Note: Descriptions are shown in the official language in which they were submitted.
CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
PROMOTION OR INHIBITION OF ANGIOGENESIS AND
CARDIOVASCULARIZATION
Background of the Invention
Field of the Invention
The present invention relates to compositions and methods useful for promoting
or inhibiting angiogenesis
and/or cardiovascularization in mammals in need of such biological effect.
This includes the diagnosis and
treatment of cardiovascular disorders as well as oncological disorders.
Description of Background
A. Cardiac Disorders and Factors
Heart failure affects approximately five million Americans, and new cases of
heart failure number about
400,000 each year. It is the single most frequent cause of hospitalization for
people age 65 and older in the United
States. Recent advances in the management of acute cardiac diseases, including
acute myocardial infarction, are
resulting in an expanding patient population that will eventually develop
chronic heart failure. From 1979 to 1995,
hospitalizations for congestive heart failure (CHF) rose from 377,000 to
872,000 (a 130 percent increase) and CHF
deaths increased 116 percent.
CHF is a syndrome characterized by left ventricular dysfunction, reduced
exercise tolerance, impaired
quality of life, and markedly shortened life expectancy. The sine qua non of
heart failure is an inability of the heart
to pump blood at a rate sufficient to meet the metabolic needs of the body's
tissues (in other words, there is
insufficient cardiac output).
At least four major compensatory mechanisms are activated in the setting of
heart failure to boost cardiac
output, including peripheral vasoconstriction, increased heart rate, increased
cardiac contractility, and increased
plasma volume. These effects are mediated primarily by the sympathetic nervous
system and the renin-angiotensin
system. See, Eichhorn, American Journal of Medicine, 104: 163-169 (1998).
Increased output from the
sympathetic nervous system increases vascular tone, heart rate, and
contractility. Angiotensin II elevates blood
pressure by I ) directly stimulating vascular smooth muscle contraction, 2)
promoting plasma volume expansion by
stimulating aldosterone and antidiuretic hormone secretion, 3) stimulating
sympathetic-mediated vascular tone, and
4) catalyzing the degradation of bradykinin, which has vasodilatory and
natriuretic activity. See, review by Brown
and Vaughan, Circulation, 97: 1411-1420 (1998). As noted below, angiotensin II
may also have directly deleterious
effects on the heart by promoting myocyte necrosis (impairing systolic
function) and intracardiac fibrosis (impairing
diastolic and in some cases systolic function). See, Weber, Circulation, 96:
4065-4082 (1998).
A consistent feature of congestive heart failure (CHF) is cardiac hypertrophy,
an enlargement of the heart
that is activated by both mechanical and hormonal stimuli and enables the
heart to adapt to demands for increased
cardiac output. Morgan and Baker, Circulation, 83: 13-25 (1991). This
hypertrophic response is frequently
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
associated with a variety of distinct pathological conditions such as
hypertension, aortic stenosis, myocardial
infarction, cardiomyopathy, valvular regurgitation, and intracardiac shunt,
all of which result in chronic
hemodynamic overload.
Hypertrophy is generally defined as an increase in size of an organ or
structure independent of natural
growth that does not involve tumor formation. Hypertrophy of the heart is due
either to an increase in the mass of
the individual cells (myocytes), or to an increase in the number of cells
making up the tissue (hyperplasia), or both.
While the enlargement of an embryonic heart is largely dependent on an
increase in myocyte number (which
continues until shortly after birth), post-natal cardiac myocytes lose their
proliferative capacity. Further growth
occurs through hypertrophy of the individual cells.
Adult myocyte hypertrophy is initially beneficial as a short term response to
impaired cardiac function by
permitting a decrease in the load on individual muscle fibers. With severe,
long-standing overload, however, the
hypertrophied cells begin to deteriorate and die. Katz, "Heart Failure", in:
Katz A.M. ed., PhysioloQy of the Heart
(New York: Raven Press,1992) pp. 638-668. Cardiac hypertrophy is a significant
risk factor for both mortality and
morbidity in the clinical course of heart failure. Katz, Trends Cardiovasc.
Med., 5: 37-44 (1995). For further
details of the causes and pathology of cardiac hypertrophy see, e.g., Heart
Disease. A Textbook of Cardiovascular
Medicine, Braunwald, E. ed. (W.B. Saunders Co., 1988), Chapter 14,
"Pathophysiology of Heart Failure."
On a cellular level, the heart is composed of myocytes and surrounding support
cells, generically called
non-myocytes. While non-myocytes are primarily fibroblast/mesenchymal cells,
they also include endothelial and
smooth muscle cells. Indeed, although myocytes make up most of the adult
myocardial mass, they represent only
about 30% of the total cell numbers present in heart. In response to hormonal,
physiological, hemodynamic, and
pathological stimuli, adult ventricular muscle cells can adapt to increased
workloads through the activation of a
hypertrophic process. This response is characterized by an increase in myocyte
cell size and contractile protein
content of individual cardiac muscle cells, without concomitant cell division
and activation of embryonic genes,
including the gene for atrial natriuretic peptide (ANP). Chien et al., FASEB
J., 5: 3037-3046 ( 1991 ); Chien et al.,
Annu. Rev. Physiol., 55: 77-95 (1993). An increment in myocardial mass as a
result of an increase in myocyte size
that is associated with an accumulation of interstitial collagen within the
extracellular matrix and around
intramyocardial coronary arteries has been described in left ventricular
hypertrophy secondary to pressure overload
in humans. Caspari et al., Cardiovasc. Res., I 1: 554-558 (1977); Schwarz et
al., Am. J. Cardiol., 42: 895-903
(1978); Hess et al., Circulation, 63: 360-371 (1981); Pearlman et al., Lab.
Invest., 46: 158-164 (1982).
It has also been suggested that paracrine factors produced by non-myocyte
supporting cells may
additionally be involved in the development of cardiac hypertrophy, and
various non-myocyte derived hypertrophic
factors, such as, leukocyte inhibitory factor (LIF) and endothelin, have been
identified. Metcalf, Growth Factors,
7: 169-173 (1992); Kurzrock et al., Endocrine Reviews, 12: 208-217 (1991);
moue et al., Proc. Natl. Acad. Sci.
USA, 86: 2863-2867 (1989); Yanagisawa and Masaki, Trends Pharm. Sci., 10: 374-
378 (1989); U.S. Patent No.
5,573,762 (issued November 12, 1996). Further exemplary factors that have been
identified as potential mediators
of cardiac hypertrophy include cardiotrophin-I (CT-1 ) (Pennica et al., Proc.
Nat. Acad. Sci. USA, 92: 1142-1 146
(1995)), catecholamines, adrenocorticosteroids, angiotensin, and
prostaglandins.
At present, the treatment of cardiac hypertrophy varies depending on the
underlying cardiac disease.
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Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, LIF,
endothelin (including endothelin-1, -2,
and -3 and big endothelin), and CT-1 are among the factors identified as
potential mediators of hypertrophy. For
example, beta-adrenergic receptor blocking drugs (beta-blockers, e.g.,
propranolol, timolol, tertalolol, carteolol,
nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol,
carvedilol, etc.) and verapamil have been used
extensively in the treatment of hypertrophic cardiomyopathy. The beneficial
effects of beta-Mockers on symptoms
(e.g., chest pain) and exercise tolerance are largely due to a decrease in the
heart rate with a consequent prolongation
of diastole and increased passive ventricular filling. Thompson et al., Br.
Heart J., 44: 488-98 ( 1980); Harrison et
al., Circulation, 29: 84-98 (1964). Verapamil has been described to improve
ventricular filling and probably
reducing myocardial ischemia. Bonow et al., Circulation, 72: 853-64 (1985).
Nifedipine and diltiazem have also been used occasionally in the treatment of
hypertrophic
cardiomyopathy. Lorell etal., Circulation, 65: 499-507 (1982); Betocchi etal.,
Am. J. Cardiol., 78:451-457 (1996).
However, because of its potent vasodilating properties, nifedipine may be
harmful, especially in patients with
outflow obstruction. Disopyramide has been used to relieve symptoms by virtue
of its negative inotropic properties.
Pollick, N. Enal. J. Med., 307: 997-999 (1982). In many patients, however, the
initial benefits decrease with time.
Wigle et al., Circulation, 92: 1680-1692 ( 1995). Antihypertensive drug
therapy has been reported to have beneficial
effects on cardiac hypertrophy associated with elevated blood pressure.
Examples of drugs used in antihypertensive
therapy, alone or in combination, are calcium antagonists, e.g., nitrendipine;
adrenergic receptor blocking agents,
e.g., those listed above; angiotensin converting enzyme (ACE) inhibitors such
as quinapril, captopril, enalapril,
ramipril, benazepril, fosinopril, and lisinopril; diuretics, e.g.,
cMorothiazide, hydrochlorothiazide,
hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide,
acetazolamide, and indapamide; and
calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, and
nicardipine.
For example, treatment of hypertension with diltiazem and captopril showed a
decrease in left ventricular
muscle mass, but the Doppler indices of diastolic function did not normalize.
Szlachcic et al., Am. J. Cardiol., 63:
198-201 (1989); Shahi et al., Lancet, 336: 458-461 (1990). These findings were
interpreted to indicate that
excessive amounts of interstitial collagen may remain after regression of left
ventricular hypertrophy. Rossi et al.,
Am. Heart J., 124: 700-709 (1992). Rossi et al., supra, investigated the
effect of captopril on the prevention and
regression of myocardial cell hypertrophy and interstitial fibrosis in
pressure overload cardiac hypertrophy, in
experimental rats.
Agents that increase cardiac contractility directly (iontropic agents) were
initially thought to benefit
patients with heart failure because they improved cardiac output in the short
teen. However, all positive inotropic
agents except digoxigenin have been found to result in increased long-term
mortality, in spite of short-term
improvements in cardiac performance. Massie, Curr. Op. in Cardiology, 12: 209-
217 ( 1997); Reddy et al., Curr.
Opin. Cardiol., 12: 233-241 ( 1997). Beta-adrenergic receptor blockers have
recently been advocated for use in heart
failure. Evidence from clinical trials suggests that improvements in cardiac
function can be achieved without
increased mortality, though documented improvements patient survival have not
yet been demonstrated. See also,
U.S. Pat. Nos. 5,935,924, 5,624,806; 5,661,122; and 5,610,134 and WO 95/28173
regarding the use of
cardiotropin-1 or antagonists thereof, or growth hormone and/or insulin-like
growth factor-I in the treatment of
CHF. Another treatment modality is heart transplantation, but this is limited
by the availability of donor hearts.
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Endothelia is a vasoconstricting peptide comprising 21 amino acids, isolated
from swine arterial
endothelial culture supernatant and structurally determined. Yanagisawa et
al., Nature, 332: 411-415 (1988).
Endothelia was later found to exhibit various actions, and endothelia
antibodies as endothelia antagonists have
proven effective in the treatment of myocardial infarction, renal failure, and
other diseases. Since endothelia is
present in live bodies and exhibits vasoconstricting action, it is expected to
be an endogenous factor involved in
the regulation of the circulatory system, and may be associated with
hypertension, cardiovascular diseases such as
myocardial infarction, and renal diseases such as acute renal failure.
Endothelia antagonists are described, for
example, in U.S. Pat. No. 5,773,414; JP Pat. Publ. 3130299/1991, EP 457,195;
EP 460,679; and EP 552,489. A
new endothelia B receptor for identifying endothelia receptor antagonists is
described in U.S. Pat. No. 5,773,223.
Current therapy for heart failure is primarily directed to using angiotensin-
converting enzyme (ACE)
inhibitors, such as captopril, and diuretics. These drugs improve hemodynamic
profile and exercise tolerance and
reduce the incidence of morbidity and mortality in patients with CHF. Kramer
et al., Circulation, 67(4): 807-816
(1983); Captopril Multicenter Research Group, J.A.C.C., ~: 755-763 (1983); The
CONSENSUS Trial Study
Group, N. Engl. J. Med., 316(23): 1429-1435 (1987); The SOLVD Investigators,
N. EnQI. J. Med., 32_ 5(5): 293-302
(1991). Further, they are useful in treating hypertension, left ventricular
dysfunction, atherosclerotic vascular
disease, and diabetic nephropathy. Brown and Vaughan, supra. However, despite
proven efficacy, response to
ACE inhibitors has been limited. For example, while prolonging survival in the
setting of heart failure, ACE
inhibitors appear to slow the progression towards end-stage heart failure, and
substantial numbers of patients on
ACE inhibitors have functional class III heart failure.
Moreover, improvement of functional capacity and exercise time is only small
and mortality, although
reduced, continues to be high. The CONSENSUS Trial Study Group, N. E~1. J.
Med., 316(23): 1429-1453 ( 1987);
The SOLVD Investigators, N. En~l. J. Med., 325(5): 293-302 (1991); Cohn et
al., N. Enal. J. Med., 325(5:
303-310 ( 1991 ); The Captopril-Digoxin Multicenter Research Group, JAMA,
259(4): 539-544 ( 1988). Hence, ACE
inhibitors consistently appear unable to relieve symptoms in more than 60% of
heart failure patients and reduce
mortality of heart failure only by approximately 15-20%. For further adverse
effects, see Brown and Vaughan,
supra.
An alternative to ACE inhibitors is represented by specific AT1 receptor
antagonists. Clinical studies are
planned to compare the efficacy of these two modalities in the treatment of
cardiovascular and renal disease.
However, animal model data suggests that the ACE/Ang II pathway, while clearly
involved in cardiac hypertrophy,
is not the only, or even the primary pathway active in this role. Mouse
genetic "knockout" models have been made
to test individual components of the pathway. In one such model, the primary
cardiac receptor for Ang II, AT sub
1 A, has been genetically deleted; these mice do not develop hypertrophy when
Ang II is given experimentally
(confirming the basic success of the model in eliminating hypertrophy
secondary to Ang II). However, when the
aorta is constricted in these animals (a model of hypertensive cardiac
stress), the hearts still become hypertrophic.
This suggests that alternative signaling pathways, not depending on this
receptor (.AT sub l A), are activated in
hypertension. ACE inhibitors would presumably not be able to inhibit these
pathways. Sec, Harada et al.,
Circulation, 97: 1952-1959 (1998). See also, Homcy, Circulation, 97: 1890-1892
(1998) regarding the enigma
associated with the process and mechanism of cardiac hypertrophy.
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About 750,000 patients suffer from acute myocardial infarction (AMI) annually,
and approximately
one-fourth of all deaths in the United States are due to AMI. In recent years,
thrombolytic agents, e.g.,
streptokinase, urokinase, and in particular tissue plasminogen activator (t-
PA) have significantly increased the
survival of patients who suffered myocardial infarction. When administered as
a continuous intravenous infusion
over 1.5 to 4 hours, t-PA produces coronary patency at 90 minutes in 69% to
90°l0 of the treated patients. Topol
etal., Am. J. Cardiol., 61: 723-728 (1988); Neuhaus etal., J. Am. Coll.
Cardiol., 12: 581-587 (1988); Neuhaus et
al., J. Am. Coll. Cardiol., 14: 1566-1569 ( 1989). The highest patency rates
have been reported with high dose or
accelerated dosing regimens. Topol, J. Am. Coll. Cardiol., 15: 922-924 (
1990). t-PA may also be administered as
a single bolus, although due to its relatively short half-life, it is better
suited for infusion therapy. Tebbe et al., Am.
J. Cardiol., 64: 448-453 (1989). A t-PA variant, specifically designed to have
longer half-life and very high fibrin
specificity, TNK t-PA (a T103N, N117Q, KHRR(296-299)AAAA t-PA variant, Keyt et
al., Proc. Natl. Acad. Sci.
USA, 91: 3670-3674 ( 1994)) is particularly suitable for bolus administration.
However, despite all these advances,
the long-term prognosis of patient survival depends greatly on the post-
infarction monitoring and treatment of the
patients, which should include monitoring and treatment of cardiac
hypertrophy.
B. Growth Factors
Various naturally occurring polypeptides reportedly induce the proliferation
of endothelial cells. Among
those polypeptides are the basic and acidic fibroblast growth factors (FGF)
(Burgess and Maciag, Annual Rev.
Biochem., 58: 575 (1989)), platelet-derived endothelial cell growth factor (PD-
ECGF) (Ishikawa etal., Nature, 338:
557 (1989)), and vascular endothelial growth factor (VEGF). Leung et al.,
Science, 246: 1306 (1989); Ferrara and
Henzel, Biochem. Biophys. Res. Commun., 161: 851 (1989); Tischer et al.,
Biochem. Bionhys. Res. Commun.,
165: 1198 (1989); EP 471,754B granted July 31, 1996.
Media conditioned by cells transfected with the human VEGF (hVEGF) cDNA
promoted the proliferation
of capillary endothelial cells, whereas control cells did not. Leung et al.,
Science, 246: 1306 (1989). Several
additional cDNAs were identified in human cDNA libraries that encode 121-, 189-
, and 206-amino acid isoforms
of hVEGF (also collectively referred to as hVEGF-related proteins). The 121-
amino acid protein differs from
hVEGF by virtue of the deletion of the 44 amino acids between residues 116 and
159 in hVEGF. The 189-amino
acid protein differs from hVEGF by virtue of the insertion of 24 amino acids
at residue 116 in hVEGF, and
apparently is identical to human vascular permeability factor (hVPF). The 206-
amino acid protein differs from
hVEGF by virtue of an insertion of 41 amino acids at residue 116 in hVEGF.
Houck etal., Mol. Endocrin., 5: 1806
( 1991 ); Ferrara et al., J. Cell. Biochem., 47: 21 l ( 1991 ): Ferrara et
al., Endocrine Reviews, 13: 18 ( 1992); Keck
et al., Science, 246: 1309 (1989); Connolly et al., J. Biol. Chem., 264: 20017
(1989); EP 370,989 published May
30, 1990.
It is now well established that angiogenesis, which involves the formation of
new blood vessels from
preexisting endothelium, is implicated in the pathogenesis of a variety of
disorders. These include solid tumors and
metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic
inflammation, intraocular neovascular
syndromes such as proliferative retinopathies, e.g., diabetic retinopathy, age-
related macular degeneration (AMD),
neovascular glaucoma, immune rejection of transplanted corneal tissue and
other tissues, rheumatoid arthritis, and
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psoriasis. Folkman etal., J. Biol. Chem., 267: 10931-10934 (1992); Klagsbrun
etal., Annu. Rev. Physiol., 53: 217-
239 ( 1991 ); and Garner A., "Vascular diseases ", In: Pathobiolow of Ocular
Disease. A Dynamic Approach, Garner
A., Klintworth GK, eds., 2nd Edition (Marcel Dekker, NY, 1994), pp 1625-1710.
In the case of tumor growth, angiogenesis appears to be crucial for the
transition from hyperplasia to
neoplasia, and for providing nourishment to the growing solid tumor. Folkman
et al., Nature, 339: 58 ( 1989). The
neovascularization allows the tumor cells to acquire a growth advantage and
proliferative autonomy compared to
the normal cells. Accordingly, a correlation has been observed between density
of microvessels in tumor sections
and patient survival in breast cancer as well as in several other tumors.
Weidner et al., N. EnQI. J. Med, 324: 1-6
(1991); Horak etal., Lancet, 340: 1120-1124 (1992); Macchiarini etal., Lancet,
340: 145-146 (1992).
The search for positive regulators of angiogenesis has yielded many
candidates, including aFGF, bFGF, TGF-
a, TGF-Vii, HGF, TNF-a, angiogenin, IL-8, etc. Folkman et al., J.B.C., supra,
and Klagsbrun et al., supra. The
negative regulators so far identified include thrombospondin (Good et al.,
Proc. Natl. Acad. Sci. USA., 87: 6624-
6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et
al., Endocrinology, 133: 1292-1299
(1993)), angiostatin (O'Reilly etal., Cell, 79: 3I5-328 (1994)), and
endostatin. O'Reilly etal., Cell, 88: 277-285
(1996).
Work done over the last several years has established the key role of VEGF,
not only in stimulating vascular
endothelial cell proliferation, but also in inducing vascular permeability and
angiogenesis. Ferrara et al., Endocr.
Rev., 18: 4-25 ( 1997). The finding that the loss of even a single VEGF allele
results in embryonic lethality points
to an irreplaceable role played by this factor in the development and
differentiation of the vascular system.
Furthermore, VEGF has been shown to be a key mediator of neovascularization
associated with tumors and
intraocular disorders. Ferrara et al., Endocr. Rev., supra. The VEGF mRNA is
overexpressed by the majority of
human tumors examined. Berkman et al., J. Clin. Invest., 91: 153-159 (1993);
Brown et al., Human Pathol., 26:
86-91 (1995); Brown etal., Cancer Res., 53: 4727-4735 (1993); Mattern etal.,
Brit. J. Cancer, 73: 931-934 (1996);
Dvorak et al., Am. J. Pathol.. 146: 1029-1039 (1995).
Also, the concentration levels of VEGF in eye fluids are highly correlated to
the presence of active
proliferation of blood vessels in patients with diabetic and other ischemia-
related retinopathies. Aiello et al., N.
Enol. J. Med., 331: 1480-1487 (1994). Furthermore, recent studies have
demonstrated the localization of VEGF
in choroidal neovascular membranes in patients affected by AMD. Lopez et al.,
Invest. Onhthalmol. Vis. Sci., 37:
855-868 ( 1996).
Anti-VEGF neutralizing antibodies suppress the growth of a variety of human
tumor cell lines in nude mice
(Kim et al., Nature, 362: 841-844 (1993); Warren et al., J. Clin. Invest., 95:
1789-1797 ( 1995): Borgstrom et al.,
Cancer Res., 56: 4032-4039 (1996); Melnyk etal., Cancer Res.. 56: 921-924
(1996)) and also inhibit intraocular
angiogenesis in models of ischemic retinal disorders. Adamis et al., Arch.
Ophthalmol., 114: 66-71 (1996).
Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action
are promising candidates for the
treatment of solid tumors and various intraocular neovascular disorders. Such
antibodies are described, for
example, in EP 817,648 published January 14, 1998 and in PCT/US 98/06724 filed
April 3, 1998.
There exist several other growth factors and mitogens, including transforming
oncogenes, that are capable of
rapidly inducing a complex set of genes to be expressed by certain cells. Lau
and Nathans, Molecular Aspects of
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Cellular Reeulation, 6: 165-202 ( 1991 ). These genes, which have been named
immediate-early- or early-response
genes, are transcriptionally activated within minutes after contact with a
growth factor or mito~en, independent of
de novo protein synthesis. A group of these intermediate-early genes encodes
secreted, extracellular proteins that
are needed for coordination of complex biological processes such as
differentiation and proliferation, regeneration,
and wound healing. Ryseck et al., Cell Growth Differ., 2: 235-233 (1991 ).
