pseudorepeat
of GITR corresponds to the first TNFR cysteine-rich motif
(Nocentini, G., et al, Proc. Natl. Acad. Sci. USA 94:6216-6221
(1997)).
[0586] The expression of TR-11 mRNA was investigated in multiple
human tissues by Northern blot hybridization. 1.25-kb mRNA was
detected in lymph node, PBL, and, weakly, in spleen. We also tested
a variety of tumor cell lines for expression of TR-11 mRNA. 1.25-kb
message was detected only in the colorectal adenocarcinoma cell
line, SW480, among the cell lines tested. The expression of
virtually all members of the TNFR superfamily is enhanced by
antigen stimulation/lymphocyte activation (Smith, C. A., et al,
Cell 76:959-962 (1994)). Consistent with this idea, TR-11
expression was upregulated in PBMC after stimulation. No TR-11
message was detectable in unstimulated PBMC when we used a
sensitive RT-PCR method. TR-11 expression was clearly induced
within 24 h by typical PBMC stimulation such as treatment with PMA
plus ionomycin or soluble anti-CD3 plus anti-CD28 mAbs. FACS
analysis for TR-11 expression, however, showed that a small
population of activated PBMC expressed TR-11 on the cell surface at
48 h after stimulation, suggesting that a prolonged period of
stimulation is required for maximum expression of TR-11 (BK,
unpublished data). Expression of TR-11 was not induced by treatment
with dexamethasone. This property was different from that of GITR
(Nocentini, G., et al, Proc. Natl. Acad. Sci. USA 94:6216-6221
(1997)).
[0587] Recently it has been shown that 4-1BB molecules associate
with TRAF1, TRAF2, and TRAF3 (Arch, R. H., et al, Mol. Cell. Biol.
18:558-565 (1998), Jang, I. K., et al, Biochem. Biophys. Res. Com.
242:613-620 (1998), and Saoulli, K., et al, J. Exp. Med.
187:1849-1862 (1998)). Because TR-11's cytoplasmic domain is
similar to that of 4-1 BB, its ability to co-precipitate five of
the six known TRAFs that were overexpressed in HEK293 EBNA cells
was tested. An interaction of TR-11 with TRAF1, TRAF2, and TRAF3
was observed but not with TRAF5 and TRAF6. The association of TR-11
with TRAF2 suggested that, like other members of the TNFR
superfamily (Arch, R. H., et al, Mol. Cell. Biol. 18:558-565
(1998), Jang, I. K., et al, Biochem. Biophys. Res. Come 242:613-620
(1998), Akiba, H., et al, J. Biol. Chem. 273:13353-13358 (1998),
Rothe, M., et al, Science 269:1421-1427 (1995), Cheng, G., et al,
Science 267:1494-1498 (1995), Duckett, C. S., et al, Mol. Cell.
Biol. 17:1535-1542 (1997), and VanArsdale, T. L., et al, Proc.
Natl. Acad. Sci. USA 94:2460-2465 (1996)), TR-11 might mediate
NF-.kappa.B activation through TRAF2. To test this possibility, an
NF-.kappa.B reporter system in HEK293 EBNA cells was used (Rothe,
M., et al, Science 269:1421-1427 (1995)). Co-transfection with the
TR-11 expression vector typically induced greater than 3-fold
higher luciferase activity when compared with the vector
transfection control. When co-expressed with TRAF2, TR-11 induced
greater luciferase activity than did TRAF2 alone. More importantly,
overexpression of dominant-negative TRAF2, which lacked the RING
and zinc finger motifs (Rothe, M., et al, Science 269:1421-1427
(1995)), abrogated the luciferase activity induced by TR-11. This
indicates that TRAF2 is an important mediator of NF-.kappa.B
activation for TR-11. A similar observation was made when the
activity of NIK, which was thought to lie downstream of TRAF2 in
the NF-.kappa.B signaling pathway, was blocked by overexpression of
the dominant-negative NIK (Song, H. Y., et al, Proc. Natl. Acad.
Sci. USA 94:9792-9796 (1997)), which lacked the two lysine residues
of catalytic domain. Taken together, these data indicate that TR-11
mediates NF-.kappa.B activation through the TRAF2/NIK pathway.
Since TRAF1 and TRAF3 were found to associate with TR-11 in HEK293
EBNA cells, the effects of TRAF1 and TRAF3 on NF-.kappa.B
activation induced by TR-11 was examined. The introduction of TRAF3
nearly abolished the luciferase activity induced by TR-11
overexpression. To a lesser extent, TRAF1 overexpression diminished
TR-11-induced NF-.kappa.B activation. These data suggest that TRAF1
and especially TRAF3 downregulate TR-11-induced NF-.kappa.B
activation.
[0588] To identify TR-11 ligand, a panel of Flag-tagged candidate
TNF ligand proteins for binding to TR-11-Fc fusion protein was
screened by immunoprecipitation. TR-11-Fc selectively bound
endokine-.alpha.-Flag among Flag-tagged TNF ligand proteins tested.
In our experimental conditions, 4-1BB and TR2 (HVEM) bound their
cognate ligands, 4-1BBL and LIGHT (Mauri, D. N., et al, Immunity
8:21-30 (1998)), respectively. Furthermore, this data clearly
showed that endokine-.alpha.-Flag protein bound TR-11 transiently
expressed on the cell surface of HEK293 EBNA cells and TR-11
constitutively expressed on the cell surface of Jurkat cell. Since
endokine-.alpha. is a transmembrane protein (see below), flow
cytometry to was used determine whether TR-11-Fc fusion protein was
able to bind HEK293 EBNA cells that were stably transfected with
full length endokine-.alpha.. The results demonstrate that TR-11-Fc
protein was capable of binding endokine-.alpha. expressed on HEK293
EBNA cells.
[0589] Next, it was determined whether interactions between TR-11
and endokine-.alpha. would result in NF-.kappa.B activation. In an
NF-.kappa.B reporter assay, ligand-dependent NF-.kappa.B activation
was demonstrated by cotransfecting transmembrane endokine-.alpha.
with TR-11 or transfecting endokine-.alpha.-expressing HEK293 EBNA
cells. In addition, when TR-11 was transiently transfected into
HEK293 EBNA cells which constitutively secreted soluble
endokine-.alpha. protein, NF-.kappa.B activation markedly increased
as compared to empty vector-transfected HEK293 EBNA cells.
Similarly, higher NF-.kappa.B activation was induced by treating
with soluble endokine-.alpha. protein HEK293 cells which were
transiently transfected with TR-11. This indicates that
endokine-.alpha. is able to trigger TR-11-specific activation of
NF-.kappa.B. It appears that higher induction of NF-.kappa.B by
endokine-.alpha. is correlated with a stronger association of TR-11
with TRAF2 in HEK293 EBNA cells, since stronger association of
TR-11 with TRAF2 was observed in cells which were cotransfected
with endokine-.alpha. than in cells which were transfected with
TR-11 alone.
[0590] Endokine-.alpha. was one of the TNF ligand proteins
initially identified by an EST database search. Hydrophilicity
analysis of a full-length endokine-.alpha. clone from a brain cDNA
library predicts a single hydrophobic transmembrane domain and the
absence of a signal sequence. Endokine-.alpha. contains two
potential glycosylation sites in the C-terminal region. These
features suggest that endokine-.alpha. is a type II membrane
protein with the C-terminal region extracellular. Northern blot
analysis of human tissue RNAs revealed expression of a single
2.4-kb endokine-.alpha. mRNA in pancreas. Various human cell lines
and PBMC were also examined for endokine-.alpha. expression. No
message was detectable in either unstimulated or stimulated T-cell
lines (CEM-6 and Jurkat), B-cell lines (Priess and Frev),
promyelocytic cell line (HL-60), monocytic cell line (THP-1), and
PBMC by RT-PCR. In contrast, HUVEC cells constitutively expressed
endokine-.alpha. and its expression was upregulated after
stimulation with LPS. Therefore, it is believed that TR-11 and its
ligand are important for interactions between activated T
lymphocytes and blood vessels.
[0591] TR-11 has 55% identity with murine GITR at the amino acid
level. The high sequence conservation between human and mouse
provides evidence that TR-11 is the human homologue of murine GITR.
At this point, however, the possibility remains that these two
receptors may serve distinct functions from one another, based on
the following facts: (1) There is a mismatch in the first
cysteine-rich pseudorepeat between GITR and TR-11; (2) in contrast
to GITR, TR-11 is not inducible by dexamethasone.
[0592] In summary, a novel protein of the TNFR superfamily, TR-11,
which activates NF-.kappa.B through a TRAF2-mediated mechanism has
identified. Expression of TR-11 is activation-inducible. The ligand
for TR-11, endokine-.alpha., is a member of the TNF ligand family
and is constitutively expressed in an endothelial cell line. This
indicates that TR-11 and its ligand may be involved in activated
T-cell trafficking.
Example 6
The Effects of Endokine Alpha on Monocytes
[0593] These studies disclose that treatment with endokine-.alpha.
induced TNF-.alpha., MCP-1, IL-8 and IL-10 release from monocytes
and inhibited the production of IL-12 in monocytes. (data not
shown).
Methods
[0594] Monocyte purification. Peripheral blood mononuclear cells
(PBMC) were purified from single donor leukopacks (American Red
Cross, Baltimore, Md.) by centrifugation through a Histopaque
gradient (Sigma). Monocytes were isolated from PBMC by counterflow
centrifugal elutriation.
[0595] ELISA. Human monocytes were incubated at a density of
5.times.10.sup.5 cells/ml with increasing concentrations of
endokine-.alpha.. For IL-12 production, the cells were primed
overnight with IFN-.gamma. (100 U/ml) in presence of
Endokine-.alpha.. LPS (10 ng/ml) was then added. Conditioned media
was collected after 24 h and kept frozen until use. ELISA kits for
the measurement of TNF-.alpha., IL-10, MCP-1 and IL-8 were
purchased from R & D Systems (Minneapolis, Minn.). Each value
was the mean of triplicate samples.+-.standard deviation.
[0596] Oxidative burst. Purified monocytes were plated in 96-well
plate at 2-10.times.10.sup.5 cell/well. Increasing concentrations
of Endokine-.alpha. are added to the wells in a total volume of 0.2
ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics).
After 3 days incubation, the plates are centrifuged and the medium
is removed from the wells. To the macrophage monolayers, 0.2 ml per
well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate
buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of
HRPO) was added, together with the stimulant (200 nM PMA). The
plates were incubated at 37.degree. C. for 2 hours and the reaction
was stopped by adding 20 .mu.l 1N NaOH per well. The absorbance was
read at 610 nm. To calculate the amount of H.sub.2O.sub.2 produced
by the macrophages, a standard curve of a H.sub.2O.sub.2 solution
of known molarity was done for each experiment.
TABLE-US-00002 Effect of Endokine-a treatment on IL-12 secretion by
monocytes Treatment IL-12 Inhibition (mg/ml) (pg/ml) % -- 513 TL-6
0.2 600 0 TL-6 1 421 28 TL-6 5 54 89
[0597] Monocytes (5.times.10.sup.5/ml) were incubated with IFN-g
(100 U/ml) and TL-6. After 16 hours, LPS (10 ng/ml) was added to
the cultures. Conditioned media was collected 24 hours following
LPS addition and analyzed in ELISA for IL-12 content.
Example 7
Assays to Detect Stimulation or Inhibition of B cell Proliferation
and Differentiation
Background
[0598] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL5, IL6, IL-7, IL10, IL-13, IL14 and IL15.