Highly-related proteins that belong to this group include cef 10 (Simmons et
al., Proc. Natl. Acad. Sci. USA,
86: 1178-1182 (1989)), cyr 61, which is rapidly activated by serum- or
platelet-derived growth factor (PDGF)
(O'Brien et al., Mol. Cell Biol., 10: 3569-3577 (1990), human connective
tissue growth factor (CTGF) (Bradham
et al., J. Cell. Biol., 114: 1285-1294 ( 1991)), which is secreted by human
vascular endothelial cells in high levels
after activation with transforming growth factor beta (TGF-(3), exhibits PDGF-
like biological and immunological
activities, and competes with PDGF for a particular cell surface receptor,
fisp-I2 (Ryseck et al., Cell Growth
Differ., 2: 235-233 (1991)), human vascular IBP-like growth factor (VIGF) (WO
96/17931), and nov, normally
arrested in adult kidney cells, which was found to be overexpressed in
myeloblastosis-associated-virus-type-1-
induced nephroblastomas. Joloit et al., Mol. Cell. Biol., 12: 10-21 (1992).
The expression of these immediate-early genes acts as "third messengers" in
the cascade of events triggered
by growth factors. It is also thought that they are needed to integrate and
coordinate complex biological processes,
such as differentiation and wound healing in which cell proliferation is a
common event.
As additional mitogens, insulin-like growth factor binding proteins (IGFBPs)
have been shown, in complex
with insulin-like growth factor (IGF), to stimulate increased binding of IGF
to fibroblast and smooth muscle cell
surface receptors. Clemmons et al., J. Clin. Invest., 77: 1548 ( 1986).
Inhibitory effects of IGFBP on various IGF
actions in vitro include stimulation of glucose transport by adipocytes,
sulfate incorporation by chondrocytes, and
thymidine incorporation in fibroblast. Zapf etal., J. Clin. Invest., 63: 1077
(1979). In addition, inhibitory effects
of IGFBPs on growth factor-mediated mitogen activity in normal cells have been
shown.
C. Need for Further Treatments
In view of the role of vascular endothelial cell growth and angiogenesis in
many diseases and disorders, it is
desirable to have a means of reducing or inhibiting one or more of the
biological effects causing these processes.
It is also desirable to have a means of assaying for the presence of
pathogenic polypeptides in normal and diseased
conditions, and especially cancer. Further, in a specific aspect, as there is
no generally applicable therapy for the
treatment of cardiac hypertrophy, the identification of factors that can
prevent or reduce cardiac myocyte
hypertrophy is of primary importance in the development of new therapeutic
strategies to inhibit pathophysiological
cardiac growth. While there are several treatment modalities for various
cardiovascular and oncologic disorders,
there is still a need for additional therapeutic approaches.
Summary of the Invention
A. Embodiments
Accordingly, the present invention concerns compositions and methods for
promoting or inhibiting
angiogenesis and/or cardiovascularization in mammals. The present invention is
based on the identification of
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
proteins that test positive in various cardiovascular assays that test
promotion or inhibition of certain biological
activities. Accordingly, the proteins are believed to be useful drugs for the
diagnosis and/or treatment (including
prevention) of disorders where such effects are desired, such as the promotion
or inhibition of angiogenesis,
inhibition or stimulation of vascular endothelial cell growth, stimulation of
growth or proliferation of vascular
S endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-
dependent tissue growth, stimulation of
angiogenesis-dependent tissue growth, inhibition of cardiac hypertrophy and
stimulation of cardiac hypertrophy,
e.g., for the treatment of congestive heart failure.
In one embodiment, the present invention provides a composition comprising a
PRO polypeptide in admixture
with a pharmaceutically acceptable carrier. In one aspect, the composition
comprises a therapeutically effective
amount of the polypeptide. In another aspect, the composition comprises a
further active ingredient, namely, a
cardiovascular, endothelial or angiogenic agent or an angiostatic agent,
preferably an angiogenic or angiostatic
agent. Preferably, the composition is sterile. The PRO polypeptide may be
administered in the form of a liquid
pharmaceutical formulation, which may be preserved to achieve extended storage
stability. Preserved liquid
pharmaceutical formulations might contain multiple doses of PRO polypeptide,
and might, therefore, be suitable
for repeated use.
In a further embodiment, the present invention provides a method for preparing
such a composition useful for
the treatment of a cardiovascular, endothelial or angiogenic disorder
comprising admixing a therapeutically effective
amount of a PRO polypeptide with a pharmaceutically acceptable carrier.
In another embodiment, the present invention provides a composition comprising
an agonist or antagonist of
a PRO polypeptide in admixture with a pharmaceutically acceptable carrier. In
one aspect, the composition
comprises a therapeutically effective amount of the agonist or antagonist. In
another aspect, the composition
comprises a further active ingredient, namely, a cardiovascular, endothelial
or angiogenic agent or an angiostatic
agent, preferably an angiogenic or angiostatic agent. Preferably, the
composition is sterile. The PRO polypeptide
agonist or antagonist may be administered in the form of a liquid
pharmaceutical formulation, which may be
preserved to achieve extended storage stability. Preserved liquid
pharmaceutical formulations might contain
multiple doses of a PRO polypeptide agonist or antagonist, and might,
therefore, be suitable for repeated use.
In a further embodiment, the present invention provides a method for preparing
such a composition useful for
the treatment of a cardiovascular, endothelial or angiogenic disorder
comprising admixing a therapeutically effective
amount of a PRO polypeptide agonist or antagonist with a pharmaceutically
acceptable carrier.
In yet another embodiment, the present invention concerns a composition
comprising an anti-PRO antibody
in admixture with a pharmaceutically acceptable carrier. In one aspect, the
composition comprises a therapeutically
effective amount of the antibody. In another aspect, the composition comprises
a further active ingredient, namely,
a cardiovascular, endothelial or angiogenic agent or an angiostatic agent,
preferably an angiogenic or angiostatic
agent. Preferably, the composition is sterile. The composition may be
administered in the form of a liquid
pharmaceutical formulation, which may be preserved to achieve extended storage
stability. Preserved liquid
pharmaceutical formulations might contain multiple doses of the anti-PRO
antibody, and might, therefore, be
suitable for repeated use. In preferred embodiments, the antibody is a
monoclonal antibody, an antibody fragment,
a humanized antibody, or a single-chain antibody.
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
In a further embodiment, the present invention provides a method for preparing
such a composition useful for
the treatment of a cardiovascular, endothelial or angiogenic disorder
comprising admixing a therapeutically effective
amount of an anti-PRO antibody with a pharmaceutically acceptable carrier.
In a still further aspect, the present invention provides an article of
manufacture comprising:
(a) a composition of matter comprising a PRO polypeptide or agonist or
antagonist thereof;
(b) a container containing said composition; and
(c) a label affixed to said container, or a package insert included in said
container referring to the use of said
PRO polypeptide or agonist or antagonist thereof in the treatment of a
cardiovascular, endothelial or angiogenic
disorder, wherein the agonist or antagonist may be an antibody which binds to
the PRO polypeptide. The
composition may comprise a therapeutically effective amount of the PRO
polypeptide or the agonist or antagonist
thereof.
In another embodiment, the present invention provides a method for identifying
an agonist of a PRO
polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions
suitable for the induction of a
cellular response normally induced by a PRO polypeptide; and
(b) determining the induction of said cellular response to determine if the
test compound is an effective
agonist, wherein the induction of said cellular response is indicative of said
test compound being an effective
agonist.
In another embodiment, the present invention provides a method for identifying
an agonist of a PRO
polypeptide comprising:
(a) contacting cells and a test compound to be screened under conditions
suitable for the stimulation of cell
proliferation by a PRO polypeptide; and
(b) measuring the proliferation of said cells to determine if the test
compound is an effective agonist, wherein
the stimulation of cell proliferation is indicative of said test compound
being an effective agonist.
In another embodiment, the invention provides a method for identifying a
compound that inhibits the activity
of a PRO polypeptide comprising contacting a test compound with a PRO
polypeptide under conditions and for a
time sufficient to allow the test compound and polypeptide to interact and
determining whether the activity of the
PRO polypeptide is inhibited. In a specific preferred aspect, either the test
compound or the PRO polypeptide is
immobilized on a solid support. In another preferred aspect. the non-
immobilized component carries a detectable
label. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a
PRO polypeptide under conditions
suitable for the induction of a cellular response normally induced by a PRO
polypeptide; and
(b) determining the induction of said cellular response to determine if the
test compound is an effective
antagonist.
In another preferred aspect, this process comprises the steps of:
(a) contacting cells and a test compound to be screened in the presence of a
PRO polypeptide under conditions
suitable for the stimulation of cell proliferation by a PRO polypeptide; and
(b) measuring the proliferation of the cells to determine if the test compound
is an effective antagonist.
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In another embodiment, the invention provides a method for identifying a
compound that inhibits the
expression of a PRO polypeptide in cells that normally expresses the
polypeptide, wherein the method comprises
contacting the cells with a test compound and determining whether the
expression of the PRO polypeptide is
inhibited. In a preferred aspect, this method comprises the steps of:
(a) contacting cells and a test compound to be screened under conditions
suitable for allowing expression of
the PRO polypeptide; and
(b) determining the inhibition of expression of said polypeptide.
In a still further embodiment, the invention provides a compound that inhibits
the expression of a PRO
polypeptide, such as a compound that is identified by the methods set forth
above.
Another aspect of the present invention is directed to an agonist or an
antagonist of a PRO polypeptide which
may optionally be identified by the methods described above.
One type of antagonist of a PRO polypeptide that inhibits one or more of the
functions or activities of the PRO
polypeptide is an antibody. Hence, in another aspect, the invention provides
an isolated antibody that binds a PRO
polypeptide. In a preferred aspect, the antibody is a monoclonal antibody,
which preferably has non-human
complementarity-determining-region (CDR) residues and human framework-region
(FR) residues. The antibody
may be labeled and may be immobilized on a solid support. In a further aspect,
the antibody is an antibody
fragment, a single-chain antibody, or a humanized antibody. Preferably, the
antibody specifically binds to the
polypeptide.
In a still further aspect, the present invention provides a method for
diagnosing a disease or susceptibility to
a disease which is related to a mutation in a PRO polypeptide-encoding nucleic
acid sequence comprising
determining the presence or absence of said mutation in the PRO polypeptide
nucleic acid sequence, wherein the
presence or absence of said mutation is indicative of the presence of said
disease or susceptibility to said disease.
In a still further aspect, the invention provides a method of diagnosing a
cardiovascular, endothelial or
angiogenic disorder in a mammal which comprises analyzing the level of
expression of a gene encoding a PRO
polypeptide (a) in a test sample of tissue cells obtained from said mammal,
and (b) in a control sample of known
normal tissue cells of the same cell type, wherein a higher or lower
expression level in the test sample as compared
to the control sample is indicative of the presence of a cardiovascular,
endothelial or angiogenic disorder in said
mammal. The expression of a gene encoding a PRO polypeptide may optionally be
accomplished by measuring
the level of mRNA or the polypeptide in the test sample as compared to the
control sample.
In a still further aspect, the present invention provides a method of
diagnosing a cardiovascular, endothelial
or angiogenic disorder in a mammal which comprises detecting the presence or
absence of a PRO polypeptide in
a test sample of tissue cells obtained from said mammal, wherein the presence
or absence of said PRO polypeptide
in said test sample is indicative of the presence of a cardiovascular,
endothelial or an'~iogenic disorder in said
mammal.
In a still further embodiment, the invention provides a method of diagnosing a
cardiovascular, endothelial or
angiogenic disorder in a mammal comprising (a) contacting an anti-PRO antibody
with a test sample of tissue cells
obtained from the mammal, and (b) detecting the formation of a complex between
the antibody and the PRO
polypeptide in the test sample, wherein the formation of said complex is
indicative of the presence of a
WO 00/53757 CA 02361849 2001-07-30 pC'T/US00/05004
cardiovascular, endothelial or angiogenic disorder in the mammal. The
detection may be qualitative or quantitative.
and may be performed in comparison with monitoring the complex formation in a
control sample of known normal
tissue cells of the same cell type. A larger or smaller quantity of complexes
formed in the test sample indicates the
presence of a cardiovascular, endothelial or angiogenic dysfunction in the
mammal from which the test tissue cells
were obtained. The antibody preferably carries a detectable label. Complex
formation can be monitored, for
example, by light microscopy, flow cytometry, fluorimetry, or other techniques
known in the art. The test sample
is usually obtained from an individual suspected to have a cardiovascular,
endothelial or angiogenic disorder.
In another embodiment, the invention provides a method for determining the
presence of a PRO polypeptide
in a sample comprising exposing a sample suspected of containing the PRO
polypeptide to an anti-PRO antibody
and determining binding of said antibody to a component of said sample. In a
specific aspect, the sample comprises
a cell suspected of containing the PRO polypeptide and the antibody binds to
the cell. The antibody is preferably
detectably labeled and/or bound to a solid support.
In further aspects, the invention provides a cardiovascular, endothelial or
angiogenic disorder diagnostic kit
comprising an anti-PRO antibody and a carrier in suitable packaging.
Preferably, such kit further comprises
instructions for using said antibody to detect the presence of the PRO
polypeptide. Preferably, the carrier is a
buffer, for example. Preferably, the cardiovascular, endothelial or angiogenic
disorder is cancer.
In yet another embodiment, the present invention provides a method for
treating a cardiovascular, endothelial
or angiogenic disorder in a mammal comprising administering to the mammal an
effective amount of a PRO
polypeptide. Preferably, the disorder is cardiac hypertrophy, trauma such as
wounds or burns, or a type of cancer.
In a further aspect, the mammal is further exposed to angioplasty or a drug
that treats cardiovascular, endothelial
or angiogenic disorders such as ACE inhibitors or chemotherapeutic agents if
the cardiovascular, endothelial or
angiogenic disorder is a type of cancer. Preferably, the mammal is human,
preferably one who is at risk of
developing cardiac hypertrophy and more preferably has suffered myocardial
infarction.
In another preferred aspect, the cardiac hypertrophy is characterized by the
presence of an elevated level of
PGF~G. Alternatively, the cardiac hypertrophy may be induced by myocardial
infarction, wherein preferably the
administration of the PRO polypeptide is initiated within 48 hours, more
preferably within 2-1 hours, following
myocardial infarction.
In another preferred embodiment, the cardiovascular, endothelial or angiogenic
disorder is cardiac hypertrophy
and said PRO polypeptide is administered together with a cardiovascular,
endothelial or angiogenic agent. The
preferred cardiovascular, endothelial or angiogenic agent for this purpose is
selected from the group consisting of
an antihypertensive drug, an ACE inhibitor, an endothelin receptor antagonist
and a thrombolytic agent. If a
thrombolytic agent is administered, preferably the PRO polypeptide is
administered following administration of
such agent. More preferably, the thrombolytic agent is recombinant human
tissue plasminogen activator.
In another preferred aspect, the cardiovascular, endothelial or angiogenic
disorder is cardiac hypertrophy and
the PRO polypeptide is administered following primary angioplasty for the
treatment of acute myocardial infarction,
preferably wherein the mammal is further exposed to angioplasty or a
cardiovascular, endothelial, or angiogenic
agent.
In another preferred embodiment, the cardiovascular, endothelial or angiogenir
disorder is a cancer and the
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
PRO polypeptide is administered in combination with a chemotherapeutic agent,
a growth inhibitory agent or a
cytotoxtc agent.
In a further embodiment, the invention concerns a method for treating a
cardiovascular, endothelial or
angiogenic disorder in a mammal comprising administering to the mammal an
effective amount of an agonist of a
PRO polypeptide. Preferably, the cardiovascular, endothelial or angiogenic
disorder is cardiac hypertrophy, trauma,
a cancer, or age-related macular degeneration. Also preferred is where the
mammal is human, and where an
effective amount of an angiogenic or angiostatic agent is administered in
conjunction with the agonist.
In a further embodiment, the invention concerns a method for treating a
cardiovascular, endothelial or
angiogenic disorder in a mammal comprising administering to the mammal an
effective amount of an antagonist
of a PRO polypeptide. Preferably, the cardiovascular, endothelial or
angiogenic disorder is cardiac hypertrophy,
trauma, a cancer, or age-related macular degeneration. Also preferred is where
the mammal is human, and where
an effective amount of an angiogenic or angiostatic agent is administered in
conjunction with the antagonist.
In a further embodiment, the invention concerns a method for treating a
cardiovascular, endothelial or
angiogenic disorder in a mammal comprising administering to the mammal an
effective amount of an anti-PRO
antibody. Preferably, the cardiovascular, endothelial or angiogenic disorder
is cardiac hypertrophy, trauma, a
cancer, or age-related macular degeneration. Also preferred is where the
mammal is human, and where an effective
amount of an angiogenic or angiostatic agent is administered in conjunction
with the antibody.
In still further embodiments, the invention provides a method for treating a
cardiovascular, endothelial or
angiogenic disorder in a mammal that suffers therefrom comprising
administering to the mammal a nucleic acid
molecule that codes for either (a) a PRO polypeptide, (b) an agonist of a PRO
polypeptide or (c) an antagonist of
a PRO polypeptide, wherein said agonist or antagonist may be an anti-PRO
antibody. In a preferred embodiment,
the mammal is human. In another preferred embodiment, the gene is administered
via ex vivo gene therapy. In a
further preferred embodiment, the gene is comprised within a vector, more
preferably an adenoviral,
adeno-associated viral, lentiviral, or retroviral vector.
In yet another aspect, the invention provides a recombinant retroviral
particle comprising a retroviral vector
consisting essentially of a promoter, nucleic acid encoding (a) a PRO
polypeptide, (b) an agonist polypeptide of a
PRO polypeptide, or (c) an antagonist polypeptide of a PRO polypeptide, and a
signal sequence for cellular
secretion of the polypeptide, wherein the retroviral vector is in association
with retroviral structural proteins.
Preferably, the signal sequence is from a mammal, such as from a native PRO
polypeptide.
In a still further embodiment, the invention supplies an ex vivo producer cell
comprising a nucleic acid
construct that expresses retroviral structural proteins and also comprises a
retroviral vector consisting essentially
of a promoter, nucleic acid encoding (a) a PRO polypeptide, (b) an agonist
polypeptide of a PRO polypeptide or
(c) an antagonist polypeptide of a PRO polypeptide, and a signal sequence for
cellular secretion of the polypeptide,
wherein said producer cell packages the retroviral vector in association with
the structural proteins to produce
recombinant retroviral particles.
In yet another embodiment, the invention provides a method for inhibiting
endothelial cell growth in a mammal
comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist
of a PRO polypeptide, or (c) an
antagonist of a PRO polypeptide, wherein endothelial cell growth in said
mammal is inhibited, and wherein said
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agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is
human and the endothelial cell
growth is associated with a tumor or a retinal disorder.
In yet another embodiment, the invention provides a method for stimulating
endothelial cell growth in a
mammal comprising administering to the mammal (a) a PRO polypeptide, (b) an
agonist of a PRO polypeptide,
or (c) an antagonist of a PRO polypeptide, wherein endothelial cell growth in
said mammal is stimulated, and
wherein said agonist or antagonist may be an anti-PRO antibody. Preferably,
the mammal is human.
In yet another embodiment, the invention provides a method for inhibiting
cardiac hypertrophy in a mammal
comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist
of a PRO polypeptide, or (c) an
antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is
inhibited, and wherein said
agonist or antagonist may be an anti-PRO antibody . Preferably, the mammal is
human and the cardiac hypertrophy
has been induced by myocardial infarction.
In yet another embodiment, the invention provides a method for stimulating
cardiac hypertrophy in a mammal
comprising administering to the mammal (a) a PRO polypeptide, (b) an agonist
of a PRO polypeptide, or (c) an
antagonist of a PRO polypeptide, wherein cardiac hypertrophy in said mammal is
stimulated, and wherein said
agonist or antagonist may be an anti-PRO antibody. Preferably, the mammal is
human who suffers from congestive
heart failure.
In yet another embodiment, the invention provides a method for inhibiting
angiogenesis induced by a PRO
polypeptide in a mammal comprising administering a therapeutically effective
amount of an anti-PRO antibody to
the mammal. Preferably, the mammal is a human, and more preferably the mammal
has a tumor or a retinal
disorder.
In yet another embodiment, the invention provides a method for stimulating
angiogenesis induced by a PRO
polypeptide in a mammal comprising administering a therapeutically effective
amount of a PRO polypeptide to the
mammal. Preferably, the mammal is a human, and more preferably angiogeneisis
would promote tissue regeneration
or wound healing.
In yet another embodiment, the invention provides a method for inhibiting
endothelial cell growth in a mammal
comprising administering to the mammal a PR0333, PR0364, PR0877, PR0879,
PR0882 or PR0885 polypeptide
or agonist thereof, wherein endothelial cell growth in said mammal is
inhibited.
In yet another embodiment, the invention provides a method for stimulating
endothelial cell growth in a
mammal comprising administering to the mammal a PR0179, PR0321, PR0840,
PR0844, PR0846, PR0878 or
PR0879 polypeptide or agonist thereof, wherein endothelial cell growth in said
mammal is stimulated.
In yet another embodiment, the invention provides a method for inhibiting
endothelial cell growth in a mammal
comprising administering to the mammal an antagonist of a PR0179, PR0321,
PR0840, PR0844, PR0846,
PR0878 or PR0879 polypeptide, wherein endothelial cell growth in said mammal
is inhibited.
In yet another embodiment, the invention provides a method for stimulating
endothelial cell growth in a
mammal comprising administering to the mammal an antagonist of a PR0333,
PR0364, PR0877, PR0879,
PR0882 or PR0885 polypeptide, wherein endothelial cell growth in said mammal
is stimulated.
In yet another embodiment, the invention provides a method for inducing
cardiac hypertrophy in a mammal
comprising administering to the mammal a PR0205, PR0882 or PR0887 polypeptide
or agonist thereof, wherein
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
cardiac hypertrophy in said mammal is induced.
In yet another embodiment, the invention provides a method for reducing
cardiac hypertrophy in a mammal
comprising administering to the mammal a PR0238, PR0878 or PR01760 polypeptide
or agonist thereof, wherein
cardiac hypertrophy in said mammal is reduced.
In yet another embodiment, the invention provides a method for inducing
cardiac hypertrophy in a mammal
comprising administering to the mammal an antagonist of a PR0238, PR0878 or
PROI 760 polypeptide, wherein
cardiac hypertrophy in said mammal is induced.
In yet another embodiment, the invention provides a method for reducing
cardiac hypertrophy in a mammal
comprising administering to the mammal an antagonist of a PR0205, PR0882 or
PR0887 polypeptide, wherein
cardiac hypertrophy in said mammal is reduced.
In yet another embodiment, the invention provides a method for inhibiting
angiogenesis induced by a PRO 179,
PR0321, PR0840, PR0844, PR0846, PR0878 or PR0879 polypeptide comprising
administering a therapeutically
effective amount of an anti-PRO 179, anti-PR0321, anti-PR0840, anti-PR0844,
anti-PR0846, anti-PR0878 or anti-
PR0879 antibody to the mammal, wherein said angiogenesis is inhibited.
In yet another embodiment, the invention provides a method for stimulating
angiogenesis induced by a
PR0179, PR0321, PR0840, PR0844, PR0846, PR0878 or PR0879 polypeptide
comprising administering a
therapeutically effective amount of said polypeptide to the mammal, wherein
said angiogenesis is stimulated.
B. Additional Embodiments
In other embodiments of the present invention, the invention provides an
isolated nucleic acid molecule
comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about 80%
nucleic acid sequence identity, alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least
about 82% nucleic acid sequence identity, alternatively at least about 83%
nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85 i~ nucleic acid
sequence identity, alternatively at least about 86°70 nucleic acid
sequence identity, alternatively at least about 87°lc
nucleic acid sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at least
about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid sequence identity,
alternatively at least about 92 ie nucleic acid
sequence identity, alternatively at least about 93% nucleic acid sequence
identity, alternatively at least about 94~1c
nucleic acid sequence identity, alternatively at least about 95% nucleic acid
sequence identity, alternatively at least
about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99 is nucleic acid
sequence identity to (a) a DNA molecule encodin; a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular domain
of a transmembrane protein, with or without the signal peptide, as disclosed
herein or any other specifically defined
fragment of the full-length amino acid sequence as disclosed herein, or (b)
the complement of the DNA molecule
of (a).
1-=>~
WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about 80%
nucleic acid sequence identity, alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least
about 82% nucleic acid sequence identity, alternatively at least about 83%
nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid sequence
identity, alternatively at least about 87%
nucleic acid sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at least
about 89% nucleic acid sequence identity, alternatively at least about 90%
nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid sequence identity,
alternatively at least about 92% nucleic acid
sequence identity, alternatively at least about 93% nucleic acid sequence
identity, alternatively at least about 94%
nucleic acid sequence identity, alternatively at least about 95% nucleic acid
sequence identity, alternatively at least
about 96% nucleic acid sequence identity, alternatively at least about 97%
nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99°lo nucleic acid
sequence identity to (a) a DNA molecule comprising the coding sequence of a
full-length PRO polypeptide cDNA
as disclosed herein, the coding sequence of a PRO polypeptide lacking the
signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane PRO polypeptide,
with or without the signal
peptide, as disclosed herein or the coding sequence of any other specifically
defined fragment of the full-length
amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide sequence
having at least about 80% nucleic acid sequence identity, alternatively at
least about 81 % nucleic acid sequence
identity, alternatively at least about 82% nucleic acid sequence identity,
alternatively at least about 83% nucleic acid
sequence identity, alternatively at least about 84% nucleic acid sequence
identity, alternatively at least about 85%
nucleic acid sequence identity, alternatively at least about 86°lo
nucleic acid sequence identity, alternatively at least
about 87% nucleic acid sequence identity, alternatively at least about 88%
nucleic acid sequence identity,
alternatively at least about 89°~o nucleic acid sequence identity,
alternatively at least about 90% nucleic acid
sequence identity, alternatively at least about 91 % nucleic acid sequence
identity, alternatively at least about 92°k
nucleic acid sequence identity, alternatively at least about 93% nucleic acid
sequence identity, alternatively at least
about 94% nucleic acid sequence identity, alternatively at least about 95%
nucleic acid sequence identity,
alternatively at least about 96% nucleic acid sequence identity, alternatively
at least about 97% nucleic acid
sequence identity, alternatively at least about 98% nucleic acid sequence
identity and alternatively at least about
99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same
mature polypeptide encoded by
any of the human protein cDNAs deposited with the ATCC as disclosed herein, or
(b) the complement of the DNA
molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule
comprising a nucleotide sequence
encoding a PRO polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated,
or is complementary to such encodin~~ nucleotide sequence, wherein the
transmembrane domain(sl of such
polypeptide are disclosed herein. Therefore. soluble extracellular domains of
the herein described PRO
polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide codin~T
sequence, or the complement
WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
thereof, that may find use as, for example, hybridization probes, for encoding
fragments of a PRO polypeptide that
may optionally encode a polypeptide comprising a binding site for an anti-PRO
antibody or as antisense
oligonucleotideprobes. Such nucleic acid fragments are usually at least about
20 nucleotides in length, alternatively
at least about 30 nucleotides in length, alternatively at least about 40
nucleotides in length, alternatively at least
about 50 nucleotides in length, alternatively at least about 60 nucleotides in
length, alternatively at least about 70
nucleotides in length, alternatively at least about 80 nucleotides in length,
alternatively at least about 90 nucleotides
in length, alternatively at least about 100 nucleotides in length,
alternatively at least about 110 nucleotides in length,
alternatively at least about 120 nucleotides in length, alternatively at least
about 130 nucleotides in length,
alternatively at least about 140 nucleotides in length, alternatively at least
about 150 nucleotides in length,
alternatively at least about 160 nucleotides in length, alternatively at least
about 170 nucleotides in length,
alternatively at least about 180 nucleotides in length, alternatively at least
about 190 nucleotides in length,
alternatively at least about 200 nucleotides in length, alternatively at least
about 250 nucleotides in length,
alternatively at least about 300 nucleotides in length, alternatively at least
about 350 nucleotides in length,
alternatively at least about 400 nucleotides in length, alternatively at least
about 450 nucleotides in length,
alternatively at least about 500 nucleotides in length, alternatively at least
about 600 nucleotides in length,
alternatively at least about 700 nucleotides in length, alternatively at least
about 800 nucleotides in length,
alternatively at least about 900 nucleotides in length and alternatively at
least about 1000 nucleotides in length,
wherein in this context the term "about" means the referenced nucleotide
sequence length plus or minus 10% of that
referenced length. It is noted that novel fragments of a PRO polypeptide-
encoding nucleotide sequence may be
determined in a routine manner by aligning the PRO polypeptide-encoding
nucleotide sequence with other known
nucleotide sequences using any of a number of well known sequence alignment
programs and determining which
PRO polypeptide-encoding nucleotide sequence fragments) are novel. All of such
PRO polypeptide-encoding
nucleotide sequences are contemplated herein. Also contemplated are the PRO
polypeptide fragments encoded by
these nucleotide molecule fragments, preferably those PRO polypeptide
fragments that comprise a binding site for
an anti-PRO antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded
by any of the isolated
nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide,
comprising an amino acid sequence
having at least about 80% amino acid sequence identity, alternatively at least
about 8l % amino acid sequence
identity, alternatively at least about 82% amino acid sequence identity,
alternatively at least about 83% amino acid
sequence identity, alternatively at least about 84% amino acid sequence
identity. alternatively at least about 85%
amino acid sequence identity, alternatively at least about 86% amino acid
sequence identity, alternatively at least
about 87% amino acid sequence identity, alternatively at least about 88~'i~
amino acid sequence identity, alternatively
at least about 89% amino acid sequence identity, alternatively at least about
90% amino acid sequence identity.
alternatively at least about 91 % amino acid sequence identity, alternatively
at least about 92% amino acid sequence
identity, alternatively at least about 93% amino acid sequence identity,
alternatively at least about 94% amino acid
sequence identity, alternatively at least about 95% amino acid sequence
identity, alternatively at least about 96%
amino acid sequence identity, alternatively at least about 97% amino acid
sequence identity, alternatively at least
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about 98°~o amino acid sequence identity and alternatively at least
about 99% amino acid sequence identity to a PRO
polypeptide having a full-length amino acid sequence as disclosed herein, an
amino acid sequence lacking the signal
peptide as disclosed herein, an extracellular domain of a transmembrane
protein, with or without the signal peptide,
as disclosed herein or any other specifically defined fragment of the full-
length amino acid sequence as disclosed
herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid sequence
having at least about 80% amino acid sequence identity, alternatively at least
about 81 % amino acid sequence
identity, alternatively at least about 82% amino acid sequence identity,
alternatively at least about 83% amino acid
sequence identity, alternatively at least about 84% amino acid sequence
identity, alternatively at least about 85%
amino acid sequence identity, alternatively at least about 86% amino acid
sequence identity, alternatively at least
about 87% amino acid sequence identity, alternatively at least about 88% amino
acid sequence identity, alternatively
at least about 89% amino acid sequence identity, alternatively at least about
90% amino acid sequence identity,
alternatively at least about 91 % amino acid sequence identity, alternatively
at least about 92% amino acid sequence
identity, alternatively at least about 93% amino acid sequence identity,
alternatively at least about 94% amino acid
sequence identity, alternatively at least about 95% amino acid sequence
identity, alternatively at least about 96%
amino acid sequence identity, alternatively at least about 97% amino acid
sequence identity, alternatively at least
about 98% amino acid sequence identity and alternatively at least about 99%
amino acid sequence identity to an
amino acid sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid sequence
scoring at least about 80% positives, alternatively at least about 81 %
positives, alternatively at least about 82%
positives, alternatively at least about 83% positives, alternatively at least
about 84% positives, alternatively at least
about 85% positives, alternatively at least about 86% positives, alternatively
at least about 87010 positives.
alternatively at least about 88% positives, alternatively at least about 89%
positives, alternatively at least about 90%
positives, alternatively at least about 91 % positives, alternatively at least
about 92% positives, alternatively at least
about 93% positives, alternatively at least about 94% positives, alternatively
at least about 95% positives.
alternatively at least about 96% positives, alternatively at least about 97%
positives, alternatively at least about 98%
positives and alternatively at least about 99% positives when compared with
the amino acid sequence of a PRO
polypeptide having a full-length amino acid sequence as disclosed herein, an
amino acid sequence lacking the signal
peptide as disclosed herein, an extracellular domain of a transmembrane
protein, with or without the signal peptide,
as disclosed herein or any other specifically defined fragment of the full-
length amino acid sequence as disclosed
herein.
In a specific aspect, the invention provides an isolated PRO polypeptide
without the N-terminal signal
sequence and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino acid
sequence as hereinbefore described. Processes for producing the same are also
herein described, wherein those
processes comprise culturing a host cell comprising a vector which comprises
the appropriate encoding nucleic acid
molecule under conditions suitable for expression of the PRO polypeptide and
recovering the PRO polypeptide from
the cell culture.
Another aspect the invention provides an isolated PRO polypeptide which is
either transmembrane
17
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
domain-deleted or transmembrane domain-inactivated. Processes for producing
the same are also herein described,
wherein those processes comprise culturing a host cell comprising a vector
which comprises the appropriate
encoding nucleic acid molecule under conditions suitable for expression of the
PRO polypeptide and recovering
the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists and antagonists of
a native PRO polypeptide as
defined herein. In a particular embodiment, the agonist or antagonist is an
anti-PRO antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying
agonists or antagonists to a PRO
polypeptide which comprise contacting the PRO polypeptide with a candidate
molecule and monitoring a biological
activity mediated by said PRO polypeptide. Preferably. the PRO polypeptide is
a native PRO polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PRO polypeptide,
or an agonist or antagonist of a PRO polypeptide as herein described, or an
anti-PRO antibody, in combination with
a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an agonist or
antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the
preparation of a medicament useful
in the treatment of a condition which is responsive to the PRO polypeptide, an
agonist or antagonist thereof or an
anti-PRO antibody.
In additional embodiments of the present invention, the invention provides
vectors comprising DNA encoding
any of the herein described polypeptides. Host cell comprising any such vector
are also provided. By way of
example, the host cells may be CHO cells, E. coli, yeast, or Baculovirus-
infected insect cells. A process for
producing any of the herein described polypeptides is further provided and
comprises culturing host cells under
conditions suitable for expression of the desired polypeptide and recovering
the desired polypeptide from the cell
culture.
In other embodiments, the invention provides chimeric molecules comprising any
of the herein described
polypeptides fused to a heterologous polypeptide or amino acid sequence.
Example of such chimeric molecules
comprise any of the herein described polypeptides fused to an epitope tag
sequence or a Fc region of an
immunoglobulin.
In yet another embodiment, the invention provides an antibody which
specifically binds to any of the above
or below described polypeptides. Optionally, the antibody is a monoclonal
antibody, humanized antibody, antibody
fragment or single-chain antibody.
In yet other embodiments, the invention provides oli~~onucleotide probes
useful for isolating genomic and
cDNA nucleotide sequences or as antisense probes, wherein those probes may be
derived from any of the above
or below described nucleotide sequences.
Brief Description of the Drawings
Figure 1 shows a nucleotide sequence (SEQ ID NO:I ) of a native sequence
PR0179 cDNA, wherein SEQ
ID NO:1 is a clone designated herein as "DNA16451-1388".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding
sequence of SEQ ID NO: l
shown in Figure I .
18
WO 00/53757 CA 02361849 2001-07-30 pC'T/jJS00/05004
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) of a native sequence PR0238
cDNA, wherein SEQ
ID N0:3 is a clone designated herein as "DNA35600-1162".
Figure 4 shows the amino acid sequence (SEQ ID N0:4) derived from the coding
sequence of SEQ ID N0:3
shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID N0:5) of a native sequence PR0364
cDNA, wherein SEQ
ID N0:5 is a clone designated herein as "DNA47365-1206".
Figure 6 shows the amino acid sequence (SEQ ID N0:6) derived from the coding
sequence of SEQ ID N0:5
shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) of a native sequence PR0844
cDNA, wherein SEQ
ID N0:7 is a clone designated herein as "DNA59838-1462".
Figure 8 shows the amino acid sequence (SEQ ID N0:8) derived from the coding
sequence of SEQ ID N0:7
shown in Figure 7.
Figure 9 shows a nucleotide sequence (SEQ ID N0:9) of a native sequence PR0846
cDNA, wherein SEQ
ID N0:9 is a clone designated herein as "DNA44196-1353".
Figure 10 shows the amino acid sequence (SEQ ID NO:10) derived from the coding
sequence of SEQ ID
N0:9 shown in Figure 9.
Figure 11 shows a nucleotide sequence (SEQ ID NO:I 1 ) of a native sequence
PRO 1760 cDNA, wherein SEQ
ID NO:11 is a clone designated herein as "DNA76532-1702".
Figure 12 shows the amino acid sequence (SEQ ID N0:12) derived from the coding
sequence of SEQ ID
NO:I 1 shown in Figure I 1.
Figure 13 shows a nucleotide sequence (SEQ ID NO:I 3) of a native sequence
PR0205 cDNA, wherein SEQ
ID N0:13 is a clone designated herein as "DNA30868".
Figure 14 shows the amino acid sequence (SEQ ID N0:14) derived from the coding
sequence of SEQ ID
N0:13 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID N0:15) of a native sequence
PR0321 cDNA, wherein SEQ
ID N0:15 is a clone designated herein as "DNA34433".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding
sequence of SEQ ID
N0:15 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) of a native sequence
PR0333 cDNA, wherein SEQ
ID N0:17 is a clone designated herein as "DNA41374".
Figure 18 shows the amino acid sequence (SEQ ID N0:18) derived from the coding
sequence of SEQ ID
N0:17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ ID N0:19) of a native sequence
PR0840 cDNA, wherein SEQ
ID N0:19 is a clone designated herein as "DNA53987".
Figure 20 shows the amino acid sequence (SEQ ID N0:20) derived from the coding
sequence of SEQ ID
N0:19 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID N0:21 ) of a native sequence
PR0877 cDNA, wherein SEQ
ID N0:21 is a clone designated herein as "DNA58120".
19
WO 00/53757 CA 02361849 2001-07-30 pCT/[JS00/05004
Figure 22 shows the amino acid sequence (SEQ ID N0:22) derived from the coding
sequence of SEQ ID
N0:21 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ ID N0:23) of a native sequence
PR0878 cDNA, wherein SEQ
ID N0:23 is a clone designated herein as "DNA58121 ".
Figure 24 shows the amino acid sequence (SEQ ID N0:24) derived from the coding
sequence of SEQ ID
N0:23 shown in Figure 23.
Figure 25 shows a nucleotide sequence (SEQ ID N0:25) of a native sequence
PR0879 cDNA, wherein SEQ
ID N0:25 is a clone designated herein as "DNA58122".
Figure 26 shows the amino acid sequence (SEQ ID N0:26) derived from the coding
sequence of SEQ ID
N0:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) of a native sequence
PR0882 cDNA, wherein SEQ
ID N0:27 is a clone designated herein as "DNA58125".
Figure 28 shows the amino acid sequence (SEQ ID N0:28) derived from the coding
sequence of SEQ ID
N0:27 shown in Figure 27.
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) of a native sequence
PR0885 cDNA, wherein SEQ
ID N0:29 is a clone designated herein as "DNA58128".
Figure 30 shows the amino acid sequence (SEQ ID N0:30) derived from the coding
sequence of SEQ ID
N0:29 shown in Figure 29.
Figure 31 shows a nucleotide sequence (SEQ ID N0:31 ) of a native sequence
PR0887 cDNA, wherein SEQ
ID N0:31 is a clone designated herein as "DNA58130".
Figure 32 shows the amino acid sequence (SEQ ID N0:32) derived from the coding
sequence of SEQ ID
N0:31 shown in Figure 31.
Detailed Description of the Invention
I. Definitions
The phrases "cardiovascular. endothelial and angiogenic disorder",
"cardiovascular, endothelial and
angiogenic dysfunction", "cardiovascular, endothelial or angiogenic disorder"
and "cardiovascular, endothelial or
angiogenic dysfunction" are used interchangeably and refer in part to systemic
disorders that affect vessels, such
as diabetes mellitus, as well as diseases of the vessels themselves, such as
of the arteries, capillaries, veins, and/or
lymphatics. This would include indications that stimulate angiogenesis and/or
cardiovascularization, and those that
inhibit angiogenesis and/or cardiovascularization. Such disorders include, for
example, arterial disease, such as
atherosclerosis, hypertension, inflammatory vasculitides, Reynaud's disease
and Reynaud's phenomenon, aneurysms.
and arterial restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema;
and other vascular disorders such as peripheral vascular disease, cancer such
as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, heman~~ioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphan'zioma, and
lymphangiosarcoma, tumor
angiogenesis, trauma such as wounds, burns, and other injured tissue, implant
fixation, scarring, ischemia
reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal
diseases such as acute renal failure, and
WO 00/53757 CA 02361849 2001-07-30 pCT~S00/05004
osteoporosis. This would also include angina, myocardial infarctions such as
acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as CHF.
"Hypertrophy", as used herein, is defined as an increase in mass of an organ
or structure independent of
natural growth that does not involve tumor formation. Hypertrophy of an organ
or tissue is due either to an increase
in the mass of the individual cells (true hypertrophy), or to an increase in
the number of cells making up the tissue
(hyperplasia), or both. Certain organs, such as the heart, lose the ability to
divide shortly after birth. Accordingly,
"cardiac hypertrophy" is defined as an increase in mass of the heart, which,
in adults, is characterized by an increase
in myocyte cell size and contractile protein content without concomitant cell
division. The character of the stress
responsible for inciting the hypertrophy, (e.g., increased preload, increased
afterload, loss of myocytes, as in
myocardial infarction, or primary depression of contractility), appears to
play a critical role in determining the nature
of the response. The early stage of cardiac hypertrophy is usually
characterized morphologically by increases in
the size of myofibrils and mitochondria, as well as by enlargement of
mitochondria and nuclei. At this stage, while
muscle cells are larger than normal, cellular organization is largely
preserved. At a more advanced stage of cardiac
hypertrophy, there are preferential increases in the size or number of
specific organelles, such as mitochondria, and
new contractile elements are added in localized areas of the cells, in an
irregular manner. Cells subjected to long-
standing hypertrophy show more obvious disruptions in cellular organization,
including markedly enlarged nuclei
with highly lobulated membranes, which displace adjacent myofibrils and cause
breakdown of normal Z-band
registration. The phrase "cardiac hypertrophy" is used to include all stages
of the progression of this condition,
characterized by various degrees of structural damage of the heart muscle,
regardless of the underlying cardiac
disorder. Hence, the term also includes physiological conditions instrumental
in the development of cardiac
hypertrophy, such as elevated blood pressure, aortic stenosis, or myocardial
infarction.
"Heart failure" refers to an abnormality of cardiac function where the heart
does not pump blood at the rate
needed for the requirements of metabolizing tissues. The heart failure can be
caused by a number of factors,
including ischemic, congenital, rheumatic, or idiopathic forms.
"Congestive heart failure" (CHF) is a progressive pathologic state where the
heart is increasingly unable to
supply adequate cardiac output (the volume of blood pumped by the heart over
time) to deliver the oxygenated blood
to peripheral tissues. As CHF progresses, structural and hemodynamic damages
occur. While these damages have
a variety of manifestations, one characteristic symptom is ventricular
hypertrophy. CHF is a common end result
of a number of various cardiac disorders.
"Myocardial infarction" generally results from atherosclerosis of the coronary
arteries, often with
superimposed coronary thrombosis. It may be divided into ovo major types:
transmural infarcts, in which
myocardial necrosis involves the full thickness of the ventricular wall, and
subendocardial (nontransmural) infarcts,
in which the necrosis involves the subendocardium, the intramural myocardium,
or both, without extending all the
way through the ventricular wall to the epicardium. Myocardial infarction is
known to cause both a change in
hemodynamic effects and an alteration in structure in the damaged and healthy
zones of the heart. Thus, for
example, myocardial infarction reduces the maximum cardiac output and the
stroke volume of the heart. Also
associated with myocardial infarction is a stimulation of the DNA synthesis
occurring in the interstice as well as
an increase in the formation of collagen in the areas of the heart not
affected.
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
As a result of the increased stress or strain placed on the heart in prolonged
hypertension due, for example,
to the increased total peripheral resistance, cardiac hypertrophy has long
been associated with "hypertension". A
characteristic of the ventricle that becomes hypertrophic as a result of
chronic pressure overload is an impaired
diastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506 (
1984); Smith et al., J. Am. Coll. Cardiol.,
5: 869-874 ( 1985). A prolonged left ventricular relaxation has been detected
in early essential hypertension, in spite
of normal or supranormal systolic function. Hartford et al., Hypertension, 6:
329-338 (1984). However, there is
no close parallelism between blood pressure levels and cardiac hypertrophy.
Although improvement in left
ventricular function in response to antihypertensive therapy has been reported
in humans, patients variously treated
with a diuretic (hydrochlorothiazide), a (3-Mocker (propranolol), or a calcium
channel Mocker (diltiazem), have
shown reversal of left ventricular hypertrophy, without improvement in
diastolic function. Inouye et al., Am. J.
Cardiol., 53: 1583-7 (1984).
Another complex cardiac disease associated with cardiac hypertrophy is
"hypertrophic cardiomyopathy". This
condition is characterized by a great diversity of morphologic, functional,
and clinical features (Maron et al., N.
En~l. J. Med., 316: 780-789 ( 1987); Spirito etal., N. Enal. J. Med., 320: 749-
755 ( 1989); Louie and Edwards, Prop.
Cardiovasc. Dis., 36: 275-308 (1994); Wigle etal., Circulation, 92: 1680-1692
(1995)), the heterogeneity of which
is accentuated by the fact that it afflicts patients of all ages. Spirito et
al., N. Enal. J. Med., 336: 775-785 ( 1997).