Interestingly, these signals are by themselves weak effectors but
can, in combination with various co-stimulatory proteins, induce
activation, proliferation, differentiation, homing, tolerance and
death among B cell populations. One of the best studied classes of
B-cell co-stimulatory proteins is the TNF-superfamily. Within this
family CD40, CD27, and CD30 along with their respective ligands CD
154, CD70, and CD 153 have been found to regulate a variety of
immune responses. Assays which allow for the detection and/or
observation of the proliferation and differentiation of these
B-cell populations and their precursors are valuable tools in
determining the effects various proteins may have on these B-cell
populations in terms of proliferation and differentiation. Listed
below are two assays designed to allow for the detection of the
differentiation, proliferation, or inhibition of B-cell populations
and their precursors.
Experimental Procedure
[0599] In vitro assay. Purified Endokine-.alpha. protein, or
truncated forms thereof, is assessed for its ability to induce
activation, proliferation, differentiation or inhibition and/or
death in B-cell populations and their precursors. The activity of
Endokine-.alpha. protein on purified human tonsillar B cells,
measured qualitatively over the dose range from 0.1 to 10,000
ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay
in which purified tonsillar B cells are cultured in the presence of
either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second
signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220). Various
dilutions of each sample are placed into individual wells of a
96-well plate to which are added 10.sup.5 B-cells suspended in
culture medium (RPMI 1640 containing 10% FBS, 5.times.10.sup.-5M
2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10.sup.-5
dilution of SAC) in a total volume of 150 ul. Proliferation or
inhibition is quantitated by a 20 h pulse (1 uCi/well) with
.sup.3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition.
The positive and negative controls are IL2 and medium
respectively.
[0600] In vivo assay. BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of endokine-.alpha. protein, or
truncated forms thereof. Mice receive this treatment for 4
consecutive days, at which time they are sacrificed and various
tissues and serum collected for analyses. Comparison of H&E
sections from normal and endokine-.alpha. protein-treated spleens
identify the results of the activity of endokine-.alpha. protein on
spleen cells, such as the diffusion of peri-arterial lymphatic
sheaths, and/or significant increases in the nucleated cellularity
of the red pulp regions, which may indicate the activation of the
differentiation and proliferation of B-cell populations.
Immunohistochemical studies using a B cell marker,
anti-CD45R(B220), are used to determine whether any physiological
changes to splenic cells, such as splenic disorganization, are due
to increased B-cell representation within loosely defined B-cell
zones that infiltrate established T-cell regions.
[0601] Flow cytometric analyses of the spleens from
endokine-.alpha. protein-treated mice is used to indicate whether
endokine-.alpha. protein specifically increases the proportion of
ThB+, CD45R(B220)dull B cells over that which is observed in
control mice.
[0602] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and Endokine-.alpha. protein-treated mice.
Example 8
Assays to Detect Stimulation or Inhibition of T cell Proliferation
and Differentiation
[0603] The anti-CD3 and/or PHA costimulation assay is used to
detected the stimulation or inhibition of T cell proliferation and
differentiation.
Assay Parameters
TABLE-US-00003 [0604] Cells: PBMC per well: .sup. 10.sup.5 PBMC
recovered per donor: 200 .times. 10.sup.6 Total plates per day: 20
Supernatants per plate: 48 (each assayed in duplicate)
Total supernatants per day per donor: 960 (two donors per day) Need
an additional 4 units of blood/week to accommodate new assay.
[0605] Reagents:
anti-human CD3 mAb (25 pg/mL final concentration in each well)
PHA
[0606] rhIL-2 (positive control)
.sup.3H-thymidine (0.5 .mu.Ci/well, 6.7 Ci/mmole)
[0607] 96-well plates
[0608] Protocol.
Purify PBMC.
[0609] Prepare plates with appropriate controls. Incubate at
37.degree. C. for 3-4 days. Add .sup.3H-TdR and return to incubator
for an additional 20-24 hours. Harvest and count.
Outcomes
[0610] This assay allows the determination of whether
Endokine-.alpha. enhances or inhibits anti-CD3-dependent
proliferation of PBMCs and whether Endokine-.alpha. stimulates PBMC
proliferation in the absence of costimulatory signals.
Example 9
Isolation of Antibody Fragments Directed Against Polypeptides of
the Present Invention from a Library of scFvs
[0611] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against polypeptides of the present invention
to which the donor may or may not have been exposed (see e.g., U.S.
Pat. No. 5,885,793 incorporated herein in its entirety by
reference).
[0612] Rescue of the library. A library of scFvs is constructed
from the RNA of human PBLs as described in WO92/01047. To rescue
phage displaying antibody fragments, approximately 10.sup.9 E. coli
harbouring the phagemid are used to inoculate 50 ml of 2.times.TY
containing 1% glucose and 100 ug/ml of ampicillin
(2.times.TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five
ml of this culture is used to innoculate 50 ml of
2.times.TY-AMP-GLU. Next 2.times.10.sup.8 TU of delta gene 3 helper
phage (M13 delta gene III, see WO 92/01047) are added and the
culture incubated at 37.degree. C. for 45 minutes without shaking
and then at 37.degree. C. for 45 minutes with shaking. The culture
is centrifuged at 4000 r.p.m. for 10 minutes and the pellet
resuspended in 2 liters of 2.times.TY containing 100 ug/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in WO 92/01047.