The causative factors of hypertrophic cardiomyopathy are also diverse and
little understood. In general, mutations
in genes encoding sarcomeric proteins are associated with hypertrophic
cardiomyopathy. Recent data suggest that
(3-myosin heavy chain mutations may account for approximately 30 to 40 percent
of cases of familial hypertrophic
cardiomyopathy. Watkins etal., N. Enal. J. Med., 326: 1108-1114 (1992);
Schwartz etal, Circulation, 91: 532-540
(1995); Marian and Roberts, Circulation, 92: 1336-1347 (1995); Thierfelder et
al., Cell, 77: 701-712 (1994);
Watkins et al., Nat. Gen., 11: 434-437 ( 1995). Besides (3-myosin heavy chain,
other locations of genetic mutations
include cardiac troponin T, alpha topomyosin, cardiac myosin binding protein
C, essential myosin light chain, and
regulatory myosin light chain. See, Malik and Watkins, Curr. O~n. Cardiol.,
12: 295-302 (1997).
Supravalvular "aortic stenosis'' is an inherited vascular disorder
characterized by narrowing of the ascending
aorta, but other arteries, including the pulmonary arteries, may also be
affected. Untreated aortic stenosis may lead
to increased intracardiac pressure resulting in myocardial hypertrophy and
eventually heart failure and death. The
pathogenesis of this disorder is not fully understood, but hypertrophy and
possibly hyperplasia of medial smooth
muscle are prominent features of this disorder. It has been reported that
molecular variants of the elastin gene are
involved in the development and pathogenesis of aortic stenosis. U.S. Patent
No. 5,650,282 issued July 22, 1997.
''Valvular regurgitation" occurs as a result of heart diseases resulting in
disorders of the cardiac valves.
Various diseases, like rheumatic fever, can cause the shrinking or pulling
apart of the valve orifice, while other
diseases may result in endocarditis, an inflammation of the endocardium or
lining membrane of the atrioventricular
orifices and operation of the heart. Defects such as the narrowing of the
valve stenosis or the defective closing of
the valve result in an accumulation of blood in the heart cavity or
regurgitation of blood past the valve. If
uncorrected, prolonged valvular stenosis or insufficiency may result in
cardiac hypertrophy and associated damage
to the heart muscle, which may eventually necessitate valve replacement.
The treatment of all these, and other cardiovascular, endothelial and
angiogenic disorders, which may or may
WO 00/53757 CA 02361849 2001-07-30 pCT/[jS00/05004
not be accompanied by cardiac hypertrophy, is encompassed by the present
invention.
The terms "cancer", "cancerous", and "malignant" refer to or describe the
physiological condition in mammals
that is typically characterized by unregulated cell growth. Examples of cancer
include but are not limited to,
carcinoma including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and
leukemia. More particular
examples of such cancers include squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer,
gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic
cancer, glioblastoma, cervical cancer,
ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder
cancer, breast cancer, colon cancer,
colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney
cancer such as renal cell carcinoma and
Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer,
thyroid cancer, testicular cancer,
esophageal cancer, and various types of head and neck cancer. The preferred
cancers for treatment herein are
breast, colon, lung, melanoma, ovarian, and others involving vascular tumors
as noted above.
The term "cytotoxic agent" as used herein refers to a substance that inhibits
or prevents the function of cells
and/or causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., '3'I, ''-5I, y°Y, and
issRe), chemotherapeutic agents, and toxins such as enzymatically active
toxins of bacterial, fungal, plant, or animal
origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of
cancer. Examples of
chemotherapeutic agents include alkylating agents, folic acid antagonists,
anti-metabolites of nucleic acid
metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine
nucleosides, amines, amino acids,
triazol nucleosides, or corticosteroids. Specific examples include Adriamycin,
Doxorubicin, 5-Fluorouracil,
Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,
Taxol, Toxotere, Methotrexate,
Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin
C, Mitoxantrone, Vincreistine,
Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin.
Dactinomycin, Mitomycins,
Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan, and other related
nitrogen mustards. Also included in this
definition are hormonal agents that act to regulate or inhibit hormone action
on tumors, such as tamoxifen and
onapristone.
A "growth-inhibitory agent" when used herein refers to a compound or
composition that inhibits growth of
a cell, such as an Wnt-overexpressing cancer cell, either in vitro or in-
vivo. Thus, the growth-inhibitory agent is
one which significantly reduces the percentage of malignant cells in S phase.
Examples of growth-inhibitory agents
include agents that block cell cycle progression (at a place other than S
phase), such as agents that induce G 1 arrest
and M-phase arrest. Classical M-phase Mockers include the vincas (vincristine
and vinblastine), taxol, and topo
II inhibitors such as doxorubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G 1 also spill
over into S-phase arrest, for example, DNA alkylating agents such as
tamoxiten, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled
"Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami etal. (WB Saunders: Philadelphia, 1995),
especially p. 13. Additional examples
include tumor necrosis factor (TNF). an antibody capable of inhibiting or
neutralizin~~ the an~_iogenic activity of
acidic or basic FGF or hepatocyte growth factor (HGF), an antibody capable of
inhibiting or neutralizing the
coagulant activities of tissue factor, protein C, or protein S (gee, WO 91
/01753, published 21 February 1991 ), or
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WO 00/53757 CA 02361849 2001-07-30 pC'T/[JS00/05004
an antibody capable of binding to HER2 receptor (WO 89/06692), such as the 4D5
antibody (and functional
equivalents thereof) (e.g., WO 92/22653).
"Treatment" is an intervention performed with the intention of preventing the
development or altering the
pathology of a cardiovascular, endothelial, and angiogenic disorder. The
concept of treatment is used in the
broadest sense, and specifically includes the prevention (prophylaxis),
moderation, reduction, and curing of
cardiovascular, endothelial, and angiogenic disorders of any stage.
Accordingly, "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down
(lessen) a cardiovascular, endothelial, and angiogenic disorder such as
hypertrophy. Those in need of treatment
include those already with the disorder as well as those prone to have the
disorder or those in whom the disorder
is to be prevented. The disorder may result from any cause, including
idiopathic, cardiotrophic, or myotrophic
causes, or ischemia or ischemic insults, such as myocardial infarction.
"Chronic" administration refers to administration of the agents) in a
continuous mode as opposed to an acute
mode, so as to maintain the initial effect, such as an anti-hypertrophic
effect, for an extended period of time.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans, domestic
and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats,
cows, sheep, pigs, etc. Preferably, the
mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
The phrase "cardiovascular, endothelial or angiogenic agents" refers
generically to any drug that acts in
treating cardiovascular, endothelial, and angiogenic disorders. Examples of
cardiovascular agents are those that
promote vascular homeostasis by modulating blood pressure, heart rate, heart
contractility, and endothelial and
smooth muscle biology, all of which factors have a role in cardiovascular
disease. Specific examples of these
include angiotensin-II receptor antagonists; endothelin receptor antagonists
such as, for example, BOSENTANT"'
and MOXONODINT"'; interferon-gamma (IFN-y); des-aspartate-angiotensin I;
thrombolytic agents, e.n.,
streptokinase, urokinase, t-PA, and a t-PA variant specifically designed to
have longer half-life and very high fibrin
specificity, TNK t-PA (a T103N, N117Q, KHRR(296-299)AAAA t-PA variant, Keyt et
al., Proc. Natl. Acad. Sci.
USA, 91: 3670-3674 (1994)); inotropic or hypertensive agents such as
digoxigenin and (3-adrenergic receptor
blocking agents, e.g., propranolol, timolol, tertalolol, carteolol, nadolol,
betaxolol, penbutolol, acetobutolol,
atenolol, metoprolol, and carvedilol; angiotensin converting enzyme (ACE)
inhibitors, e.g., quinapril, captopril,
enalapril, ramiprii, benazepril, fosinopril, and lisinopril; diuretics, e.g.,
chlorothiazide, hydrochlorothiazide,
hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide,
acetazolamide, and indapamide; and
calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, nicardipine.
One preferred category of this type
is a therapeutic agent used for the treatment of cardiac hypertrophy or of a
physiological condition instrumental in
the development of cardiac hypertrophy, such as elevated blood pressure.
aortic stenosis, or myocardial infarction.
"Angiogenic agents" and "endothelial agents" are active agents that promote
angiogenesis and/or endothelial
cell growth, or, if applicable, vasculogenesis. This would include factors
that accelerate wound healing, such as
growth hormone, insulin-like growth factor-I (IGF-I), VEGF, VIGF, PDGF,
epidermal ,rowth factor (EGF), CTGF
and members of its family, FGF, and TGF-a and TGF-~3.
24
WU 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
"Angiostatic agents" are active agents that inhibit angiogenesis or
vasculogenesis or otherwise inhibit or
prevent growth of cancer cells. Examples include antibodies or other
antagonists to angiogenic agents as defined
above, such as antibodies to VEGF. They additionally include cytotherapeutic
agents such as cytotoxic agents,
chemotherapeutic agents, growth-inhibitory agents, apoptotic agents, and other
agents to treat cancer, such as anti-
s HER-2, anti-CD20, and other bioactive and organic chemical agents.
In a pharmacological sense, in the context of the present invention, a
"therapeutically effective amount" of
an active agent such as a PRO polypeptide or agonist or antagonist thereto or
an anti-PRO antibody, refers to an
amount effective in the treatment of a cardiovascular, endothelial or
angiogenic disorder in a mammal and can be
determined empirically.
As used herein, an "effective amount" of an active agent such as a PRO
polypeptide or agonist or antagonist
thereto or an anti-PRO antibody, refers to an amount effective for carrying
out a stated purpose, wherein such
amounts may be determined empirically for the desired effect.
The terms "PRO polypeptide" and "PRO" as used herein and when immediately
followed by a numerical
designation refer to various polypeptides, wherein the complete designation
(i.e., PRO/number) refers to specific
polypeptide sequences as described herein. The terms "PRO/number polypeptide"
and "PRO/number" wherein the
term "number" is provided as an actual numerical designation as used herein
encompass native sequence
polypeptides and polypeptide variants (which are further defined herein). The
PRO polypeptides described herein
may be isolated from a variety of sources, such as from human tissue types or
from another source, or prepared by
recombinant or synthetic methods.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequence as the
corresponding PRO polypeptide derived from nature. Such native sequence PRO
polypeptides can be isolated from
nature or can be produced by recombinant or synthetic means. The term "native
sequence PRO polypeptide"
specifically encompasses naturally-occurring truncated or secreted forms of
the specific PRO polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms (e.g.,
alternatively spliced forms) and
naturally-occurring allelic variants of thepolypeptide. In various embodiments
of the invention, the native sequence
PRO polypeptides disclosed herein are mature or full-length native sequence
polypeptides comprising the full-length
amino acids sequences shown in the accompanying figures. Start and stop codons
are shown in bold font and
underlined in the figures. However, while the PRO polypeptide disclosed in the
accompanying figures are shown
to begin with methionine residues designated herein as amino acid position 1
in the figures, it is conceivable and
possible that other methionine residues located either upstream or downstream
from the amino acid position 1 in
the figures may be employed as the starting amino acid residue for the PRO
polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the
PRO polypeptide which is
essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a
PRO polypeptide ECD will have less
than 1 ~/o of such transmembrane and/or cytoplasmic domains and preferably,
will have less than 0.5 ~~ of such
domains. It will be understood that any transmembrane domains identified for
the PRO polypeptides of the present
invention are identified pursuant to criteria routinely employed in the art
for identifying that type of hydrophobic
domain. The exact boundaries of a transmembrane domain may vary but most
likely by no more than about 5 amino
acids at either end of the domain as initially identified herein. Optionally,
therefore, an extracellular domain of a
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
PRO polypeptide may contain from about ~ or fewer amino acids on either side
of the transmembrane
domain/extracellular domain boundary as identified in the Examples or
specification and such polypeptides, with
or without the associated signal peptide, and nucleic acid encoding them, are
contemplated by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are shown
in the present specification and/or the accompanying figures. It is noted,
however, that the C-terminal boundary
of a signal peptide may vary, but most likely by no more than about 5 amino
acids on either side of the signal
peptide C-terminal boundary as initially identified herein, wherein the C-
terminal boundary of the signal peptide
may be identified pursuant to criteria routinely employed in the art for
identifying that type of amino acid sequence
element (e.g., Nielsen et al., Prot. Ena., 10:1-6 (1997) and von Heinje et
al., Nucl. Acids Res., 14:4683-4690
(1986)). Moreover, it is also recognized that, in some cases, cleavage of a
signal sequence from a secreted
polypeptide is not entirely uniform, resulting in more than one secreted
species. These mature polypeptides, where
the signal peptide is cleaved within no more than about 5 amino acids on
either side of the C-terminal boundary of
the signal peptide as identified herein, and the polynucleotides encoding
them, are contemplated by the present
invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or
below having at least about
80% amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein,
a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO
polypeptide, with or without the signal peptide, as disclosed herein or any
other fragment of a full-length PRO
polypeptide sequence as disclosed herein. Such PRO polypeptide variants
include, for instance, PRO polypeptides
wherein one or more amino acid residues are added, or deleted, at the N- or C-
terminus of the full-length native
amino acid sequence. Ordinarily, a PRO polypeptide variant will have at least
about 80% amino acid sequence
identity, alternatively at least about 81 % amino acid sequence identity,
alternatively at least about 82% amino acid
sequence identity, alternatively at least about 83% amino acid sequence
identity, alternatively at least about 84%
amino acid sequence identity, alternatively at least about 85% amino acid
sequence identity, alternatively at least
about 86% amino acid sequence identity, alternatively at least about 87% amino
acid sequence identity. alternatively
at least about 88% amino acid sequence identity, alternatively at least about
89% amino acid sequence ide-ntity,
alternatively at least about 90% amino acid sequence identity, alternatively
at least about 91 % amino acid sequence
identity, alternatively at least about 92% amino acid sequence identity,
alternatively at least about 93% amino acid
sequence identity, alternatively at least about 94% amino acid sequence
identity, alternatively at least about 95%
amino acid sequence identity, alternatively at least about 96% amino acid
sequence identity, alternatively at least
about 97% amino acid sequence identity, alternatively at least about 98% amino
acid sequence identity and
alternatively at least about 99% amino acid sequence identity to a full-length
native sequence PRO polypeptide
sequence as disclosed herein, a PRO polypeptide sequence lacking the signal
peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the signal peptide,
as disclosed herein or any other
specifically defined fragment of a full-length PRO polypeptide sequence as
disclosed herein. Ordinarily, PRO
variant polypeptides are at least about 10 amino acids in length,
alternatively at least about 20 amino acids in length,
alternatively at least about 30 amino acids in length, alternatively at least
about 40 amino acids in length,
alternatively at least about 50 amino acids in length, alternatively at least
about 60 amino acids in length,
26
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
alternatively at least about 70 amino acids in length, alternatively at least
about 80 amino acids in length,
alternatively at least about 90 amino acids in length, alternatively at least
about 100 amino acids in length,
alternatively at least about 150 amino acids in length, alternatively at least
about 200 amino acids in length,
alternatively at least about 300 amino acids in length, or more.
As shown below, Table 1 provides the complete source code for the ALIGN-2
sequence comparison computer
program. This source code may be routinely compiled for use on a UNIX
operating system to provide the ALIGN-2
sequence comparison computer program.
In addition, Tables 2A-2D show hypothetical exemplifications for using the
below described method to
determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid
sequence identity (Tables 2C-2D)
using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid sequence
of a hypothetical PRO polypeptide of interest, "Comparison Protein" represents
the amino acid sequence of a
polypeptide against which the "PRO" polypeptide of interest is being compared,
"PRO-DNA" represents a
hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA"
represents the nucleotide
sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid
molecule of interest is being
compared, "X", "Y", and "Z" each represent different hypothetical amino acid
residues and "N", "L" and "V" each
represent different hypothetical nucleotides.
27
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/*
Table 1
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is M; stop-stop = 0; J (joker) match = 0
*/
#define M -8 /* value of a match with a stop */
int day[26][26] _ {
/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z */
/* A */ { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, O, M, 1, 0,-2, I, 1, 0, 0,-6,
0,-3, 0},
/* B */ { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2, M,-1, 1, 0, 0, 0, 0,-2,-5,
0,-3, 1},
/* C */ {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4, M,-3,-5,-4, 0,-2, 0,-2,-8,
0, 0,-5},
/* D */ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 2},
/* E *J { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 3},
/* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-5,-4,-3,-3, 0,-1, 0,
0, 7,-5},
/* G */ { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-1,-3, 1, 0, 0,-1,-7,
0,-5, 0},
/* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2,-1,-1, 0,-2,-3,
0, 0, 2},
/* I */ {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2,_M,-2,-2,-2,-1, 0, 0, 4,-5,
0,-I,-2},
/* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, M, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0},
/* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, M,-1, 1, 3, 0, 0, 0,-2,-3,
0,-4, 0},
/* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3, M,-3,-2,-3,-3,-1, 0, 2,-2,
0,-I,-2},
/* M */ {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1, 0, 2,-4,
0,-2,-1},
/* N */ { 0, 2,-4, 2, I,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0, 1, 0, 0,-2,-4,
0,-2, 1},
/* O */ { M,_M, M, M,_M,_M, M,_M,_M,_M,_M, M,_M,_M, O, M, M, M,
M,_M,_M,_M,_M,_M,_M,_M},
/* P */ { 1,-1,-3,-1,-I,-5,-I, 0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0, 1, 0, 0,-1,-6,
0,-5, 0},
/* Q */ { 0, 1,-5, 2, 2,-S,-1, 3,-2, 0, 1,-2,-1, 1, M, 0, 4, 1,-1,-1, 0,-2,-5,
0,-4, 3},
/* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, O, M, 0, 1, 6, 0,-1, 0,-2, 2,
0,-4, 0},
/* S */ { I, 0, 0, 0, 0,-3, I,-I,-I, 0, 0,-3,-2, 1, M, 1,-1, 0, 2, 1, 0,-1,-2,
0,-3, 0},
/* T */ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, M, 0,-1,-1, 1, 3, 0, 0,-5,
0,-3, 0},
/*U*/ {0,0,0,0,0,0,0,0,0,0,0,0,O,O, M,0,0,0,0,0,0,0,0,0,0,0},
/* V */ { 0,-2,-2,-2,-2,-I,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1, 0, 0, 4,-6,
0,-2,-2},
/* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4, M,-6,-5, 2,-2,-5, 0,-6,17,
0, 0,-6},
/* x */ { o, o, o, o, o, o, o, o, o, o, o, o, o, o, M, o, o, o, o, o, o, o, o,
o, o, o},
/* Y */ {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,-4},
/* Z */ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1, M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4}
};
Page 1 of day.h
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/*
*/
#include < stdio.
h >
#include < ctype.
h >
#define MAXJMP 16 /* max jumps in a diag */
#define MAXGAP 24 /* don't continue to penalize gaps larger
than this */
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases
since last jmp */
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap */
#define DINSl 1 /* penalty per base */
#de6ne PINSO 8 /* penalty for a gap */
#define PINS1 4 /* penalty per residue */
struct jmp {
short n[MAXJMP]; /* size of jmp (neg for dely) */
unsigned short x[MAXJMP]; /* base no. of jmp in seq x */
}; /* limits seq to 2''16 -1 */
struct diag
{
int score; /* score at last jmp */
long offset;/* offset of prey block */
short ijmp;/* current jmp index */
struct jmp jp; /* list of jmps */
};
struct path
{
int spc; /*
number
of
leading
spaces
*/
short n[JMPS]; /* size of jmp (gap) */
int x[JMPS]; /* loc of jmp (last elem before gap) */
};
char *ofile; /* output file name */
char *namex[2]; /* seq names: getseqsQ */
char *prog; /* prog name for err msgs */
char *seqx[2]; /* seqs: getseqs(1 */
int dmax; /* best diag: nwQ *!
int dmax0; /* tlnal diag */
int dna; /* set if dna: main() */
int endgaps; /* set if penalizing end gaps */
int gapx, gapy; /* total gaps in seqs */
int len0, lenl; /* seq lens */
int ngapx, ngapy; 1* total size of gaps */
int smax; /* max score: nw() */
int *xbm; /* bitmap for matching */
long offset; /* current offset in jmp file */
struct diag /* holds diagonals *!