[0613] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harbouring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are pelleted (IEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100 ug
ampicillin/ml and 25 ug kanamycin/ml (2.times.TY-AMP-KAN) and grown
overnight, shaking at 37.degree. C. Phage particles are purified
and concentrated from the culture medium by two PEG-precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through
a 0.45 um filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
[0614] Panning of the library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 mg/ml or 10 mg/ml of a
polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 10.sup.13 TU of phage are applied to the tube
and incubated for 30 minutes at room temperature tumbling on an
over and under turntable and then left to stand for another 1.5
hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10
times with PBS. Phage are eluted by adding 1 ml of 100 mM
triethylamine and rotating 15 minutes on an under and over
turntable after which the solution is immediately neutralized with
0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage are then used to infect 10
ml of mid-log E. coli TG1 by incubating eluted phage with bacteria
for 30 minutes at 37.degree. C. The E. coli are then plated on TYE
plates containing 1% glucose and 100 ug/ml ampicillin. The
resulting bacterial library is then rescued with delta gene 3
helper phage as described above to prepare phage for a subsequent
round of selection. This process is then repeated for a total of 4
rounds of affinity purification with tube-washing increased to 20
times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3
and 4.
[0615] Characterization of binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see e.g., WO92/01047) and then
by sequencing.
Example 10
Method of Determining Alterations in the Endokine Alpha Gene
[0616] RNA is isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease). cDNA
is then generated from these RNA samples using protocols known in
the art. (see, Sambrook.) The cDNA is then used as a template for
PCR, employing primers surrounding regions of interest in SEQ ID
NO: 1. Suggested PCR conditions consist of 35 cycles at 95.degree.
C. for 30 seconds; 60-120 seconds at 52-58.degree. C.; and 60-120
seconds at 70.degree. C., using buffer solutions described in
Sidransky, D., et al., Science 252:706 (1991).
[0617] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase (Epicentre Technologies). The intron-exon borders of
selected exons of endokine alpha are also determined and genomic
PCR products analyzed to confirm the results. PCR products
harboring suspected mutations in endokine alpha are then cloned and
sequenced to validate the results of the direct sequencing.
[0618] PCR products of endokine alpha are cloned into T-tailed
vectors as described in Holton, T. A. and Graham, M. W., Nucleic
Acids Research, 19:1156 (1991) and sequenced with T7 polymerase
(United States Biochemical). Affected individuals are identified by
mutations in endokine alpha not present in unaffected
individuals.
[0619] Genomic rearrangements are also observed as a method of
determining alterations in the endokine alpha gene. Genomic clones
isolated using techniques known in the art are nick-translated with
digoxigenindeoxy-uridine 5'-triphosphate (Boehringer Manheim), and
FISH performed as described in Johnson, C. G. et al., Methods Cell
Biol. 35:73-99 (1991). Hybridization with the labeled probe is
carried out using a vast excess of human cot-1 DNA for specific
hybridization to the endokine alpha genomic locus.
[0620] Chromosomes are counterstained with
4,6-diamino-2-phenylidole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson, C. V. et al., Genet. Anal. Tech.
Appl., 8:75 (1991).) Image collection, analysis and chromosomal
fractional length measurements are performed using the ISee
Graphical Program System. (Inovision Corporation, Durham, N.C.)
Chromosome alterations of the genomic region of endokine alpha
(hybridized by the probe) are identified as insertions, deletions,
and translocations. These endokine alpha alterations are used as a
diagnostic marker for an associated disease.
Example 11
Method of Detecting Abnormal Levels of Endokine Alpha in a
Biological Sample
[0621] Endokine alpha polypeptides can be detected in a biological
sample, and if an increased or decreased level of endokine alpha is
detected, this polypeptide is a marker for a particular phenotype.
Methods of detection are numerous, and thus, it is understood that
one skilled in the art can modify the following assay to fit their
particular needs.
[0622] For example, antibody-sandwich ELISAs are used to detect
endokine alpha in a sample, preferably a biological sample. Wells
of a microtiter plate are coated with specific antibodies to
endokine alpha, at a final concentration of 0.2 to 10 ug/ml. The
antibodies are either monoclonal or polyclonal and are produced
using technique known in the art. The wells are blocked so that
non-specific binding of endokine alpha to the well is reduced.
[0623] The coated wells are then incubated for >2 hours at RT
with a sample containing endokine alpha. Preferably, serial
dilutions of the sample should be used to validate results. The
plates are then washed three times with deionized or distilled
water to remove unbounded endokine alpha.
[0624] Next, 50 .mu.l of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[0625] Seventy-five ul of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution is then added to
each well and incubated 1 hour at room temperature to allow
cleavage of the substrate and flourescence. The flourescence is
measured by a microtiter plate reader. A standard curve is prepared
using the experimental results from serial dilutions of a control
sample with the sample concentration plotted on the X-axis (log
scale) and fluorescence or absorbance on the Y-axis (linear scale).
The endokine alpha polypeptide concentration in a sample is then
interpolated using the standard curve based on the measured
flourescence of that sample.
Example 12
Method of Treating Decreased Levels of Endokine Alpha
[0626] The present invention also relates to a method for treating
an individual in need of an increased level of endokine alpha
biological activity in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of endokine alpha or an agonist thereof.
[0627] Moreover, it will be appreciated that conditions caused by a
decrease in the standard or normal expression level of endokine
alpha in an individual can be treated by administering endokine
alpha, preferably in a soluble and/or secreted form. Thus, the
invention also provides a method of treatment of an individual in
need of an increased level of endokine alpha polypeptide comprising
administering to such an individual a pharmaceutical composition
comprising an amount of endokine alpha to increase the biological
activity level of endokine alpha in such an individual.
[0628] For example, a patient with decreased levels of endokine
alpha polypeptide receives a daily dose 0.1-100 .mu.g/kg of the
polypeptide for six consecutive days. Preferably, the polypeptide
is in a soluble and/or secreted form.
Example 13
Method of Treating Increased Levels of Endokine Alpha
[0629] The present invention relates to a method for treating an
individual in need of a decreased level of endokine alpha
biological activity in the body comprising, administering to such
an individual a composition comprising a therapeutically effective
amount of endokine alpha antagonist. Preferred antagonists for use
in the present invention are endokine alpha-specific antibodies or
endokine alpha antisense polynucleotides.