*dx;
struct path /* holds path for seqs *!
pp[2];
char *callocQ, , , *strcpyQ;
*mallocQ *indexQ
char *getseqQ,
*g callocQ;
Page 1 of nw.h
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/* Needleman-Wunsch alignment program
* usage: progs filel filet
* where filel and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with '; ' >' or ' <' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
#include "nw.h"
#include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
static pbval[26] _ {
I, 2~(1 < <('D'-'A'))~(1 < <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1«15, 1«16, I«17, 1«18, 1«19, 1«20, 1«21, I«22,
1«23, 1«24, 1«25(1«('E'-'A'))~(1«('Q'-'A'))
};
main(ac, av) main
int ac;
char *av[];
{
prop = av[0];
if (ac ! = 3) {
fprintf(stderr,"usage: %s filel filet\n", prog);
fprintf(stderr,"where filel and filet are two dna or two protein
sequences.\n");
fprintf(stderr, "The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr,"Output is in the file \"align.out\"\n");
exit(1);
{
namex[0] = av(1];
namex[1] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : ~bval;
endgaps = 0; /* I to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; /* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
print(); /* print scats, alignment */
cleanup(0); /* unlink any tmp files */
Page 1 of nw.c
WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
/* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*/
nw() riW
{
char *px, * py; /* seqs and ptrs */
int *ndely, /* keep track of dely */
*dely;
int ndelx, /* keep track of delx */
delx;
int *tmp; /* for swapping row0, rowl */
int mis; /* score for each type */
int ins0, insl;/* insertion penalties */
registerid; /* diagonal index */
registerij; /* jmp index */
register*col0, *coll; /* score for curr, last row */
registerxx, yy;
/* index
into seqs
*1
dx = (struct diag *)g calloc("to get diags", IenO+lenl+1, sizeof(struct
diag));
ndely = (int *)g calloc("to get ndely", lenl + l, sizeof(int));
dely = (int *)g calloc("to get dely", lenl+l, sizeof(int));
col0 = (int *)g calloc("to get col0", lenl+l, sizeof(int));
col l = (int *)g calloc("to get coi l ", lenl + 1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
ins 1 = (dna)? DINS 1 : PINS 1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[0] _ -ins0, yy = l; yy < = lenl; yy++) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
col0[0] = 0; /* Waterman Bull Math Biol 84 */
else
for (yy = 1; yy < = lent; yy++)
dely[yy] _ -ins0;
/* fill in match matrix
*/
for (px = seqx[0], xx = 1; xx < = IenO; px++, xx++) {
l* initialize first entry in col
*/
if (endgaps) {
if (xx == 1)
coil[0) = delx = -(ins0+insl);
else
coil[0] = deli = col0(0] - insl;
ndelx = xx;
else {
coil[0] = 0;
delx = -ins0;
ndelx = 0;
Page 2 of nw.c
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for (py = seqx(1], yy = l; yy < = lent; py++, yy++) {
mis = col0[yy-1];
if (dna)
mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis += day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
*/
if (endgaps ~ ~ ndely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = dely[yy]) {
dely[yy] = col0[yy] - (ins0+insl);
ndely[yy] = 1;
} else {
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (col0[yy] - (ins0+insl) > = dely[yy]) {
dely[yy) = col0[yy] - (ins0+insl);
ndely[yy] = 1;
} else
}
ndely[yy] + +;
/* update penalty for del in y seq;
* favor new del over ongong del
*/
if (endgaps ~ ~ ndelx < MAXGAP) {
if (coil[yy-1] - ins0 > = delx) {
delx = coil[yy-1] - (ins0+insl);
ndelx = 1;
} else {
delx - = insl;
ndelx+ +;
}
} else {
if (coil[yy-1] - (ins0+insl) > = delx) {
delx = colt[yy-1] - (ins0+insl);
ndelx = l;
} else
ndelx+ +;
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
*/
...nw
Page 3 of nw.c
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id=xx-yy+lenl-1;
if (mis > = delx && mis > = dely[yy])
tol l [yy] = mis;
else if (delx > = dely[yy]) {
tol l [yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ( ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
else {
toll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ( ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] _ -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
if (xx == len0 && yy < lent) {
/* last col
*/
if (endgaps)
toll[yy] -= ins0+insl*(lenl-yy);
if (col l [yy] > smax) {
smax = toll[yy];
dmax = id;
if (endgaps && xx < IenO)
coil[yy-1] -= ins0+insl*(len0-xx);
if (toll[yy-1] > smax) {
smax = toll[yy-1];
dmax = id;
tmp = col0; col0 = coi l ; coi l = tmp;
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll);
...nw
Page 4 of nw.c
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/*
* print() -- only routine visible outside this module
* static:
* getmatQ -- trace back best path, count matches: print()
* pr align() -- print alignment of described in array p[]: print()
* dumpblock() -- dump a block of lines with numbers, stars: pr align()
* numsQ -- put out a number line: dumpblock()
* putlineQ -- put out a line (name, [num], seq, [num]): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* stripnameQ -- strip any path and prefix from a seqname
*/
#include "nw.h"
#define SPC 3
#define P LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq */
extern day[26][26J;
int olen; /* set output line length */
FILE *fx; /* output file */
print
print()
{
int Ix, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(1);
fprintf(fx, " < first sequence: % s (length = %d)\n", namex[0], len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], lenl);
olen = 60;
lx = len0;
ly = lent;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[0].spc = tirstgap = lent - dmax - 1;
ly -= pp[0].spc;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lenl - 1);
Ix -= pp[1].spc;
if (dmax0 < len0 - 1) { /* trailing gap in x */
lastgap = len0 - dmax0 -1;
lx -= lastgap;
else if (dmax0 > IenO - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - 1);
ly -= lastgap;
getmat((x, ly, firstgap, lastgap);
pr alignQ;
Page 1 of nwprint.c
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/*
* trace back the best path, count matches
*/
static
getmat(Ix, ly, firstgap, lastgap) getrilat
int lx, ly; /* "core" (minus endgaps) *l
int firstgap, lastgap; /* leading trailing overlap */
{
int nm, i0, il, siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *p 1;
/* get total matches, score
*/
i0 = il = siz0 = sizl = 0;
p0 = seqx[0] + pp[1].spc;
pl = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + 1;
nm = 0;
while ( *p0 && *pl ) {
if (siz0) {
pl++;
nl++;
siz0--;
else if (sizl) {
p0++;
n0++;
sizl--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
run++;
if (n0++ _= pp[0].x[i0])
siz0 = pp[0].n[i0++];
if (nl++ _= pp(1].x[il))
sizl = pp[1).n(il++];
p0++;
pl++;
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
*/
if (endgaps)
lx = (len0 < lenl)? len0 : lenl;
else
Ix = (lx < ly)? lx : ly;
pct = 100.*(double)nm/(double)Ix;
fprintf(fx, "\n");
fprintf(fx, "< %d match%s in an overlap of %od: %.2f percent similarity\n",
nm, (nm == 1)~ "' . "es", lx, pct);
Pale 2 of nwprint.c
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fprintf(fx, " < gaps in first sequence: %d", gapx); ...getrilat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx = = I)? "':"s");
fprintf(fx,"%s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapy) {
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dna)? "base" : "residue", (ngapy = = 1 )? "' : "s");
fprintf(fx,"%s", outx);
if (dna)
fprintf(fx,
"\n < score: %d (match = % d, mismatch = % d, gap penalty = % d + % d per
base)\n",
smax, DMAT, DMIS, DINSO, DINSI);
else
fprintf(fx,
"\n< score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
"<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
firstgap, (dna)? "base" : "residue", (firstgap == I)? "' . "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "' . "s");
else
fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core
-- for checking */
static lmax; /* lengths of stripped
file names */
static ij[2]; /* jmp index for a
path */
static nc[2]; /* number at start
of current line */
static ni[2]; /* current elem number
-- for gapping */
static siz[2];
static *ps[2]; /* ptr to current element
char */
static *po[2]; /* ptr to next output
char char slot *!
static out[2][P /* output line */
char LINE];
static star[P LINE];/* set by stars() */
char
/*
* print alignment of described in struct path pp[]
*/
static
pr align
pr align()
{
int nn; /* char count */
int more;
register i;
for (i = 0, lmax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > Imax)
lmax = nn;
nc[i] = 1;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i];
Page 3 of nwprint.c
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
for (nn = nm = 0, more = 1; more; ) { ...pr align
for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence'?
*/
if (!*ps[i])
continue;
more+ +;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ,
pp[i] . spc--;
else if (siz[i]) { /* in a gap */
*po[i]++ _ ,
siz[i]__~
else { /* we're putting a seq element
*/
*Po[il _ *Ps[il;
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i] + +;
/*
* are we at next gap for this seq?
*/
if (ni[i] _= pp[i].x[ij[i]]) {
/*
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _= pp[i].x[ij[ill)
siz[i] += PP[il.n[iJ[il++l;
ni[i)++;
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
po[i] = out[i];
nn = 0;
/*
* dump a block of lines, including numbers, stars: pr align()
*/
static
dumpblockQ ClllrilpblOCli
{
register i;
for (i = 0; i < 2; i++)
*po[i]__ _ '\0';
Page 4 of nwprint.c
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
... dumpblock
(void) putc('\n', fx);
for (i = 0; i < 2; i++) {
if (*out[i] && (*out[i] ! _ ' ' ~ ~ *(po[i]) ! _ ' ')) {
if (i = = 0)
nums(i);
if (i == 0 && *out[1])
starsQ;
putline(i);
if (i == 0 && *out[1])
fprintf(fx, star);
if (i == 1)
nums(i);
/*
* put out a number line: dumpblock()
*/
static
nums(ix) nums
int ix; /* index in out[] holding seq line */
{
char mine[P LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i = 0; i < Imax+P SPC; i++, pn++)
*pn =
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if (*py =- ' ' ~ ~ *PY =_ -')
*Pn = >
else {
if (i% 10 == 0 ~ ~ (i == 1 && nc[ix] != 1)) {
j = (i < 0)? _i : i;
for (px = pn; j; j /= 10, px--)
*px=j%10+'0';
if (i < 0)
*Px = ,
else
*Pn = , ,
~++;
*Pn = '\0';
nc[ix] = i;
for (pn = mine; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
/*
* put our a line (name, [num], seq, [num]): dumpblockQ
*/
static
putline(ix) puthrie
int ix;
{
Page 5 of nwprint.c
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
... putline
int i;
register char *px;
for (px = namex(ix], i = 0; *px && *px ! _ ':'; px++, i++)
(void) putt(*px, fx);
for (; i < Imax+P SPC; i++)
(void) putt(' ', fx);
/* these count from 1:
* ni(] is current element (from 1)
* nc[] is number at start of current line
*/
for (px = out[ix]; *px; px++)
(void) putt(*px&Ox7F, fx);
(void) putt('\n', fx);
/*
* put a line of stars (seqs always in out[0], out(1]): dumpblockQ
*/
static
stars() stars
int i;
register char *p0, *pl, cx, *px;
if (!*out[0] ~ ~ (*out[0] _- ' && *(po[0]) _- ' ') ~ ~
!*out[1] ~ ~ l*out[1] _- ' ' && *(po[1]) _- ' '))
return;
px = star;
for (i = lmax+P SPC; i; i--)
*px++ _ ' ,
for (p0 = out[0], pl = out[1]; *p0 && *pl; p0++, pl++) {
if (isalpha(*p0) && isalpha(*pl)) {
if (xbm[*p0-'A']&xbm[*p1-'A']) {
cx = '*';
nm++:
else if (!dna && day[*p0-'A'][*pl-'A'] > 0)
cx = . ,
else
cx = '
else
cx=' ,
*px++ = cx;
*px++ _ '\n';
*Px = '\0';
Page 6 of nwprint. c
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WO 00/53757 CA 02361849 2001-07-30 pCT/jJS00/05004
/*
* strip path or prefix from pn, return len: pr align()
*1
static
stripname(pn) stripname
char *pn; /* file name (may be path) */
f
register char *px, *py;
PY = ~>
for (px = pn; *px; px++)
if (*px =- '/')
py=px+1;
if (py)
(void) strcpy(pn, py);
return(strlen(pn));
Page 7 of nwprint.c
WO 00/53757 CA 02361849 2001-07-30 pCT~s00/05004
/*
* cleanup() -- cleanup any tmp file
* getseq() -- read in seq, set dna, len, maxlen
* g callocQ -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmpsQ -- write a filled array of jmps to a tmp file: nwQ
*/
#include "nw.h"
#include <sys/file.h>
char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */
FILE *fj;
int cleanupQ; /* cleanup tmp file */
long lseekQ;
/*
* remove any tmp file if we blow
*/
cleanup(i) cleanup
int i;
if (fj)
(void) unlink(jname);
exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; , ' <', or ' >'
* seq in upper or lower case
*/
char
getseq(file, len) getseq
char *file; /* file name */
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file, "r")) _ = 0) {
fprintf(stderr,"%s: can't read %s\n", prog, file);
exit(I);
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >'1
continue;
for (px = line; *px ! _ '\n'; px++)
if (isupper(*px) ~ ~ islower(*px))
tlen++;
if ((pseq = malloc((unsigned)(tlen+6))) _ = 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6,
tile);
exit(1);
pseq[0] = pseq[1] = pseq[2] = pseq[3] _ '\0';
Page 1 of nwsubr.c
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WO 00/53757 CA 02361849 2001-07-30 pCT/[JS00/05004
...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >')
continue;
for (px = line; *px ! _ '\n'; px++) {
if (isupper(*px))
*py + + _ *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU",*(py-1)))
natgc+ +;
*py + + _ '\0';
*pY = ~\0';
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq +4);
char
g calloc(msg, nx, sz) g Ca110C
char *msg; 1* program, calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *callocQ;
if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "%s: g callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(1);
return(px);
/*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
*/
readjmpsQ readjmps
{
int fd = -1;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open() %s\n", prog, jname);
cleanup( 1 );
for (i = i0 = il = 0, dmax0 = dmax, xx = len0; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j~ > = xx; j--)
Page 2 of nwsubr. c
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WO 00/53757 CA 02361849 2001-07-3o pCT/US00/05004
...readjmps
if (j < 0 && dx[dmax].offset && ~) {
(void) Iseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx(dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
else
break;
if (i > = JMPS) {
fprintf(stderr, "%s: too many gaps in alignment\n", prog);
cleanup(1);
if (j >=0){
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x(j];
dmax + = siz;
if (siz < 0) { /* gap in second seq */
pp[1].n[i1J = -siz;
xx + = siz;
/*id=xx-yy+lenl-1
*/
pp[lJ.x[il] = xx - dmax + lent - 1;
gapy + + ;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ~ endgaps)? -siz : MAXGAP;
il++;
else if (siz > 0) { /* gap in first seq */
pp[0].n[i0] = siz;
pp[0].x[i0] = xx;
gapx++;
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
else
break;
/* reverse the order of jmps
*/
for (j = 0, i0--; j < i0; j++, i0--) {
~ = PP[0].n~]; PP[0].n~] = PP[0].n[iOJ; PP[0].n[i0] = i;
i = PP[OJ.x[j]; PP[OJ.x[j] = PP[O].x[i0]; PP[0].x[i0] = i;
for (j = 0, il--; j < il; j++, il--) {
~ = PP[lJ.n~]; PP[1].n~] = PP[1].n[il]; PP[1].n[i1] = i;
i = PP[lJ.x~J; PP[1].xU] = PP[1].x[il]; PP[1].x[il] _- i;
if (fd > = 0)
(void) close(fd);
if (tj) {
(void) unlink(jname);
fj = 0;
offset = 0;
Page 3 of nwsubr. c
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
/*
* write a filled jmp struct offset of the prev one (if any): nwQ
*/
writejmps(ix) WrltejrilpS
int ix;
{
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp() %s\n", prog, jname);
cleanup(1);
if ((fj = fopen(jname, "w")) _ = 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit(1);
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ixJ.offset), l, fj);
Page 4 of nwsubr.c
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WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
T..L7.. ~1 A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
divided by 15 = 33.3
4~
WO 00/53757 CA 02361849 2001-07-30 pCT~S00/05004
Table 2B
PRO XXXXXXXXXX
(Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
divided by 10 = 50
46
WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
Table 2C
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
6 divided by 14 = 42.9
47
WO 00/53757 CA 02361849 2001-07-3o PCT/US00/05004
Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
4 divided by 12 = 33.3 %
48
WO 00/53757 CA 02361849 2001-07-30 pCT/LTS00/05004
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide
sequences identified herein
is defined as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid
residues in a PRO sequence, after aligning the sequences and introducing gaps,
if necessary, to achieve the
maximum percent sequence identity, and not considering any conservative
substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various
ways that are within the skill in the art, for instance, using publicly
available computer software such as BLAST,
BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the
art can determine
appropriate parameters for measuring alignment, including any algorithms
needed to achieve maximal alignment
over the full-length of the sequences being compared. For purposes herein,
however, % amino acid sequence
identity values are obtained as described below by using the sequence
comparison computer program ALIGN-2,
wherein the complete source code for the ALIGN-2 program is provided in Table
1. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc., and the source
code shown in Table 1 has been
filed with user documentation in the U.S. Copyright Office, Washington D.C.,
20559, where it is registered under
U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech,
Inc., South San Francisco, California or may be compiled from the source code
provided in Table 1. The ALIGN-2
program should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.OD. All sequence
comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given amino acid
sequence A to, with, or against
a given amino acid sequence B (which can alternatively be phrased as a given
amino acid sequence A that has or
comprises a certain % amino acid sequence identity to, with, or against a
given amino acid sequence B) is calculated
as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in B.
It will be appreciated that where the length of amino acid sequence A is not
equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of B
to A. As examples of % amino acid sequence identity calculations, Tables 2A-2B
demonstrate how to calculate
the % amino acid sequence identity of the amino acid sequence designated
"Comparison Protein" to the amino acid
sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as
described above using the ALIGN-2 sequence comparison computer program.
However, °lo amino acid sequence
identity may also be determined using the sequence comparison program NCBI-
BLAST2 (Altschul et al.. Nucleic
Acids Res., 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program
may be downloaded from
http://www.ncbi.nlm.nih.Qov. or otherwise obtained from the National Institute
of Health, Bethesda, MID. NCBI-
3S BLAST2 uses several search parameters, wherein all of those search
parameters are set to default values includin'"
for example, unmask = yes, strand = all, expected occurrences = 10, minimum
low complexity length = 1 i />, multi-
pass e-value = 0.01, constant for multi-pass = 25, dropoff for final upped
alignment = 25 and scoring matrix =
49
WO 00/$3757 CA 02361849 2001-07-30 PCT/US00/05004
BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino acid
sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which can
alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid sequence
identity to, with, or against a given amino acid sequence B) is calculated as
follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues
in B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of B
to A.
In addition, % amino acid sequence identity may also be determined using the
WU-BLAST-2 computer
program (Altschul et al., Methods in EnzvmoloQy, 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not set to default values, i.
e., the adjustable parameters, are set with
the following values: overlap span = 1, overlap fraction = 0.125, word
threshold (T) = 11, and scoring matrix =
BLOSUM62. For purposes herein, a % amino acid sequence identity value is
determined by dividing (a) the
number of matching identical amino acids residues between the amino acid
sequence of the PRO polypeptide of
interest having a sequence derived from the native PRO polypeptide and the
comparison amino acid sequence of
interest (i.e., the sequence against which the PRO polypeptide of interest is
being compared which may be a PRO
variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of
amino acid residues of the PRO
polypeptide of interest. For example, in the statement "a polypeptide
comprising an amino acid sequence A which
has or having at least 80% amino acid sequence identity to the amino acid
sequence B", the amino acid sequence
A is the comparison amino acid sequence of interest and the amino acid
sequence B is the amino acid sequence of
the PRO polypeptide of interest.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a
nucleic acid molecule which
encodes an active PRO polypeptide as defined below and which has at least
about 80% nucleic acid sequence
identity with a nucleotide acid sequence encoding a full-length native
sequence PRO polypeptide sequence as
disclosed herein, a full-length native sequence PRO polypeptide sequence
lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any
other fragment of a full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, a PRO variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
alternatively at least about 8l % nucleic
acid sequence identity, alternatively at least about 82% nucleic acid sequence
identity, alternatively at least about
83% nucleic acid sequence identity, alternatively at least about 84% nucleic
acid sequence identity, alternatively
at least about 85% nucleic acid sequence identity, alternatively at least
about 86% nucleic acid sequence identity,
alternatively at least about 87% nucleic acid sequence identity, alternatively
at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid sequence
identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 9l % nucleic acid
sequence identity, alternatively at least
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
about 92% nucleic acid sequence identity, alternatively at least about 93%
nucleic acid sequence identity,
alternatively at least about 94% nucleic acid sequence identity, alternatively
at least about 95% nucleic acid
sequence identity, alternatively at least about 96% nucleic acid sequence
identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid
sequence identity and alternatively at
least about 99% nucleic acid sequence identity with a nucleic acid sequence
encoding a full-length native sequence
PRO polypeptide sequence as disclosed herein, a full-length native sequence
PRO polypeptide sequence lacking
the signal peptide as disclosed herein, an extracellular domain of a PRO
polypeptide, with or without the signal
sequence, as disclosed herein or any other fragment of a full-length PRO
polypeptide sequence as disclosed herein.
Variants do not encompass the native nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at least about
60 nucleotides in length, alternatively at least about 90 nucleotides in
length, alternatively at least about 120
nucleotides in length, alternatively at least about 150 nucleotides in length,
alternatively at least about 180
nucleotides in length, alternatively at least about 210 nucleotides in length,
alternatively at least about 240
nucleotides in length, alternatively at least about 270 nucleotides in length,
alternatively at least about 300
nucleotides in length, alternatively at least about 450 nucleotides in length,
alternatively at least about 600
nucleotides in length, alternatively at least about 900 nucleotides in length,
or more.
"Percent (%) nucleic acid sequence identity" with respect to the PRO
polypeptide-encoding nucleic acid
sequences identified herein is defined as the percentage of nucleotides in a
candidate sequence that are identical
with the nucleotides in a PRO polypeptide-encoding nucleic acid sequence,
after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved in various
ways that are within the skill in the
art, for instance, using publicly available computer software such as BLAST,
BLAST-2, ALIGN, ALIGN-2 or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring
alignment, including any algorithms needed to achieve maximal alignment over
the full-length of the sequences
being compared. For purposes herein, however, % nucleic acid sequence identity
values are obtained as described
below by using the sequence comparison computer program ALIGN-2, wherein the
complete source code for the
ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence comparison
computer program was authored
by Genentech, Inc., and the source code shown in Table 1 has been filed with
user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco,
California or may be compiled from the source code provided in Table 1. The
ALIGN-2 program should be
compiled for use on a UNIX operating system, preferably digital UNIX V4.OD.
All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % nucleic acid sequence identity of a given nucleic
acid sequence C to, with, or
against a given nucleic acid sequence D (which can alternatively be phrased as
a given nucleic acid sequence C that
has or comprises a certain % nucleic acid sequence identity to, with, or
against a given nucleic acid sequence D)
is calculated as follows:
100 times the fraction W/Z
51
WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2
in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be appreciated
that where the length of nucleic acid sequence C is not equal to the length of
nucleic acid sequence D, the % nucleic
acid sequence identity of C to D will not equal the Qlo nucleic acid sequence
identity of D to C. As examples of %
nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to
calculate the % nucleic acid sequence
identity of the nucleic acid sequence designated "Comparison DNA" to the
nucleic acid sequence designated "PRO-
DNA".
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained as
described above using the ALIGN-2 sequence comparison computer program.
However, % nucleic acid sequence
identity may also be determined using the sequence comparison program NCBI-
BLAST2 (Altschul et al., Nucleic
Acids Res., 25:3389-3402 ( 1997)). The NCBI-BLAST2 sequence comparison program
may be downloaded from
http://www.ncbi.nlm.nih.gov. or otherwise obtained from the National Institute
of Health, Bethesda, MD. NCBI-
BLAST2 uses several search parameters, wherein all of those search parameters
are set to default values including,
for example, unmask = yes, strand = all, expected occurrences = I 0, minimum
low complexity length = 15/5, multi-
pass e-value = 0.01, constant for mufti-pass = 25, dropoff for final gapped
alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence
identity of a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can
alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid sequence
identity to, with, or against a given nucleic acid sequence D) is calculated
as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-
BLAST2 in that program's alignment of C and D, and where Z is the total number
of nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence D,
the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated
using the WU-BLAST-2
computer program (Altschul et al., Methods in Enzymoloay> 266:460-480 (1996)).