[0630] Antisense technology is used to inhibit production of
endokine alpha. This technology is one example of a method of
decreasing levels of endokine alpha polypeptide, preferably a
soluble and/or secreted form, due to a variety of etiologies, such
as cancer.
[0631] For example, a patient diagnosed with abnormally increased
levels of endokine alpha is administered intravenously antisense
polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21
days. This treatment is repeated after a 7-day rest period if the
is determined to be well tolerated.
Example 14
Method of Treatment Using Gene Therapy--Ex vivo
[0632] One method of gene therapy transplants fibroblasts, which
are capable of expressing soluble and/or mature endokine alpha
polypeptides, onto a patient. Generally, fibroblasts are obtained
from a subject by skin biopsy. The resulting tissue is placed in
tissue-culture medium and separated into small pieces. Small chunks
of the tissue are placed on a wet surface of a tissue culture
flask, approximately ten pieces are placed in each flask. The flask
is turned upside down, closed tight and left at room temperature
over night. After 24 hours at room temperature, the flask is
inverted; the chunks of tissue remain fixed to the bottom of the
flask and fresh media (e.g., Ham's F12 media, with 10% FBS,
penicillin and streptomycin) is added. The flasks are then
incubated at 37.degree. C. for approximately one week.
[0633] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[0634] pMV-7 (Kirschmeier, P. T. et al., DNA 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0635] The cDNA encoding endokine alpha can be amplified using PCR
primers which correspond to the 5' and 3' end encoding sequences
respectively. Preferably, the 5' primer contains an EcoRI site and
the 3' primer includes a HindIII site. Equal quantities of the
Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is then used to transform E. coli HB101, which are then plated onto
agar containing kanamycin for the purpose of confirming that the
vector contains properly inserted endokine alpha.
[0636] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the endokine alpha gene is
then added to the media and the packaging cells transduced with the
vector. The packaging cells now produce infectious viral particles
containing the endokine alpha gene (the packaging cells are now
referred to as producer cells).
[0637] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether endokine alpha protein is
produced.
[0638] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 15
Method of Treatment Using Gene Therapy--In Vivo
[0639] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) endokine alpha
sequences into an animal to increase or decrease the expression of
the endokine alpha polypeptide. The endokine alpha polynucleotide
may be operatively linked to a promoter or any other genetic
elements necessary for the expression of the endokine alpha
polypeptide by the target tissue. Such gene therapy and delivery
techniques and methods are known in the art, see, for example, WO
90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151,
5,580,859; Tabata H. et al, Cardiovasc. Res. 35:470-479 (1997);
Chao J. et al., Pharmacol. Res. 35:517-522 (1997); Wolff J. A.
Neuromuscul. Disord. 7:314-318 (1997); Schwartz B. et al., Gene
Ther. 3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290
(1996) (incorporated herein by reference).
[0640] The endokine alpha polynucleotide constructs may be
delivered by any method that delivers injectable materials to the
cells of an animal, such as, injection into the interstitial space
of tissues (heart, muscle, skin, lung, liver, intestine and the
like). The endokine alpha polynucleotide constructs can be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0641] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, the endokine alpha
polynucleotides may also be delivered in liposome formulations
(such as those taught in Felgner P. L. et al. Ann. NY Acad. Sci.
772:126-139 (1995), and Abdallah B. et al. Biol. Cell 85:1-7
(1995)) which can be prepared by methods well known to those
skilled in the art.
[0642] The endokine alpha polynucleotide vector constructs used in
the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Any strong promoter known to those skilled
in the art can be used for driving the expression of DNA. Unlike
other gene therapy techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory
nature of the polynucleotide synthesis in the cells. Studies have
shown that non-replicating DNA sequences can be introduced into
cells to provide production of the desired polypeptide for periods
of up to six months.
[0643] The endokine alpha polynucleotide construct can be delivered
to the interstitial space of tissues within an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0644] For the naked endokine alpha polynucleotide injection, an
effective dosage amount of DNA or RNA will be in the range of from
about 0.05 .mu.g/kg body weight to about 50 mg/kg body weight.
Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
Of course, as the artisan of ordinary skill will appreciate, this
dosage will vary according to the tissue site of injection. The
appropriate and effective dosage of nucleic acid sequence can
readily be determined by those of ordinary skill in the art and may
depend on the condition being treated and the route of
administration. The preferred route of administration is by the
parenteral route of injection into the interstitial space of
tissues. However, other parenteral routes may also be used, such
as, inhalation of an aerosol formulation particularly for delivery
to lungs or bronchial tissues, throat or mucous membranes of the
nose. In addition, naked endokine alpha polynucleotide constructs
can be delivered to arteries during angioplasty by the catheter
used in the procedure.
[0645] The dose response effects of injected endokine alpha
polynucleotide in muscle in vivo are determined as follows.
Suitable endokine alpha template DNA for production of mRNA coding
for endokine alpha polypeptide is prepared in accordance with a
standard recombinant DNA methodology. The template DNA, which may
be either circular or linear, is either used as naked DNA or
complexed with liposomes. The quadriceps muscles of mice are then
injected with various amounts of the template DNA.
[0646] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The endokine alpha
template DNA is injected in 0.1 ml of carrier in a 1 cc syringe
through a 27 gauge needle over one minute, approximately 0.5 cm
from the distal insertion site of the muscle into the knee and
about 0.2 cm deep. A suture is placed over the injection site for
future localization, and the skin is closed with stainless steel
clips.
[0647] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 .mu.m cross-section of the individual quadriceps muscles
is histochemically stained for endokine alpha protein. A time
course for endokine alpha protein expression may be done in a
similar fashion except that quadriceps from different mice are
harvested at different times. Persistence of endokine alpha DNA in
muscle following injection may be determined by Southern blot
analysis after preparing total cellular DNA and HIRT supernatants
from injected and control mice. The results of the above
experimentation in mice can be use to extrapolate proper dosages
and other treatment parameters in humans and other animals using
endokine alpha naked DNA.