Most of the WU-BLAST-2
search parameters are set to the default values. Those not set to default
values, i.e., the adjustable parameters, are
set with the following values: overlap span = I, overlap fraction = 0.125,
word threshold (T) = 11, and scoring
matrix =BLOSUM62. For purposes herein, a % nucleic acid sequence identity
value is determined by dividing (a)
the number of matching identical nucleotides between the nucleic acid sequence
of the PRO polypeptide-encodin~~
nucleic acid molecule of interest having a sequence derived from the native
sequence PRO polypeptide-encoding
nucleic acid and the comparison nucleic acid molecule of interest (i.e., the
sequence against which the PRO
polypeptide-encoding nucleic acid molecule of interest is bein~~ compared
which may be a variant PRO
3S polynucleotide) as determined by WU-BLAST-2 by (b) the total number of
nucleotides of the PRO polypeptide-
encoding nucleic acid molecule of interest. For example, in the statement "an
isolated nucleic acid molecule
comprising a nucleic acid sequence A which has or having at least 80% nucleic
acid sequence identity to the nucleic
52
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
acid sequence B", the nucleic acid sequence A is the comparison nucleic acid
molecule of interest and the nucleic
acid sequence B is the nucleic acid sequence of the PRO polypeptide-encoding
nucleic acid molecule of interest.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active PRO
polypeptide and which are capable of hybridizing, preferably under stringent
hybridization and wash conditions,
to nucleotide sequences encoding the full-length PRO polypeptide shown in
Figure 2 (SEQ ID N0:2), Figure 4
(SEQ ID N0:4), Figure 6 (SEQ ID N0:6), Figure 8 (SEQ ID N0:8) , Figure 10 (SEQ
ID NO:10), Figure 12 (SEQ
ID N0:12), Figure 14 (SEQ ID N0:14), Figure 16 (SEQ ID N0:16), Figure 18 (SEQ
ID N0:18), Figure 20 (SEQ
ID N0:20), Figure 22 (SEQ ID N0:22), Figure 24 (SEQ ID N0:24), Figure 26 (SEQ
ID N0:26), Figure 28 (SEQ
ID N0:28), Figure 30 (SEQ ID N0:30), and Figure 32 (SEQ ID N0:32),
respectively. PRO variant polypeptides
may be those that are encoded by a PRO variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as described
above, includes amino acid residues in the sequences compared that are not
only identical, but also those that have
similar properties. Amino acid residues that score a positive value to an
amino acid residue of interest are those
that are either identical to the amino acid residue of interest or are a
preferred substitution (as defined in Table 3
below) of the amino acid residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A
to, with, or against a given
amino acid sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises
a certain % positives to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value by the
sequence alignment program ALIGN-
2 in that program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be
appreciated that where the length of amino acid sequence A is not equal to the
length of amino acid sequence B,
the % positives of A to B will not equal the % positives of B to A.
"Isolated", when used to describe the various polypeptides disclosed herein,
means a polypeptide that has been
identified and separated and/or recovered from a component of its natural
environment. Preferably, the isolated
polypeptide is free of association with all components with which it is
naturally associated. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic
uses for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified ( 1 ) to a
degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to homogeneity
by SDS-PAGE under non-reducing or reducing conditions usin~~ Coomassie blue
or, preferably, silver stain.
Isolated polypeptide includes polypeptide ifs. situ within recombinant cells,
since at least one component of the PRO
natural environment will not be present. Ordinarily, however, isolated
polypeptide will be prepared by at least one
purification step.
An "isolated" nucleic acid molecule encoding a PRO polypeptide or an
"isolated" nucleic acid molecule
encoding an anti-PRO antibody is a nucleic acid molecule that is identified
and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily associated in
the natural source of the PRO-encoding
53
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
nucleic acid or the natural source of the anti-PRO-encoding nucleic acid.
Preferably, the isolated nucleic acid is
free of association with all components with which it is naturally associated.
An isolated PRO-encoding nucleic
acid molecule or an isolated anti-PRO-encoding nucleic acid molecule is other
than in the form or setting in which
it is found in nature. Isolated nucleic acid molecules therefore are
distinguished from the PRO-encoding nucleic
acid molecule or from the anti-PRO-encoding nucleic acid molecule as it exists
in natural cells. However, an
isolated nucleic acid molecule encoding a PRO polypeptide or an isolated
nucleic acid molecule encoding an anti-
PRO antibody includes PRO-nucleic acid molecules or anti-PRO-nucleic acid
molecules contained in cells that
ordinarily express PRO polypeptides or anti-PRO antibodies where, for example,
the nucleic acid molecule is in
a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked
coding sequence in a particular host organism. The control sequences that are
suitable for prokaryotes, for example,
include a promoter, optionally an operator sequence, and a ribosome binding
site. Eukaryotic cells are known to
utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid
sequence. For example, DNA for a presequence or secretory leader is operably
linked to DNA for a PRO
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is positioned so as
to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are contiguous,
and, in the case of a secretory leader,
contiguous and in reading phase. However, enhancers do not have to be
contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers
are used in accordance with conventional practice.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and generally
is an empirical calculation dependent upon probe length, washing temperature,
and salt concentration. In general,
longer probes require higher temperatures for proper annealing, while shorter
probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA to reanneal
when complementary strands are
present in an environment below their melting temperature. The higher the
degree of desired homology between
the probe and hybridizable sequence, the higher the relative temperature that
can be used. As a result, it follows
that higher relative temperatures would tend to make the reaction conditions
more stringent, while lower
temperatures less so. For additional details and explanation of stringency of
hybridization reactions, see, Ausubel
et al., Current Protocols in Molecular Bioloay (Whey Interscience Publishers,
1995).
"Stringent conditions" or "high-stringency conditions", as defined herein, may
be identified by those that: ( 1 )
employ low ionic strength and high temperature for washing, for example, O.OlS
M sodium chloride/0.0015 M
sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ during
hybridization a denaturing went, such as
formamide, for example, 50% (v/v) formamide with 0.10lo bovine serum
albumin/0.1 % Ficoll/0.1 °~o
polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium
chloride, 75 mM sodium
citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI,
0.075 M sodium citrate). 50 mM sodium
phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution,
sonicated salmon sperm DNA (50
~g/ml), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at
42°C in 0.2 x SSC (sodium chloride/sodium
54
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
citrate) and 50% formamide at 55 °C, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA
at 55°C.
"Moderately-stringent conditions" may be identified as described by Sambrook
et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Press, 1989), and include the
use of washing solution and
hybridization conditions (e.g., temperature, ionic strength, and % SDS) less
stringent than those described above.
An example of moderately stringent conditions is overnight incubation at
37°C in a solution comprising: 20%
formamide, 5 x SSC ( I 50 mM NaCI, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5 x Denhardt's
solution,10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters
in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to
adjust the temperature, ionic strength, etc.
1~ as necessary to accommodate factors such as probe length and the like.
The modifier "epitope-tagged" when used herein refers to a chimeric
polypeptide comprising a PRO
polypeptide fused to a "tag polypeptide". The tag polypeptide has enough
residues to provide an epitope against
which an antibody can be made, yet is short enough such that it does not
interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly unique so
that the antibody does not substantially
cross-react with other epitopes. Suitable tag polypeptides generally have at
least six amino acid residues and usually
between about 8 and 50 amino acid residues (preferably, between about 10 and
20 amino acid residues).
"Active" or "activity" in the context of PRO variants refers to forms) of PRO
proteins that retain the biologic
and/or immunologic activities of a native or naturally-occurring PRO
polypeptide.
"Biological activity" in the context of a molecule that antagonizes a PRO
polypeptide that can be identified
2~ by the screening assays disclosed herein (e.g., an organic or inorganic
small molecule, peptide, etc.) is used to refer
to the ability of such molecules to bind or complex with the PRO polypeptide
identified herein, or otherwise
interfere with the interaction of the PRO polypeptides with other cellular
proteins or otherwise inhibits the
transcription or translation of the PRO polypeptide. Particularly preferred
biological activity includes cardiac
hypertrophy, activity that acts on systemic disorders that affect vessels,
such as diabetes mellitus, as well as diseases
of the arteries, capillaries, veins, and/or lymphatics, and cancer.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully blocks.
inhibits, or neutralizes one or more of the biological activities of a native
PRO polypeptide disclosed herein, for
example, if applicable, its mitogenic or angiogenic activity. Antagonists of a
PRO polypeptide may act by
interfering with the binding of a PRO polypeptide to a cellular receptor, by
incapacitating or killing cells that have
3~ been activated by a PRO polypeptide, or by interfering with vascular
endothelial cell activation after binding of a
PRO polypeptide to a cellular receptor. All such points of intervention by a
PRO polypeptide antagonist shall be
considered equivalent for purposes of this invention. The antagonists inhibit
the mitogenic, angiogenic, or other
biological activity of PRO polypeptides, and thus are useful for the treatment
of diseases or disorders characterized
by undesirable excessive neovascularization, including by way of example
tumors, and especially solid malignant
3J tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and
other retinopathies, retrolental fibroplasia, age-
related macular degeneration. neovascular glaucoma, hemangiomas, thyroid
hyperplasias (including Grave's
disease), corneal and other tissue transplantation, and chronic inflammation.
The antagonists also are useful for
the treatment of diseases or disorders characterized by undesirable excessive
vascular permeability, such as edema
associated with brain tumors, ascites associated with malignancies, Meigs'
syndrome, lung inflammation, nephrotic
WO 00/53757 CA 02361849 2001-07-30 PCT/US00/05004
syndrome, pericardial effusion (such as that associated with pericarditis),
and pleural effusion. In a similar manner,
the term "agonist" is used in the broadest sense and includes any molecule
that mimics a biological activity of a
native PRO polypeptide disclosed herein. Suitable agonist or antagonist
molecules specifically include agonist or
antagonist antibodies or antibody fragments, fragments, or amino acid sequence
variants of native PRO
polypeptides, peptides, small organic molecules, etc.
A "small molecule" is defined herein to have a molecular weight below about
500 daltons.
The term "PRO polypeptide receptor" as used herein refers to a cellular
receptor for a PRO polypeptide,
ordinarily a cell-surface receptor found on vascular endothelial cells, as
well as variants thereof that retain the ability
to bind a PRO polypeptide.
1~ "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the
same structural characteristics.
While antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and
other antibody-like molecules that lack antigen specificity. Polypeptides of
the latter kind are, for example,
produced at low levels by the lymph system and at increased levels by
myelomas. The term "antibody" is used in
the broadest sense and specifically covers, without limitation, intact
monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies) formed from at least
two intact antibodies, and antibody
fragments, so long as they exhibit the desired biological activity.
"Native antibodies" and "native immunoglobulins" are usually heterotetrameric
glycoproteins of about 150,000
daltons, composed of two identical light (L) chains and two identical heavy
(H) chains. Each light chain is linked
to a heavy chain by one covalent disulfide bond, while the number of disulfide
linkages varies among the heavy
2~ chains of different immunoglobulin isotypes. Each heavy and light chain
also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable domain (VH)
followed by a number of constant
domains. Each light chain has a variable domain at one end (VL) and a constant
domain at its other end; the constant
domain of the light chain is aligned with the first constant domain of the
heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain. Particular
amino acid residues are believed to form
an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in
sequence among antibodies and are used in the binding and specificity of each
particular antibody to and for its
particular antigen. However, the variability is not evenly distributed
throughout the variable domains of antibodies.
It is concentrated in three segments called complementarity-determining
regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The more highly
conserved portions of variable
domains are called the framework regions (FR). The variable domains of native
heavy and light chains each
comprise four FR regions, largely adopting a (3-sheet configuration, connected
by three CDRs, which form loops
connecting, and in some cases forming part of, the (3-sheet structure. The
CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other chain,
contribute to the formation of the
antigen-binding site of antibodies. See, Kabat et al., NIH Publ. No.91-3242,
Vol. I, pages 647-669 (1991 ). The
constant domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector
functions, such as participation of the antibody in antibody-dependent
cellular toxicity.
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen-binding or variable
region of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab')=, and Fv fragments;
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
diabodies; linear antibodies (Zapata etal., Protein EnQ., 8(J O):1057-1062 (
1995)); single-chain antibody molecules;
and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments, each
with a single antigen-binding site, and a residual "Fc" fragment, whose name
reflects its ability to crystallize readily.
Pepsin treatment yields an F(ab')= fragment that has two antigen-combining
sites and is still capable of cross-linking
antigen.
"Fv" is the minimum antibody fragment that contains a complete antigen-
recognition and -binding site. This
region consists of a dimer of one heavy- and one light-chain variable domain
in tight, non-covalent association.
It is in this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on
the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-
binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising only three
CDRs specific for an antigen) has
the ability to recognize and bind antigen, although at a lower affinity than
the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH 1 )
of the heavy chain. Fab' fragments differ from Fab fragments by the addition
of a few residues at the carboxy
terminus of the heavy chain CHl domain including one or more cysteines from
the antibody hinge region. Fab'-SH
is the designation herein for Fab' in which the cysteine residues) of the
constant domains bear a free thiol group.
F(ab')~ antibody fragments originally were produced as pairs of Fab' fragments
that have hinge cysteines between
them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one of two
clearly distinct types, called kappa (x) and lambda (~,), based on the amino
acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins can
be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IbD, IgE, IgG, and IgM;
and several of these may be further divided into subclasses (isotypes), e.g.,
IgGI , IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different classes of
immunoglobulins are called a, 8, E,
y, and ;~. respectively. The subunit structures and three-dimensional
configurations of different classes of
immunoglobulins are well known.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of
substantially homogeneous antibodies, i. e., the individual antibodies
comprising the population are identical except
for possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly
specific. being directed against a single antigenic site. Furthermore, in
contrast to conventional (polyclonal)
antibody preparations that typically include different antibodies directed
against different determinants (epitopes),
each monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity,
the monoclonal antibodies are advantageous in that they are synthesized by the
hybridoma culture, uncontaminated
by other immunoglobulins. The modifier "monoclonal" indicates the character of
the antibody as being obtained
from a substantially homogeneous population of antibodies, and is not to be
construed as requirin ;; production of
the antibody by any particular method. For example, the monoclonal antibodies
to be used in accordance with the
present invention may be made by the hybridoma method first described by
Kohler et al., Nature, 256: 495 ( 1975),
or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:
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WO 00/53757 CA 02361849 2001-07-30 pCT/US00/05004
624-628 (1991) and Marks etal., J. Mol. Biol., 222: 581-597 (1991), for
example.
The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which a
portion of the heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass, while the remainder of the
chains) is identical with or homologous to corresponding sequences in
antibodies derived from another species or
belonging to another antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the
desired biological activity. U.S. Patent No. 4,816,567; Morrison et al., Proc.
Natl. Acad. Sci. USA, 81: 6851-6855
( 1984).
"Humanized" forms of non-human (e.g., marine) antibodies are chimeric
immunoglobulins, immunoglobulin
l~ chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')Z, or other
antigen-binding subsequences of antibodies)
that contain minimal sequence derived from non-human immunoglobulin. For the
most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues from a CDR of
the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as mouse, rat
or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv FR residues of the
human immunoglobulin are replaced
by corresponding non-human residues. Furthermore, humanized antibodies may
comprise residues that are found
neither in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made
to further refine and maximize antibody performance. In general, the humanized
antibody will comprise
substantially all of at least one, and typically two, variable domains, in
which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those
2~ of a human immunoglobulin sequence. The humanized antibody preferably also
will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see
Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-
329 (1988); and Presta, Curr. On.
Struct. Biol., 2: 593-596 (1992). The humanized antibody includes a
PRIMATIZEDTM antibody wherein the
antigen-binding region of the antibody is derived from an antibody produced by
immunizing macaque monkeys with
the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and V~ domains
of an antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide
linker between the VH and VL domains that enables the sFv to form the desired
structure for antigen binding. For
a review of sFv see, Pluckthun in The Pharmacolow of Monoclonal Antibodies,
Vol. 1 13, Rosenburg and Moore,
eds. (Springer-Verlag: New York, 1994), pp. 269-315.
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain (V~) in the same
polypeptide chain (VH - Vr). By using a linker that is too short to allow
pairing between the two domains on the
same chain, the domains are forced to pair with the complementary domains of
another chain and create two
antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
An "isolated" antibody is one that has been identified and separated and/or
recovered from a component of
its natural environment. Contaminant components of its natural environment are
materials that would interfere with
diagnostic or therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or
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nonproteinaceous solutes. In preferred embodiments, the antibody will be
purified (1) to greater than 95% by
weight of antibody as determined by the Lowry method, and most preferably more
than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning
cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie
blue or, preferably, silver stain. Isolated antibody includes the antibody ira
situ within recombinant cells, since at
least one component of the antibody's natural environment will not be present.
Ordinarily, however, isolated
antibody will be prepared by at least one purification step.
The word "label" when used herein refers to a detectable compound or other
composition that is conjugated
directly or indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
1 ~ (e.g., radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration
of a substrate compound or composition that is detectable. Radionuclides that
can serve as detectable labels include,
for example, I-131, I-123, I-125, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-
109. The label may also be a non-
detectable entity such as a toxin.
By "solid phase" is meant a non-aqueous matrix to which an antibody of the
present invention can adhere.
Examples of solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled
pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,
polyvinyl alcohol and silicones. In
certain embodiments, depending on the context, the solid phase can comprise
the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography column). This
term also includes a discontinuous solid
phase of discrete particles, such as those described in U.S. Patent No.
4,275,149.
2~ A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant that is
useful for delivery of a drug (such as the PRO polypeptide or antibodies
thereto disclosed herein) to a mammal.
The components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement
of biological membranes.
As used herein, the term "immunoadhesin" designates antibody-like molecules
that combine the binding
specificity of a heteroloQous protein (an "adhesin") with the effector
functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an amino acid
sequence with the desired binding
specificity that is other than the antigen recognition and binding site of an
antibody (i.e., is "heterologous"), and
an immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a
contiguous amino acid sequence comprising at least the binding site of a
receptor or a ligand. The immunoglobulin
3~ constant domain sequence in the immunoadhesin may be obtained from any
immunoglobulin, such as IgG-1, IaG-2,
IgG-3, or IaG-4 subtypes, IbA (including IgA-1 and IgA-2), IgE, IgD, or IgM.
II. Compositions and Methods of the Invention
A. PR0179, PR0238, PR0364. PR0844, PR0846, PR01760, PR020~. PR0321, PR0333,
PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 Variants
In addition to the full-length native sequence PR0179, PR0238, PR03fi-1,
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 and
PR0887 polypeptides
described herein, it is contemplated that PR0179, PR0238, PR0364, PR08~.-1.
PR0846, PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR088~ and PR0887
variants can be
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WO 00/53757 CA 02361849 2001-07-30 pCT/[JS00/05004
prepared. PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 variants can be prepared by
introducing appropriate
nucleotide changes into the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 DNA, and/or
by synthesis of the
desired PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide. Those skilled in
the art will appreciate
that amino acid changes may alter post-translational processes of the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887, such as changing the number or position of glycosylation sites or
altering the membrane anchoring
characteristics.
Variations in the native full-length sequence PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887, or in various
domains of the PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205,
PR0321, PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 described herein, can be
made, for example, using any
of the techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S.
Patent No. 5,364,934. Variations may be a substitution, deletion or insertion
of one or more codons encoding the
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 that results in a change in the amino
acid sequence of the
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 as compared with the native sequence
PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887. Optionally the variation is by substitution of at
least one amino acid with any other
amino acid in one or more of the domains of the PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887. Guidance in
determining which amino acid residue may be inserted, substituted or deleted
without adversely affecting the desired
activity may be found by comparing the sequence of the PR0179, PR0238, PR0364,
PR0844, PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 with that of
homologous known protein molecules and minimizing the number of amino acid
sequence changes made in regions
of high homology. Amino acid substitutions can be the result of replacing one
amino acid with another amino acid
having similar structural and/or chemical properties, such as the replacement
of a leucine with a serine, i.e.,
conservative amino acid replacements. Insertions or deletions may optionally
be in the range of about 1 to 5 amino
acids. The variation allowed may be determined by systematically making
insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for activity
exhibited by the full-length or mature
native sequence.
In particular embodiments, conservative substitutions of interest are shown in
Table 3 under the heading of
preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial changes,
denominated exemplary substitutions in Table 3, or as further described below
in reference to amino acid classes,
are introduced and the products screened.
WO 00/53757 CA 02361849 2001-07-30 pCT/[JS00/05004
Table 3
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala
ala
His (H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala; phe:
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
Substantial modifications in function or immunological identity of the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide are accomplished by selecting substitutions that differ
significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or
helical conformation, (b) the charge or hydrophobicity of the molecule at the
target site, or (c) the bulk of the side
chain. Naturally occurring residues are divided into groups based on common
side-chain properties:
(1 ) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into
the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-directed)
mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res.,
13:4331 (1986); Zoller etal., Nucl. Acids Res., 10:6487 (1987)], cassette
mutagenesis [Wells etal., Gene, 34:315
(1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R.
Soc. London SerA, 317:415 ( 1986)] or
other known techniques can be performed on the cloned DNA to produce the
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
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PR0885 or PR0887 variant DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a contiguous
sequence. Among the preferred scanning amino acids are relatively small,
neutral amino acids. Such amino acids
include alanine, glycine, serine, and cysteine. Alanine is typically a
preferred scanning amino acid among this group
because it eliminates the side-chain beyond the beta-carbon and is less likely
to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine
is also typically preferred
because it is the most common amino acid. Further, it is frequently found in
both buried and exposed positions
[Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol.,
150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an isoteric amino
acid can be used.
B. Modifications of PR0179, PR0238 PR0364 PR0844 PR0846 PR01760, PR0205,
PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887
Covalent modifications of PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 and PR0887 are included
within the scope of
this invention. One type of covalent modification includes reacting targeted
amino acid residues of a PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 polypeptide with an organic derivatizing
agent that is capable of reacting
with selected side chains or the N- or C- terminal residues of the PRO 179,
PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887.
Derivatization with bifunctional agents is useful, for instance, for
crosslinking PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 to a water-insoluble support matrix or surface for use in the
method for purifying anti-
PR0179, anti-PR0238, anti-PR0364, anti-PR0844, anti-PR0846, anti-PRO 1760,
anti-PR0205, anti-PR0321,
anti-PR0333, anti-PR0840, anti-PR0877, anti-PR0878, anti-PR0879, anti-PR0882,
anti-PR0885 or anti-PR0887
antibodies, and vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-
azidosalicylic acid, homobifunctional
imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-
azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
Groups of Beryl or threonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side chains
[T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &
Co.. San Francisco, pp. 79-86
(1983)], acetylation of the N-terminal amine, and amidation of any C-terminal
carboxyl ~~roup.
Another type of covalent modification of the PR0179, PR0238, PR0364, PR08-14,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
3S included within the scope of this invention comprises altering the native
glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for purposes herein to
mean deletin;~ one or more
carbohydrate moieties found in native sequence PR0179, PR0238, PR0364, PR08-
1~, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 (either by
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WO 00/53757 PCT/US00/05004
removing the underlying glycosylation site or by deleting the glycosylation by
chemical and/or enzymatic means),
and/or adding one or more glycosylation sites that are not present in the
native sequence PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887. In addition, the phrase includes qualitative changes
in the glycosylation of the
native proteins, involving a change in the nature and proportions of the
various carbohydrate moieties present.
Addition of glycosylation sites to the PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide may be
accomplished by altering the amino acid sequence. The alteration may be made,
for example, by the addition of,
or substitution by, one or more serine or threonine residues to the native
sequence PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 (for O-linked glycosylation sites). The PR0179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
amino acid sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA
encoding the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide at preselected
bases such that codons are
generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PR0179,
PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 polypeptide is by chemical or enzymatic coupling of
glycosides to the polypeptide. Such
methods are described in the art, e.g., in WO 87/05330 published 1 I September
1987, and in Aplin and Wriston,
CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the PRO 179, PR0238, PR0364,
PR0844, PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
may be accomplished chemically or enzymatically or by mutational substitution
of codons encoding for amino acid
residues that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys.,
259:52 ( 1987) and by Edge et al., Anal.
Biochem., 1 18:131 (1981). Enzymatic cleavage of carbohydrate moieties on
polypeptides can be achieved by the
use of a variety of endo- and exo-glycosidases as described by Thotakura et
al., Meth. Enzvmol., 138:350 ( 1987).
Another type of covalent modification of PR0179, PR0238, PR0364, PR084=l,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 comprises
linking the PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide to one of a
variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in
U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179, 337.
The PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PRO 321, PR0333,
PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 of the present invention may
also be modified in a way
to form a chimeric molecule comprising PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887 fused
to another,
heteroloQous polypeptide or amino acid sequence.
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CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
In one embodiment, such a chimeric molecule comprises a fusion of the PR0179,
PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 with a tag polypeptide which provides an epitope to which an
anti-tag antibody can selectively
bind. The epitope~tag is generally placed at the amino- or carboxyl- terminus
of the PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PK0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887. The presence of such epitope-tagged forms of the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 can be detected using an antibody against the tag polypeptide. Also,
provision of the epitope tag enables
the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 to be readily purified by affinity
purification using an anti-tag
antibody or another type of affinity matrix that binds to the epitope tag.
Various tag polypeptides and their
respective antibodies are well known in the art. Examples include poly-
histidine (poly-His) or poly-histidine-
glycine (poly-His-gly) tags; the flu HA tag polypeptide and its antibody 12CA5
[Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10
antibodies thereto [Evan et al.,
Molecular and Cellular Bioloay, 5:3610-3616 (1985)]; and the Herpes Simplex
virus glycoprotein D (gD) tag and
its antibody [Paborsky et al., Protein Enaineerina, x:547-553 ( 1990)]. Other
tag polypeptides include the Flag-
peptide [Hopp et al., BioTechnolosy, 6:1204-1210 (1988)]; the KT3 epitope
peptide [Martin et al., Science,
255:192-194 (1992)]; an a-tubulin epitope peptide [Skinner etal., J. Biol.
Chem., 266:15163-15166 (1991)]; and
the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad.
Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent
form of the chimeric molecule (also referred to as an "immunoadhesin"), such a
fusion could be to the Fc region
of an IgG molecule. The Ig fusions preferably include the substitution of a
soluble (transmembrane domain deleted
or inactivated) form of a PRO 179, PR0238, PR0364, PR0844, PR0846, PR01760.
PR020_5, PR0321, PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide in place
of at least one variable
region within an Ig molecule. In a particularly preferred embodiment, the
immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI
molecule. For the production of
immunoglobulin fusions see also, US Patent No. 5,428,130 issued June 27, 1995.
C. Preparation of the PRO 179. PR0238, PR0364, PR0844, PR0846. PRO 1760.
PR0205. PR0321,
PR0333, PR0840, PR0877. PR0878. PR0879, PR0882, PR0885 and PR0887
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. In
particular, cDNAs
encoding PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides have been
identified and isolated, as
disclosed in further detail in the Examples below. It is noted that proteins
produced in separate expression rounds
may be given different PRO numbers but the UNQ number is unique for any given
DNA and the encoded protein,
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CA 02361849 2001-07-30
WO 00/53757 PCT/L1S00/05004
and will not be changed. However, for sake of simplicity, in the present
specification the protein encoded by
DNA16451-1388, DNA35600-I 162, DNA47365-1206, DNA59838-1462, DNA44196-1353,
DNA76532-1702,
DNA30868, DNA34433, DNA41374, DNA53987, DNA58120, DNA58121, DNA58122,
DNA58125, DNA58128,
or DNA58130, as well as all further native homologues and variants included in
the foregoing definition of
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887, will be referred to as "PR0179",
"PR0238", "PR0364",
"PR0844", "PR0846", "PR01760", "PR0205", "PR0321 ", "PR0333", "PR0840",
"PR0877", "PR0878",
"PR0879", "PR0882", "PR0885" or "PR0887", respectively, regardless of their
origin or mode of preparation.
The description below relates primarily to production of PR0179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
polypeptides by culturing cells transformed or transfected with a vector
containing nucleic acid encoding PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 polypeptides. It is, of course, contemplated
that alternative methods that
are well known in the art may be employed to prepare PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887. For instance,
the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide sequence, or portions
thereof, may be produced by
direct peptide synthesis using solid-phase techniques. See, e.g., Stewart
etal., Solid-Phase Peptide Synthesis (W.H.
Freeman Co.: San Francisco, CA, 1969); Merrifield, J. Am. Chem. Soc., 85: 2149-
2154 (1963). In vitro protein
synthesis may be performed using manual techniques or by automation. Automated
synthesis may be accomplished,
for instance, with an Applied Biosystems Peptide Synthesizer (Foster City, CA)
using manufacturer's instructions.
Various portions of PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205,
PR0321, PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be chemically
synthesized separately
and combined using chemical or enzymatic methods to produce the full-length
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333> PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 polypeptide.
i. Isolation of DNA Encoding PR0179 PR0238, PR0364. PR0844, PR0846, PR01760,
PR0205.
PR0321, PR0333. PR0840 PR0877 PR0878, PR0879 PR0882 PR0885 or PR0887
DNA encoding PR0179, PR0238, PR0364, PR0844, PR08=l6, PR01760, PR0205, PR0321,
PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may be
obtained from a cDNA
library prepared from tissue believed to possess the mRNA encodin~~ PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877. PR0878, PR0879,
PR0882, PR0885 or
PR0887 and to express it at a detectable level. Accordingly, DN_~s encoding
human PR0179, human PR0238,
human PR0364, human PR0844, human PR0846, human PR01760, human PR0205, human
PR0321. human
PR0333, human PR0840, human PR0877, human PR0878, human PR0879, human PR0882,
human PR0885
or human PR0887 can be conveniently obtained from cDNA libraries prepared from
human tissues, such as
described in the Examples. The gene encoding PR0179, PR02 38> PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
may also be obtained from a genomic library or by oligonucleotide synthesis.
Libraries can be screened with probes (such as antibodies to the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide or oligonucleotides of at least about 20-80 bases) designed
to identify the gene of interest or
the protein encoded by it. Screening the cDNA or genomic library with the
selected probe may be conducted using
standard procedures, such as described in Sambrook etal., supra. An
alternative means to isolate the gene encoding
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 is to use PCR methodology. Sambrook
et al., supra;
Dieffenbach et al., PCR Primer: A Laboratory Manual (New York: Cold Spring
Harbor Laboratory Press, 1995).
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon
hybridization to DNA in the library being
screened. Methods of labeling are well known in the art, and include the use
of radiolabels like 3-P-labeled ATP,
biotinylation, or enzyme labeling. Hybridization conditions, including
moderate stringency and high stringency,
are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across the
full-len~h sequence can be determined through sequence alignment using
computer software programs such as
ALIGN. DNAstar, and INHERIT, which employ various algorithms to measure
homology.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
supra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
ii. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PR0179,
PR0238. PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 production and cultured in conventional
nutrient media modified as
appropriate for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired
sequences. The culture conditions, such as media, temperature, pH, and the
like, can be selected by the skilled
artisan without undue experimentation. In general, principles, protocols, and
practical techniques for maximizing
the productivity of cell cultures can be found in Mammalian Cell
Biotechnoloay: A Practical Approach, M. Butler,
ed. (IRL Press, 1991 ) and Sambrook et al., supra.
Methods of transfection are known to the ordinarily skilled artisan, for
example, CaPO~ treatment and
3S electroporation. Depending on the host cell used, transformation is
performed using standard techniques
appropriate to such cells. The calcium treatment employing calcium chloride,
as described in Sambrook et al.,
sccpra, or electroporation is generally used for prokaryotes or other cells
that contain substantial cell-wall barriers.
Infection with Agrobactericcrn turnefaciens is used for transformation of
certain plant cells, as described by Shaw
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et al., Gene, 23: 315 ( 1983) and WO 89/05859 published 29 June 1989. For
mammalian cells without such cell
walls, the calcium phosphate precipitation method of Graham and van der Eb,
ViroloQy, 52:456-457 ( 1978) can be
employed. General aspects of mammalian cell host system transformations have
been described in U.S. Patent No.
4,399,216. Transformations into yeast are typically carried out according to
the method of Van Solingen et al., J.
Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:
3829 (1979). However, other methods
for introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial protoplast fusion with
intact cells, or polycations, e.g., polybrene or polyornithine, may also be
used. For various techniques for
transforming mammalian cells, see, Keown et al., Methods in EnzymoloQy, 185:
527-537 (1990) and Mansour et
al., Nature, 336: 348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein
include prokaryote, yeast, or higher
eukaryote cells. Suitable prokaryotes include, but are not limited to,
eubacteria, such as Gram-negative or Gram-
positive organisms, for example, Enterobacteriaceae such as E. coli. Various
E. coli strains are publicly available,
such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537);
E. coli strain W3110 (ATCC
27,325); and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells
include Enterobacteriaceae such as
Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typlZimurium,
Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as
B. subtilis and B. licheniformis (e.g.,
B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989),
Pseudomorras such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting. Strain
W3110 is one particularly preferred host
or parent host because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host
cell secretes minimal amounts of proteolytic enzymes. For example, strain
W3110 may be modified to effect a
genetic mutation in the genes encoding proteins endogenous to the host, with
examples of such hosts including E.
coli W3110 strain 1 A2, which has the complete genotype tonA ; E. coli W3110
strain 9E4, which has the complete
genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the
complete genotype tonA ptr3 phoA
EI S (argF-lac)169 degP ompT kan ; E. coli W3110 strain 37D6, which has the
complete genotype torrA ptr3 phoA
EIS (argF-lac)169 degP ornpT rbs7 ilvG kan'; E. coli W3l l0 strain 40B4, which
is strain 37D6 with a non-
kanamycin resistant degP deletion mutation; and an E. coli strain having
mutant periplasmic protease disclosed in
U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro
methods of cloning, e. ~., PCR or other
nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning or
expression hosts for vectors encoding PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887.
Sacclraronryces
cerevisiae is a commonly used lower eukaryotic host microorganism. Others
include Schiosaccharomyces pombe
(Beach and Nurse, Nature, 290: 140 [1981 ]; EP 139,383 published 2 May 1985);
Klu~~verorrayces hosts (U.S. Patent
No. 4,943,529; Fleer et al., Bio/Technolow. 9: 968-975 (1991)) such as, e.g.,
K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourtetal., J. Bacteriol., 737 [1983]), K. fragilis (ATCC
12,424). K. bulgaricus (ATCC 16,045).
K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosoplzilarum (ATCC
36,906; Van den Bera etal.,
Bio/Technoloay, 8: 135 (1990)), K . thernrotolerarrs, and K. marxian.us;
yarrowia (EP 402,226); Pichia pastoris
(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28: 265-278 [1988]);
Candida; Tr-iclroderma ree,sia (EP
244,234); Neurospora cr-assa (Case et nl.. Proc. Natl. Acad. Sci. USA, 76:
5259-5263 [ 1979]); Sclrwarnriomyces
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such as Scl2waru~iornvces occidentalis (EP 394,538 published 31 October 1990);
and filamentous fungi such as, e.g.,
Neurospora, Penicilliurn, Tolypocladiurn (WO 91 /00357 published 10 January
1991 ), and Aspergillus hosts such
as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289
[ 1983]; Tilburn et al., Gene, 26:
205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes,
EMBO J., 4: 475-479 [1985]). Methylotropic yeasts are suitable herein and
include, but are not limited to, yeast
capable of growth on methanol selected from the genera consisting of
Hansenula, Carrdida, Kloeckera, Pichia,
Saccharomyces, Torulopsi.s> and Rhodotorula. A list of specific species that
are exemplary of this class of yeasts
may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
Suitable host cells for the expression of nucleic acid encoding glycosylated
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 are derived from multicellular organisms. Examples of
invertebrate cells include insect cells
such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of
useful mammalian host cell lines
include Chinese hamster ovary (CHO) and COS cells. More specific examples
include monkey kidney CV 1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293
or 293 cells subcloned for
growth in suspension culture, Graham et al., J. Gen. Virol., 36: 59 (1977));
Chinese hamster ovary cells/-DHFR
(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol.
Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver
cells (Hep G2, HB 8065);
and mouse mammary tumor (MMT 060562, ATCC CCL51 ). The selection of the
appropriate host cell is deemed
to be within the skill in the art.
iii. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PR0179, PR0238, PR0364,
PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
may be inserted into a replicable vector for cloning (amplification of the
DNA) or for expression. Various vectors
are publicly available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage.
The appropriate nucleic acid sequence may be inserted into the vector by a
variety of procedures. In general, DNA
is inserted into an appropriate restriction endonuclease sites) using
techniques known in the art. Vector
components generally include, but are not limited to, one or more of a signal
sequence if the sequence is to be
secreted, an origin of replication, one or more marker genes, an enhances
element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing one or more
of these components employs
standard ligation techniques that are known to the skilled artisan.
The PROl79, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 may be produced recombinantly
not only directly, but
also as a fusion polypeptide with a heterologous polypeptide, which may be a
signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal
sequence may be a component of the vector, or it may be a part of the DNA
encoding PRO 179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840. PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 that is inserted into the vector. The signal sequence may be
a prokaryotic signal sequence
selected, for example, from the group of the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II
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leaders. For yeast secretion the signal sequence may be, e.g., the yeast
invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces a-factor leaders, the latter
described in U.S. Patent No. 5,010, I 82),
or acid phosphatase leader, the C. albicaus glucoamylase leader (EP 362,179
published 4 April 1990), or the signal
described in WO 90/13646 published 15 November 1990. In mammalian cell
expression, mammalian signal
sequences may be used to direct secretion of the protein, such as signal
sequences from secreted polypeptides of
the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate in one
or more selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin
of replication from the plasmid pBR322 is suitable for most Gram-negative
bacteria, the 2~ plasmid origin is
suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV,
or BPV) are useful for cloning
vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection Gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification of cells
competent to take up the nucleic acid encoding PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887, such as DHFR
or thymidine kinase. An appropriate host cell when wild-type DHFR is employed
is the CHO cell line deficient
in DHFR activity, prepared and propagated as described by Urlaub et al., Proc.
Natl. Acad. Sci. USA, 77: 4216
(1980). A suitable selection gene for use in yeast is the trpl gene present in
the yeast plasmid YRp7. Stinchcomb
etal., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper
et al., Gene, 10: 157 (1980). The
trill gene provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for
example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977).
Expression and cloning vectors usually contain a promoter operably linked to
the nucleic acid sequence
encoding PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 to direct mRNA synthesis.
Promoters recognized by
a variety of potential host cells are well known. Promoters suitable for use
with prokaryotic hosts include the (3-
lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978);
Goeddel ez al., Nature, 281: 544
(1979)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,
Nucleic Acids Res., 8: 4057 (1980);
EP 36,776), and hybrid promoters such as the tac promoter. deBoer et al.,
Proc. Natl. Acad. Sci. USA, 80: 21-25
(1983). Promoters for use in bacterial systems also will contain a Shine-
Dalgarno (S.D.) sequence operably linked
to the DNA encoding PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205,
PR0321, PR0333.
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887.
3S Examples of suitable promoting sequences for use with yeast hosts include
the promoters for 3-
phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 207 3 ( 1980))
or other glycolytic enzymes (Hess et
al., J. Adv. Enzvme ReQ., 7: 149 ( 1968); Holland, Biochemistry, 17: 4900 (
1978)), such as enolase, glyceraldehyde-
3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and
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CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
glucokinase.
Other yeast promoters that are inducible promoters having the additional
advantage of transcription controlled
by growth conditions are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization.
Suitable vectoia and promoters for
use in yeast expression are further described in EP 73,657.
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 nucleic acid transcription from
vectors in mammalian host cells
is controlled, for example, by promoters obtained from the genomes of viruses
such as polyoma virus, fowlpox virus
1~ (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2),
bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus, and Simian Virus 40
(SV40); by heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter; and by heat-
shock promoters, provided such
promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 orPR0887 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 on a promoter to increase its transcription.
Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and
insulin). Typically, however, one will
use an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the
2~ replication origin (bp I 00-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side
of the replication origin, and adenovirus enhancers. The enhancer may be
spliced into the vector at a position 5'
or 3' to the sequence coding for PR0179, PR0238, PR036-1, PR0844, PR0846,
PR01760, PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, but is
preferably located at a site
5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast. fun;>i, insect,
plant, animal, human, or nucleated cells
from other multicellular organisms) will also contain sequences necessary for
the termination of transcription and
for stabilizing the mRNA. Such sequences are commonly available from the 5'
and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide
segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA encoding
PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR03? 1, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 in recombinant vertebrate cell culture are described
in Gething et al., Nature. 293:
620-625 (1981 ); Mantei et al., Nature, 281: 40-46 (1979): EP 117,060; and EP
117,058.
iv. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly, for
example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of mRNA
(Thomas, Proc. Natl. Acad. Sci. USA,
CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
77:5201-5205 ( 1980)), dot blotting (DNA analysis), or iu situ hybridization,
using an appropriately labeled probe,
based on the sequences provided herein. Alternatively, antibodies may be
employed that can recognize specific
duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or
DNA-protein duplexes.
The antibodies in turn may be labeled and the assay may be carried out where
the duplex is bound to a surface, so
that upon the formation of duplex on the surface, the presence of antibody
bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as immunohistochemical
staining of cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of
gene product. Antibodies useful for immunohistochemical staining and/or assay
of sample fluids may be either
monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the
antibodies may be prepared
against a native-sequence PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide or
against a synthetic peptide
based on the DNA sequences provided herein or against exogenous sequence fused
to DNA encoding PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 and encoding a specific antibody epitope.
v. Purification of PolyQeptide
Forms of PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptides may be recovered
from culture medium
or from host cell lysates. If membrane-bound, it can be released from the
membrane using a suitable detergent
solution (e.g., TRITON-XTM 100) or by enzymatic cleavage. Cells employed in
expression of nucleic acid encoding
the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide can be disrupted by
various physical or chemical
means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell-
lysing agents. It may be desired to
purify the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide from recombinant
cell proteins or
polypeptides. The following procedures are exemplary of suitable purification
procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography
on silica or on a canon-exchange
resin such as DEAF; chromatofocusing; SDS-PAGE; ammonium sulfate
precipitation; gel filtration using, for
example, Sephadex G-75; protein A Sepharose columns to remove contaminants
such as IgG; and metal chelating
columns to bind epitope-tagged forms of the PR0179, PR0238, PR0364, PR0844,
PR0846, PRO 1760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide. Various
methods of protein purification may be employed and such methods are known in
the art and described, for
example, in Deutscher, Methods in Enzymolo~y, I 82 (1990); Scopes, Protein
Purification: Principles and Practice
(Springer-Verlag: New York, 1982). The purification steps) selected will
depend. for example, on the nature of
the production process used and the particular PR0179, PR0238, PR0364, PR08-
14. PR0846. PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 produced.
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D. Uses of the PR0179 PR0238 PR0364 PR0844 PR0846 PR01760, PR0205 PR0321
PR0333,
PR0840 PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887 polypeptides
i. Assays for Cardiovascular Endothelial and An~ioQenic Activity
Various assays can be used to test the polypeptide herein for cardiovascular,
endothelial, and angiogenic
activity. Such assays include those provided in the Examples below.
Assays for testing for endothelia antagonist activity, as disclosed in U.S.
Pat. No. 5,773,414, include a rat heart
ventricle binding assay where the polypeptide is tested for its ability to
inhibit iodinized endothelia-1 binding in a
receptor assay, an endothelia receptor binding assay testing for intact cell
binding of radiolabeled endothelia-1 using
rabbit renal artery vascular smooth muscle cells, an inoshol phosphate
accumulation assay where functional activity
1~ is determined in Rat-1 cells by measuring intra-cellular levels of second
messengers, an arachidonic acid release
assay that measures the ability of added compounds to reduce endothelia-
stimulated arachidonic acid release in
cultured vascular smooth muscles, in vitro (isolated vessel) studies using
endothelium from male New Zealand
rabbits, and in vivo studies using male Sprague-Dawley rats.
Assays for tissue generation activity include, without limitation, those
described in WO 95/16035 (bone,
cartilage, tendon); WO 95/05846 (nerve, neuronal), and WO 91/07491 (skin,
endothelium).
Assays for wound-healing activity include, for example, those described in W
inter, Epidermal Wound Healing,
Maibach, HI and Rovee, DT, eds. (Year Book Medical Publishers, Inc., Chicago),
pp. 71-112, as modified by the
article of Eaglstein and Mertz, J. Invest. Dermatol., 71: 382-384 (1978).
An assay to screen for a test molecule relating to a PRO polypeptide that
binds an endothelia B, (ETB,)
receptor polypeptide and modulates signal transduction activity involves
providing a host cell transformed with a
DNA encoding endothelia B, receptor polypeptide, exposing the cells to the
test candidate, and measuring
endothelia B, receptor signal transduction activity, as described, e.g., in
U.S. Pat. No. 5,773,223.
There are several cardiac hypertrophy assays. Irwitro assays include induction
of spreading of adult rat
cardiac myocytes. In this assay, ventricular myocytes are isolated from a
single (male Sprague-Dawley) rat,
essentially following a modification of the procedure described in detail by
Piper et al., "Adult ventricular rat heart
muscle cells" in Cell Culture Technigues in Heart and Vessel Research, H.M.
Piper, ed. (Benin: Springer-Verlag,
1990), pp. 36-60. This procedure permits the isolation of adult ventricular
myocytes and the long-term culture of
these cells in the rod-shaped phenotype. Phenylephrine and Prostaglandin F=a
(PGF=~) have been shown to induce
a spreading response in these adult cells. The inhibition of myocyte spreading
induced by PGF=a or PGF~a analogs
3~ (e.g., fluprostenol) and phenylephrine by various potential inhibitors of
cardiac hypertrophy is then tested.
One example of an irmiuo assay is a test for inhibiting cardiac hypertrophy
induced by fluprostenol in. vivo.
This pharmacological model tests the ability of the PRO polypeptide to inhibit
cardiac hypertrophy induced in rats
(e.g., male Wistar or Sprague-Dawley) by subcutaneous injection of
fluprostenol (an agonist analog of PGFZa). It
is known that rats with pathologic cardiac hypertrophy induced by myocardial
infarction have chronically elevated
levels of extractable PGF=a in their myocardium. Lai et al., Am J Physiol
(Heart Circ. Physiol.), 271: H2197-
H2208 (1996). Accordingly, factors that can inhibit the effects of
fluprostenol on myocardial growth irr vwo are
potentially useful for treating cardiac hypertrophy. The effects of the PRO
polypeptide on cardiac hypertrophy are
determined by measuring the weight of heart, ventricles, and left ventricle
(normalized by body weight) relative to
fluprostenol-treated rats not receiving the PRO polypeptide.
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CA 02361849 2001-07-30
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Another example of an in vivo assay is the pressure-overload cardiac
hypertrophy assay. For irt vivo testing
it is common to induce pressure-overload cardiac hypertrophy by constriction
of the abdominal aorta of test animals.
In a typical protocol, rats (e.g., male Wistar or Sprague-Dawley) are treated
under anesthesia, and the abdominal
aorta of each rat is narrowed down just below the diaphragm. Beznak M., Can.