Example 16
Gene Therapy Using Endogenous Endokine Alpha Gene
[0648] Another method of gene therapy according to the present
invention involves operably associating the endogenous endokine
alpha sequence with a promoter via homologous recombination as
described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24,
1997; International Publication Number WO 96/29411, published Sep.
26, 1996; International Publication Number WO 94/12650, published
Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438
(1989). This method involves the activation of a gene which is
present in the target cells, but which is not expressed in the
cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and
targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous endokine alpha, flanking the promoter. The
targeting sequence will be sufficiently near the 5' end of endokine
alpha so the promoter will be operably linked to the endogenous
sequence upon homologous recombination. The promoter and the
targeting sequences can be amplified using PCR. Preferably, the
amplified promoter contains distinct restriction enzyme sites on
the 5' and 3' ends. Preferably, the 3' end of the first targeting
sequence contains the same restriction enzyme site as the 5' end of
the amplified promoter and the 5' end of the second targeting
sequence contains the same restriction site as the 3' end of the
amplified promoter.
[0649] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[0650] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[0651] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous endokine alpha sequence. This results in the
expression of endokine alpha in the cell. Expression may be
detected by immunological staining, or any other method known in
the art.
[0652] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2 HPO.sub.4, 6 mM dextrose). The cells are again
centrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[0653] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the endokine
alpha locus, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is
digested with HindIII. The CMV promoter is amplified by PCR with an
XbaI site on the 5' end and a BamHI site on the 3' end. Two
endokine alpha non-coding sequences are amplified via PCR; one
endokine alpha non-coding sequence (endokine alpha fragment 1) is
amplified with a HindIII site at the 5' end and an Xba site at the
3' end; the other endokine alpha non-coding sequence (endokine
alpha fragment 2) is amplified with a BamHI site at the 5' end and
a HindIII site at the 3' end. The CMV promoter and endokine alpha
fragments are digested with the appropriate enzymes (CMV
promoter--XbaI and BamHI; endokine alpha fragment 1--XbaI; endokine
alpha fragment 2--BamHI) and ligated together. The resulting
ligation product is digested with HindIII, and ligated with the
HindIII-digested pUC18 plasmid.
[0654] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1-5.times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 msec should
be observed.
[0655] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37_C. The following day,
the media is aspirated and replaced with 10 ml of fresh media and
the cells are incubated for a further 16-24 hours.
[0656] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 17
Effect of Endokine Alpha on the Expression of MHC Class II,
Costimulatory and Adhesion Molecules and Cell Differentiation of
Monocytes and Monocyte-Derived Human Dendritic Cells
[0657] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD 1, CD80, CD86, CD40 and MHC class TI antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[0658] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of
endokine alpha or LPS (positive control), washed with PBS
containing 1% BSA and 0.02 mM sodium azide, and then incubated with
1:20 dilution of appropriate FITC- or PE-labeled monoclonal
antibodies for 30 minutes at 4.degree. C. After an additional wash,
the labeled cells are analyzed by flow cytometry on a FACScan
(Becton Dickinson).
[0659] Effect on the production of cytokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Thl helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (106/ml) are
treated with increasing concentrations of endokine alpha for 24
hours. LPS (100 ng/ml) is added to the cell culture as positive
control. Supernatants from the cell cultures are then collected and
analyzed for IL-12 content using commercial ELISA kit (e.g., R
& D Systems (Minneapolis, Minn.)). The standard protocols
provided with the kits are used.
[0660] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increase expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[0661] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of endokine alpha or LPS (positive control), washed
with PBS containing 1% BSA and 0.02 mM sodium azide, and then
incubated with 1:20 dilution of appropriate FITC- or PE-labeled
monoclonal antibodies for 30 minutes at 4.degree. C. After an
additional wash, the labeled cells are analyzed by flow cytometry
on a FACScan (Becton Dickinson).
[0662] Monocyte activation and/or increased survival Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. Endokine alpha,
agonists, or antagonists of endokine alpha can be screened using
the three assays described below. For each of these assays,
peripheral blood mononuclear cells (PBMC) are purified from single
donor leukopacks (American Red Cross, Baltimore, Md.) by
centrifugation through a Histopaque gradient (Sigma). Monocytes are
isolated from PBMC by counterflow centrifugal elutriation.
[0663] 1. Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from an internally regulated
process (apoptosis). Addition to the culture of activating factors,
such as TNF-alpha, dramatically improves cell survival and prevents
DNA fragmentation. Propidium iodide (PI) staining is used to
measure apoptosis as follows. Monocytes are cultured for 48 hours
in polypropylene tubes in serum-free medium (positive control), in
the presence of 100 ng/ml TNF-alpha (negative control), and in the
presence of varying concentrations of the compound to be tested.
Cells are suspended at a concentration of 2.times.10.sup.6/ml in
PBS containing PI at a final concentration of 5 .mu.g/ml, and then
incubated at room temperature for 5 minutes before FAC Scan
analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
[0664] 2. Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.10.sup.5 cells/ml with increasing concentrations of
endokine alpha or in the absence of endokine alpha. For IL-12
production, the cells are primed overnight with IFN-.gamma. (100
U/ml) in presence of endokine alpha. LPS (10 ng/ml) is then added.
Conditioned media is collected after 24 h and kept frozen until
use. Measurement of TNF-.alpha., IL-10, MCP-1 and IL-8 is then
performed using a commercially available ELISA kit (e.g., R & D
Systems (Minneapolis, Minn.)) applying the standard protocols
provided with the kit.
[0665] 3. Oxidative burst. Purified monocytes are plated in 96-well
plates at 2-1.times.10.sup.5 cell/well. Increasing concentrations
of endokine alpha are added to the wells in a total volume of 0.2
ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics).
After 3 days incubation, the plates are centrifuged and the medium
is removed from the wells. To the macrophage monolayers, 0.2 ml per
well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate
buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of
HRPO) is added, together with the stimulant (200 nM PMA). The
plates are incubated at 37.degree. C. for 2 hours and the reaction
is stopped by adding 20 .mu.l 1N NaOH per well. The absorbance is
read at 610 nm. To calculate the amount of H.sub.2O.sub.2 produced
by the macrophages, a standard curve of a H.sub.2O.sub.2 solution
of known molarity is performed for each experiment.
[0666] The studies described in this example tested activity in
endokine alphaprotein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of endokine
alpha polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of endokine alpha.
Example 18
Assay to Detect Stimulation or Inhibition of T Cell
Proliferation
[0667] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
.mu.l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control mAb (B33.1) overnight at 4.degree. C. (1 .mu.g/ml in 0.05 M
bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC
are isolated by F/H gradient centrifugation from human peripheral
blood and added to quadruplicate wells (5.times.10.sup.4/well) of
mAb coated plates in RPMI containing 10% FCS and P/S in the
presence of varying concentrations of endokine alpha protein (total
volume 200 .mu.l). Relevant protein buffer and medium alone are
controls. After 48 hour culture at 37.degree. C., plates are spun
for 2 min. at 1000 rpm and 100 .mu.l of supernatant is removed and
stored -20.degree. C. for measurement of IL-2 (or other cytokines)
if effect on proliferation is observed. Wells are supplemented with
100 .mu.l of medium containing 0.5 .mu.Ci of .sup.3H-thymidine and
cultured at 37.degree. C. for 18-24 hr. Wells are harvested and
incorporation of .sup.3H-thymidine used as a measure of
proliferation. Anti-CD3 alone is the positive control for
proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce
proliferation of T cells is used as the negative controls for the
effects of endokine alpha proteins.
[0668] The studies described in this example tested activity in
endokine alpha protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of
endokine alpha polynucleotides (e.g., gene therapy), agonists,
and/or antagonists of endokine alpha.
Example 19
Production of an Antibody
a) Hybridoma Technology
[0669] The antibodies of the present invention can be prepared by a
variety of methods. (see, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing Endokine alpha are
administered to an animal to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of Endokine 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.
[0670] Monoclonal antibodies specific for protein Endokine alpha
are 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.,
pp. 563-681 (1981)). In general, an animal (preferably a mouse) is
immunized with Endokine alpha polypeptide or, more preferably, with
a secreted Endokine alpha polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably 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.
[0671] 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 (SP20), available
from the ATCC. 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 Endokine alpha polypeptide.
[0672] Alternatively, additional antibodies capable of binding to
Endokine alpha polypeptide can be produced in a two-step procedure
using anti-idiotypic antibodies. Such a method makes use of the
fact that antibodies are themselves antigens, and therefore, it is
possible to obtain an antibody which binds to a second antibody. In
accordance with this method, 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 Endokine alpha
protein-specific antibody can be blocked by Endokine alpha. Such
antibodies comprise anti-idiotypic antibodies to the Endokine alpha
protein-specific antibody and are used to immunize an animal to
induce formation of further Endokine alpha protein-specific
antibodies.
[0673] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed infra.
(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).)
b) Isolation of Antibody Fragments Directed Against Endokine Alpha
from a Library of scFvs
[0674] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against Endokine alpha to which the donor may or may
not have been exposed (see e.g., U.S. Pat. No. 5,885,793
incorporated herein by reference in its entirety).
[0675] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of
0.8 with shaking. Five ml of this culture is used to innoculate 50
ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 .mu.g/ml
ampicillin and 50 .mu.g/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[0676] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[0677] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0678] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing
[0679] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0680] 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.
[0681] The disclosures of all patents, patent applications, and
publications referred to herein are hereby incorporated by
reference.
Sequence CWU 1
1
1011849DNAHomo sapiensCDS(53)..(559) 1gttttccaca gctctcattt
ctccaaaaat gtgtttgagc cacttggaaa at atg cct 58 Met Pro 1tta agc cat
tca aga act caa gga gct cag aga tca tcc tgg aag ctg 106Leu Ser His
Ser Arg Thr Gln Gly Ala Gln Arg Ser Ser Trp Lys Leu 5 10 15tgg ctc
ttt tgc tca ata gtt atg ttg cta ttt ctt tgc tcc ttc agt 154Trp Leu
Phe Cys Ser Ile Val Met Leu Leu Phe Leu Cys Ser Phe Ser 20 25 30tgg
cta atc ttt att ttt ctc caa tta gag act gct aag gag ccc tgt 202Trp
Leu Ile Phe Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu Pro Cys35 40 45
50atg gct aag ttt gga cca tta ccc tca aaa tgg caa atg gca tct tct
250Met Ala Lys Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala Ser Ser
55 60 65gaa cct cct tgc gtg aat aag gtg tct gac tgg aag ctg gag ata
ctt 298Glu Pro Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu Ile
Leu 70 75 80cag aat ggc tta tat tta att tat ggc caa gtg gct ccc aat
gca aac 346Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn
Ala Asn 85 90 95tac aat gat gta gct cct ttt gag gtg cgg ctg tat aaa
aac aaa gac 394Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys
Asn Lys Asp 100 105 110atg ata caa act cta aca aac aaa tct aaa atc
caa aat gta gga ggg 442Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile
Gln Asn Val Gly Gly115 120 125 130act tat gaa ttg cat gtt ggg gac
acc ata gac ttg ata ttc aac tct 490Thr Tyr Glu Leu His Val Gly Asp
Thr Ile Asp Leu Ile Phe Asn Ser 135 140 145gag cat cag gtt cta aaa
aat aat acc tac tgg ggt atc att tta cta 538Glu His Gln Val Leu Lys
Asn Asn Thr Tyr Trp Gly Ile Ile Leu Leu 150 155 160gca aat ccc caa
ttc atc tcc tagagacttg atttgatctc ctcattccct 589Ala Asn Pro Gln Phe
Ile Ser 165tcagcacatg tagaggtgcc agtgggtgga ttggagggag aagatattca
atttctagag 649tttgtctgtc tacaaaaatc aacacaaaca gaactcctct
gcacgtgaat tttcatctat 709catgcctatc tgaaagagac tcaggggaaa
agccaaagac ttttggttgg atctgcagag 769atacttcatt aatccatgat
aaaacaaata tggatgacag aggacatgtg cttttcaaag 829aatctttatc
taattcttga attcatgagt ggaaaaatgg agttctattc ccatggaaga
889tttacctggt atgcaaaaag gatctggggc agtagcctgg ctttgttctc
atattcttgg 949gctgctgtaa ttcattcttc tcatactccc atcttctgag
accctcccaa taaaaagtag 1009actgatagga tggccacaga tatgcctacc
ataccctact ttagatatgg tggtgttaga 1069agataaagaa caatctgaga
actattggaa tagaggtaca agtggcataa aatggaatgt 1129acgctatctg
gaaatttctc ttggttttat cttcctcagg atgcagggtg ctttaaaaag
1189ccttatcaaa ggagtcattc cgaaccctca cgtagagctt tgtgagaact
tactgttggt 1249gtgtgtgtct aaacattgct aattgtaaag aaagagtaac
cattagtaat cattaggttt 1309aaccccagaa tggtattatc attactggat
tatgtcatgt aatgatttag tatttttagc 1369tagctttcca cagtttgcaa
agtgctttcg taaaacagtt agcaattcta tgaagttaat 1429tgggcaggca
tttgggggaa aattttagtg atgagaatgt gatagcatag catagccaac
1489tttcctcaac tcataggaca agtgactaca agaggcaatg ggtagtcccc
tgcattgcac 1549tgtctcagct ttagaattgt tatttctgct atcgtgttat
aagactctaa aacttagcga 1609attcactttt caggaagcat attccccttt
agcccaaggt gagcagagtg aagctacaac 1669agatctttcc tttaccagca
cacttttttt tttttcctgc ctgaatcagg gagatccagg 1729atgctgttca
ggccttatcc caaccaaatt cccctcttca ctttgcaggg cccatcttag
1789tcaaatgtgc taacttctaa aataataaat agcactaatt caaaaaaaaa
aaaaaaaaaa 18492169PRTHomo sapiens 2Met Pro Leu Ser His Ser Arg Thr
Gln Gly Ala Gln Arg Ser Ser Trp1 5 10 15Lys Leu Trp Leu Phe Cys Ser
Ile Val Met Leu Leu Phe Leu Cys Ser 20 25 30Phe Ser Trp Leu Ile Phe
Ile Phe Leu Gln Leu Glu Thr Ala Lys Glu 35 40 45Pro Cys Met Ala Lys
Phe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala 50 55 60Ser Ser Glu Pro
Pro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu65 70 75 80Ile Leu
Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn 85 90 95Ala
Asn Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys Asn 100 105
110Lys Asp Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile Gln Asn Val
115 120 125Gly Gly Thr Tyr Glu Leu His Val Gly Asp Thr Ile Asp Leu
Ile Phe 130 135 140Asn Ser Glu His Gln Val Leu Lys Asn Asn Thr Tyr
Trp Gly Ile Ile145 150 155 160Leu Leu Ala Asn Pro Gln Phe Ile Ser
1653233PRTHomo sapiens 3Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu
Leu Ala Glu Glu Ala1 5 10 15Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly
Ser Arg Arg Cys Leu Phe 20 25 30Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45Cys Leu Leu His Phe Gly Val Ile
Gly Pro Gln Arg Glu Glu Ser Pro 50 55 60Arg Asp Leu Ser Leu Ile Ser
Pro Leu Ala Gln Ala Val Arg Ser Ser65 70 75 80Ser Arg Thr Pro Ser
Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120
125Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg
Ile Ala145 150 155 160Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser
Ala Ile Lys Ser Pro 165 170 175Cys Gln Arg Glu Thr Pro Glu Gly Ala
Glu Ala Lys Pro Trp Tyr Glu 180 185 190Pro Ile Tyr Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205Ser Ala Glu Ile Asn
Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220Gln Val Tyr
Phe Gly Ile Ile Ala Leu225 2304205PRTHomo sapiens 4Met Thr Pro Pro
Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr1 5 10 15Leu His Leu
Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30Gln Gly
Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45Arg
Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala 50 55
60Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg65
70 75 80Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser
Asn 85 90 95Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr
Ser Gln 100 105 110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala
Pro Ser Ser Pro 115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe
Ser Ser Gln Tyr Pro Phe 130 135 140His Val Pro Leu Leu Ser Ser Gln
Lys Met Val Tyr Pro Gly Leu Gln145 150 155 160Glu Pro Trp Leu His
Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr 165 170 175Gln Gly Asp
Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val 180 185 190Leu
Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 195 200
205524DNAArtificial SequenceOligonucleotide primer 5gcgccatggc
taagtttgga ccat 24627DNAArtificial SequenceOligonucleotide primer
6gcgaagcttt caagtctcta ggagatg 27726DNAArtificial
SequenceOligonucleotide primer 7gcggatcccg agactgctaa ggagcc
26831DNAArtificial SequenceOligonucleotide primer 8gcggatccct
aggagatgaa ttggggattt g 31932DNAArtificial SequenceOligonucleotide
primer 9gcgggatccg ccatcatgcc tttaagccat tc 321031DNAArtificial
SequenceOligonucleotide primer 10gcggatccct aggagatgaa ttggggattt g
31
* * * * *