J. Biochem. Physiol., 33: 985-94
(1955). The aorta is exposed through a surgical incision, and a blunted needle
is placed next to the vessel. The
aorta is constricted with a ligature of silk thread around the needle, which
is immediately removed and which
reduces the lumen of the aorta to the diameter of the needle. This approach is
described, for example, in Rossi et
al., Am. Heart J., 124: 700-709 (1992) and O'Rourke and Reibel, P.S.E.M.B.,
200: 95-100 (1992).
In yet another in vivo assay, the effect on cardiac hypertrophy following
experimentally induced myocardial
infarction (MI) is measured. Acute MI is induced in rats by left coronary
artery ligation and confirmed by
electrocardiographic examination. A sham-operated group of animals is also
prepared as control animals. Earlier
data have shown that cardiac hypertrophy is present in the group of animals
with MI, as evidenced by an 18%
increase in heart weight-to-body weight ratio. Lai et al., supra. Treatment of
these animals with candidate Mockers
of cardiac hypertrophy, e.g., PRO polypeptide, provides valuable information
about the therapeutic potential of the
candidates tested. One further such assay test for induction of cardiac
hypertrophy is disclosed in U.S. Pat. No.
5,773,415, using Sprague-Dawley rats.
For cancer, a variety of well-known animal models can be used to further
understand the role of the genes
identified herein in the development and pathogenesis of tumors, and to test
the efficacy of candidate therapeutic
agents, including antibodies and other antagonists of the native PRO
polypeptides, such as small-molecule
antagonists. The in vivo nature of such models makes them particularly
predictive of responses in human patients.
Animal models of tumors and cancers (e.g., breast cancer, colon cancer,
prostate cancer, lung cancer, etc.) include
both non-recombinant and recombinant (transgenic) animals. Non-recombinant
animal models include, for
example, rodent, e.g., murine models. Such models can be generated by
introducing tumor cells into syngeneic mice
using standard techniques, e.g., subcutaneous injection, tail vein injection,
spleen implantation, intraperitoneal
implantation, implantation under the renal capsule, or orthopin implantation,
e.g., colon cancer cells implanted in
colonic tissue. See, e.g., PCT publication No. WO 97/33551. published
September 18, 1997. Probably the most
often used animal species in oncological studies are immunodeficient mice and,
in particular, nude mice. The
observation that the nude mouse with thymic hypo/aplasia could successfully
act as a host for human tumor
xenografts has lead to its widespread use for this purpose. The autosomal
recessive n.u gene has been introduced
into a very large number of distinct congenic strains of nude mouse,
including, for example, ASW, A/He, AKR,
BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR, NFS,
NFS/N, NZB, NZC, NZW,
P, RIII, and SJL. In addition, a wide variety of other animals with inherited
immunological defects other than the
nude mouse have been bred and used as recipients of tumor xenografts. For
further details .see, e.g., The Nude
Mouse in Oncolosy Research, E. Boven and B. Winograd, eds. (CRC Press, Inc.,
1991 ).
The cells introduced into such animals can be derived from known tumor/cancer
cell lines, such as any of the
above-listed tumor cell lines, and, for example, the B 104-1-1 cell line
(stable NIH-3T3 cell line transfected with
the ~zeu protooncogene); ras-transfected NIH-3T3 cells: Caco-2 (ATCC HTB-37);
or a moderately well-
differentiated grade II human colon adenocarcinoma cell line. HT-29 (ATCC HTB-
38); or from tumors and cancers.
Samples of tumor or cancer cells can be obtained from patients undergoing
surgery, using standard conditions
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involving freezing and storing in liquid nitrogen. Karmali et al., Br. J.
Cancer, 48: 689-696 (1983).
Tumor cells can be introduced into animals such as nude mice by a variety of
procedures. The subcutaneous
(s.c.) space in mice is very suitable for tumor implantation. Tumors can be
transplanted s.c. as solid blocks, as
needle biopsies by use of a trochar, or as cell suspensions. For solid-block
or trochar implantation, tumor tissue
fragments of suitable size are introduced into the s.c. space. Cell
suspensions are freshly prepared from primary
tumors or stable tumor cell lines, and injected subcutaneously. Tumor cells
can also be injected as subdermal
implants. In this location, the inoculum is deposited between the lower part
of the dermal connective tissue and the
s.c. tissue.
Animal models of breast cancer can be generated, for example, by implanting
rat neuroblastoma cells (from
which the neu oncogene was initially isolated), or neu-transformed NIH-3T3
cells into nude mice, essentially as
described by Drebin ez al. Proc. Nat. Acad. Sci. USA, 83: 9129-9133 ( 1986).
Similarly, animal models of colon cancer can be generated by passaging colon
cancer cells in animals, e.g.,
nude mice, leading to the appearance of tumors in these animals. An orthotopic
transplant model of human colon
cancer in nude mice has been described, for example, by Wang et al., Cancer
Research, 54: 4726-4728 ( 1994) and
Too et al., Cancer Research, 55: 681-684 (1995). This model is based on the so-
called "METAMOUSET"~" sold
by Anticancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from
the in vitro cultures can then
be passaged to animals. Such tumors can serve as targets for further testing
or drug screening. Alternatively, the
tumors resulting from the passage can be isolated and RNA from pre-passage
cells and cells isolated after one or
more rounds of passage analyzed for differential expression of genes of
interest. Such passaging techniques can
be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMSS, CMS21, and WEHI-164 are chemically induced
fibrosarcomas of
BALB/c female mice (DeLeo et al., J. Exp. Med., 146: 720 (1977)), which
provide a highly controllable model
system for studying the anti-tumor activities of various agents. Palladino et
al., J. Immunol., 138: 4023-4032
( 1987). Briefly, tumor cells are propagated in vitro in cell culture. Prior
to injection into the animals, the cell lines
are washed and suspended in buffer, at a cell density of about 10x10'' to
10x10' cells/ml. The animals are then
infected subcutaneously with 10 to 100 ~cl of the cell suspension, allowing
one to three weeks for a tumor to appear.
In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most
thoroughly studied
experimental tumors, can be used as an investigational tumor model. Efficacy
in this tumor model has been
correlated with beneficial effects in the treatment of human patients
diagnosed with small-cell carcinoma of the lung
(SCCL). This tumor can be introduced in normal mice upon injection of tumor
fragments from an affected mouse
or of cells maintained in culture. Zupi et al., Br. J. Cancer, 41: suppl. 4,
30 ( 1980). Evidence indicates that tumors
can be started from injection of even a single cell and that a very high
proportion of infected tumor cells survive.
For further information about this tumor model see, Zacharski, Haemostasis,
16: 300-320 (1986).
One way of evaluating the efficacy of a test compound in an animal model with
an implanted tumor is to
measure the size of the tumor before and after treatment. Traditionally, the
size of implanted tumors has been
measured with a slide caliper in two or three dimensions. The measure limited
to two dimensions does not
accurately reflect the size of the tumor; therefore, it is usually converted
into the corresponding volume by using
a mathematical formula. However, the measurement of tumor size is very
inaccurate. The therapeutic effects of
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a drug candidate can be better described as treatment-induced growth delay and
specific growth delay. Another
omportant variable in the description of tumor growth is the tumor volume
doubling time. Computer programs for
the calculation and description of tumor growth are also available, such as
the program reported by Rygaard and
Spanb Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals, Wu and
Sheng eds. (Basel, 1989), p. 301.
S It is noted, however, that necrosis and inflammatory responses following
treatment may actually result in an increase
in tumor size, at least initially. Therefore, these changes need to be
carefully monitored, by a combination of a
morphometric method and flow cytometric analysis.
Further, recombinant (transgenic) animal models can be engineered by
introducing the coding portion of the
PRO gene identified herein into the genome of animals of interest, using
standard techniques for producing
transgenic animals. Animals that can serve as a target for transgenic
manipulation include, without limitation, mice,
rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g.,
baboons, chimpanzees and monkeys.
Techniques known in the art to introduce a transgene into such animals include
pronucleic microinjection (U.S.
Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl.
Acad. Sci. USA, 82: 6148-615 (1985)); gene targeting in embryonic stem cells
(Thompson et al., Cell, 56: 3l 3-321
(1989)); electroporation of embryos (Lo, Mol. Cell. Biol., 3: 1803-1814
(1983)); and sperm-mediated gene transfer.
Lavitrano et al., Cell, 57: 717-73 (1989). For a review, see for example, U.S.
Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those
that carry the transgene only in part
of their cells ("mosaic animals"). The transgene can be integrated either as a
single transgene, or in concatamers,
e.g., head-to-head or head-to-tail tandems. Selective introduction of a
transgene into a particular cell type is also
possible by following, for example, the technique of Lasko et al., Proc. Natl.
Acad. Sci. USA, 89: 6232-636 ( 1992).
The expression of the transgene in transgenic animals can be monitored by
standard techniques. For example,
Southern blot analysis or PCR amplification can be used to verify the
integration of the transgene. The level of
mRNA expression can then be analyzed using techniques such as in situ
hybridization, Northern blot analysis, PCR,
or immunocytochemistry. The animals are further examined for signs of tumor or
cancer development.
Alternatively, "knock-out" animals can be constructed that have a defective or
altered gene encoding a PRO
polypeptide identified herein, as a result of homologous recombination between
the endogenous Qene encoding the
PRO polypeptide and altered genomic DNA encoding the same polypeptide
introduced into an embryonic cell of
the animal. For example, cDNA encoding a particular PRO polypeptide can be
used to clone genomic DNA
encoding that polypeptide in accordance with established techniques. A portion
of the genomic DNA encoding a
particular PRO polypeptide can be deleted or replaced with another gene, such
as a gene encoding a selectable
marker that can be used to monitor integration. Typically, several kilobases
of unaltered flanking DNA (both at
the 5' and 3' ends) are included in the vector. See, e.g., Thomas and
Capecchi, Cell. 51: 503 ( 1987) for a description
of homologous recombination vectors. The vector is introduced into an
embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has homologously
recombined with the endogenous DNA
are selected. See, e.g., Li et al., Cell, 69: 915 (1992). The selected cells
are then injected into a blastocyst of an
animal (e.g., a mouse or rat) to form aggregation chimeras. See, e.g.,
Bradley, in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL: Oxford, 1987),
pp. 1 13-152. A chimeric embryo can
then be implanted into a suitable pseudopregnant female foster animal and the
embryo brought to term to create a
"knock-out" animal. Progeny harboring the homologously recombined DNA in their
germ cells can be identified
CA 02361849 2001-07-30
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by standard techniques and used to breed animals in which all cells of the
animal contain the homologously
recombined DNA. Knockout animals can be characterized, for instance, by their
ability to defend against certain
pathological conditions and by their development of pathological conditions
due to absence of the PRO polypeptide.
The efficacy of antibodies specifically binding the PRO polypeptides
identified herein, and other drug
candidates, can be tested also in the treatment of spontaneous animal tumors.
A suitable target for such studies is
the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly
invasive, malignant tumor that is the
most common oral malignancy of cats, accounting for over 60% of the oral
tumors reported in this species. It rarely
metastasizes to distant sites, although this low incidence of metastasis may
merely be a reflection of the short
survival times for cats with this tumor. These tumors are usually not amenable
to surgery, primarily because of the
anatomy of the feline oral cavity. At present, there is no effective treatment
for this tumor. Prior to entry into the
study, each cat undergoes complete clinical examination and biopsy, and is
scanned by computed tomography (CT).
Cats diagnosed with sublingual oral squamous cell tumors are excluded from the
study. The tongue can become
paralyzed as a result of such tumor, and even if the treatment kills the
tumor, the animals may not be able to feed
themselves. Each cat is treated repeatedly, over a longer period of time.
Photographs of the tumors will be taken
daily during the treatment period, and at each subsequent recheck. After
treatment, each cat undergoes another CT
scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
The data are evaluated for
differences in survival, response, and toxicity as compared to control groups.
Positive response may require
evidence of tumor regression, preferably with improvement of quality of life
and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma,
adenocarcinoma, lymphoma, chondroma,
or leiomyosarcoma of dogs, cats, and baboons can also be tested. Of these,
mammary adenocarcinoma in dogs and
cats is a preferred model as its appearance and behavior are very similar to
those in humans. However, the use of
this model is limited by the rare occurrence of this type of tumor in animals.
Other in. vitro and irr vivo cardiovascular, endothelial, and angiogenic tests
known in the art are also suitable
herein.
ii. Tissue Distribution
The results of the cardiovascular, endothelial, and angiogenic assays herein
can be verified by further studies,
such as by determining mRNA expression in various human tissues.
As noted before, gene amplification and/or gene expression in various tissues
may be measured by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA (Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 ( 1980)), dot blotting (DNA analysis), or in situ
hybridization, using an appropriately
labeled probe, based on the sequences provided herein. Alternativel~~,
antibodies may be employed that can
recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA
hybrid duplexes or
DNA-protein duplexes.
Gene expression in various tissues, alternatively, may be measured by
immunological methods, such as
immunohistochemical staining of tissue sections and assay of cell culture or
body fluids, to quantitate directly the
expression of gene product. Antibodies useful for immunohistochemical staining
and/or assay of sample fluids may
be either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be
prepared against a native-sequence PRO polypeptide or against a synthetic
peptide based on the DNA sequences
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provided herein or against exogenous sequence fused to PRO DNA and encoding a
specific antibody epitope.
General techniques for generating antibodies, and special protocols for in
situ hybridization are provided
hereinbelow.
iii. Antibody Binding Studies
$ The results of the cardiovascular, endothelial, and angiogenic study can be
further verified by antibody binding
studies, in which the ability of anti-PRO antibodies to inhibit the effect of
the PRO polypeptides on endothelial cells
or other cells used in the cardiovascular, endothelial, and angiogenic assays
is tested. Exemplary antibodies include
polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies,
the preparation of which will be
described hereinbelow.
Antibody binding studies may be carried out in any known assay method, such as
competitive binding assays,
direct and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of
Techniques (CRC Press, Inc., 1987), pp.147-158.
Competitive binding assays rely on the ability of a labeled standard to
compete with the test sample analyte
for binding with a limited amount of antibody. The amount of target protein in
the test sample is inversely
proportional to the amount of standard that becomes bound to the antibodies.
To facilitate determining the amount
of standard that becomes bound, the antibodies preferably are insolubilized
before or after the competition, so that
the standard and analyte that are bound to the antibodies may conveniently be
separated from the standard and
analyte that remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to
a different immunogenic
portion, or epitope, of the protein to be detected. In a sandwich assay, the
test sample analyte is bound by a first
antibody that is immobilized on a solid support, and thereafter a second
antibody binds to the analyte, thus forming
an insoluble three-part complex. See, e.g., US Pat No. 4,376,110. The second
antibody may itself be labeled with
a detectable moiety (direct sandwich assays) or may be measured using an anti-
immunoglobulin antibody that is
labeled with a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA
assay, in which case the detectable moiety is an enzyme.
For immunohistochemistry, the tissue sample may be fresh or frozen or may be
embedded in paraffin and fixed
with a preservative such as formalin, for example.
iv. Cell-Based Tumor Assays
Cell-based assays and animal models for cardiovascular, endothelial, and
angiogenic disorders, such as tumors,
can be used to verify the findings of a cardiovascular, endothelial, and
angiogenic assay herein, and further to
understand the relationship between the genes identified herein and the
development and pathogenesis of
undesirable cardiovascular. endothelial, and angiogenic cell growth. The role
of gene products identified herein
in the development and pathology of undesirable cardiovascular, endothelial.
and angiogenic cell growth, e.g., tumor
cells, can be tested by using cells or cells lines that have been identified
as being stimulated or inhibited by the PRO
polypeptide herein. Such cells include, for example, those set forth in the
Examples below.
In a different approach, cells of a cell type known to be involved in a
particular cardiovascular, endothelial,
and angiogenic disorder are transfected with the cDNAs herein, and the ability
of these cDNAs to induce excessive
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growth or inhibit growth is analyzed. If the cardiovascular, endothelial, and
angiogenic disorder is cancer, suitable
tumor cells include, for example, stable tumor cells lines such as the B 104-I-
1 cell line (stable NIH-3T3 cell line
transfected with the neu protooncogene) and ras-transfected NIH-3T3 cells,
which can be transfected with the
desired gene and monitored for tumorigenic growth. Such transfected cell lines
can then be used to test the ability
of poly-or monoclonal antibodies or antibody compositions to inhibit
tumorigenic cell growth by exerting cytostatic
or cytotoxic activity on the growth of the transformed cells, or by mediating
antibody-dependent cellular cytotoxicity
(ADCC). Cells transfected with the coding sequences of the genes identified
herein can further be used to identify
drug candidates for the treatment of cardiovascular, endothelial, and
angiogenic disorders such as cancer.
In addition, primary cultures derived from tumors in transgenic animals (as
described above) can be used in
the cell-based assays herein, although stable cell lines are preferred.
Techniques to derive continuous cell lines from
transgenic animals are well known in the art. See, e.g., Small et al., Mol.
Cell. Biol., 5: 642-648 (1985).
v. Gene Therapy
The PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide herein and
polypeptidyl agonists and
antagonists may be employed in accordance with the present invention by
expression of such polypeptides in vivo,
which is often referred to as gene therapy.
There are two major approaches to getting the nucleic acid (optionally
contained in a vector) into the patient's
cells: in vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the sites
where the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is required,
i.e., the site of synthesis of
the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, if known, and the site
(e.g., wound) where
biological activity of PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333,
PR0840. PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is
needed. For e.s vivo treatment,
the patient's cells are removed, the nucleic acid is introduced into these
isolated cells, and the modified cells are
administered to the patient either directly or, for example, encapsulated
within porous membranes that are implanted
into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There
are a variety of techniques available for
introducing nucleic acids into viable cells. The techniques vary depending
upon whether the nucleic acid is
transferred into cultured cells in vitro, or transferred in viro in the cells
of the intended host. Techniques suitable
for the transfer of nucleic acid into mammalian cells in vitro include the use
of liposomes, electroporation,
microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc.
Transduetion involves the association of a replication-defective, recombinant
viral (preferably retroviral) particle
with a cellular receptor, followed by introduction of the nucleic acids
contained by the particle into the cell. A
commonly used vector for ex vivo delivery of the gene is a retrovirus.
The currently preferred in vivo nucleic acid transfer techniques include
transfection with viral or non-viral
vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-
associated virus (AAV)) and lipid-based
systems (useful lipids for lipid-mediated transfer of the gene are, for
example, DOTMA, DOPE, and DC-Chol; see,
e.g., Tonkinson etal., Cancer Investigation. 14(1): 54-65 (1996)). The most
preferred vectors for use in gene
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therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. A viral vector such as a
retroviral vector includes at least one transcriptional promoter/enhancer or
locus-defining element(s), or other
elements that control gene expression by other means such as alternate
splicing, nuclear RNA export, or
post-translational modification of messenger. In addition, a viral vector such
as a retroviral vector includes a nucleic
acid molecule that, when transcribed in the presence of a gene encoding
PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide, is operably linked thereto and acts as a translation
initiation sequence. Such vector constructs
also include a packaging signal, long terminal repeats (LTRs) or portions
thereof, and positive and negative strand
primer binding sites appropriate to the virus used (if these are not already
present in the viral vector). In addition,
such vector typically includes a signal sequence for secretion of the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a
mammalian signal sequence, most preferably the native signal sequence for the
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 polypeptide. Optionally, the vector construct may also
include a signal that directs
polyadenylation, as well as one or more restriction sites and a translation
termination sequence. By way of example,
such vectors will typically include a 5' LTR, a tRNA binding site, a packaging
signal, an origin of second-strand
DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used
that are non-viral, such as cationic
lipids, polylysine, and dendrimers.
In some situations, it is desirable to provide the nucleic acid source with an
agent that targets the target cells,
such as an antibody specific for a cell-surface membrane protein or the target
cell, a ligand for a receptor on the
target cell, etc. Where liposomes are employed, proteins that bind to a cell-
surface membrane protein associated
with endocytosis may be used for targeting and/or to facilitate uptake, e.g.,
capsid proteins or fragments thereof
tropic for a particular cell type, antibodies for proteins that undergo
internalization in cycling, and proteins that
target intracellular localization and enhance intracellular half-life. The
technique of receptor-mediated endocytosis
is described, for example, by Wu et al., J. Biol. Chem., 262: 4429-4432 (
1987); and Wagner et al.. Proc. Natl. Acad.
Sci. USA, 87: 3410-3414 (1990). For a review of the currently known gene
markin~~ and gene therapy protocols,
see, Anderson et al., Science, 256: 808-813 (1992). See also WO 93/25673 and
the references cited therein.
Suitable gene therapy and methods for making retroviral particles and
structural proteins can be found in, e.g.,
U.S. Pat. No. 5,681,746.
vi. Use of Gene as Diagnostic
This invention is also related to the use of the gene encoding the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide as a diagnostic. Detection of a mutated form of the PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide will allow a diagnosis of a cardiovascular, endothelial,
and angio~ epic disease or a
susceptibility to a cardiovascular, endothelial, and angiogenic disease, such
as a tumor, since mutations in the
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321. PR0333,
PR0840, PR0877,
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PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide may cause tumors.
Individuals carrying mutations in the genes encoding a human PR0179, human
PR0238, human PR0364,
human PR0844, human PR0846, human PRO 1760, human PR0205, human PR0321, human
PR0333, human
PR0840, human PR0877, human PR0878, human PR0879, human PR0882, human PR0885
or human PR0887
polypeptide may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be
obtained from a patient's cells, such as from blood, urine, saliva, tissue
biopsy, and autopsy material. The genomic
DNA may be used directly for detection or may be amplified enzymatically by
using PCR (Saiki etal., Nature, 324:
163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same
purpose. As an example, PCR
primers complementary to the nucleic acid encoding the PRO 179, PR0238,
PR0364, PR0844, PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
can be used to identify and analyze PRO 179, PR0238, PR0364, PR0844, PR0846,
PRO 1760, PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
mutations. For
example, deletions and insertions can be detected by a change in size of the
amplified product in comparison to the
normal genotype. Point mutations can be identified by hybridizing amplified
DNA to radiolabeled RNA encoding
the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, or alternatively,
radiolabeled antisense DNA
sequences encoding the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide.
Perfectly matched sequences
can be distinguished from mismatched duplexes by RNase A digestion or by
differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection
of alteration in
electrophoretic mobility of DNA fragments in gels with or without denaturing
agents. Small sequence deletions
and insertions can be visualized by high resolution gel electrophoresis. DNA
fragments of different sequences may
be distinguished on denaturing formamidine gradient gels in which the
mobilities of different DNA fragments are
retarded in the gel at different positions according to their specific melting
or partial melting temperatures. See, e.g.,
Myers et al., Science, 230: 1242 (1985).
Sequence changes at specific locations may also be revealed by nuclease
protection assays, such as RNase
and S 1 protection or the chemical cleavage method, for example, Cotton et
al., Proc. Natl. Acad. Sci. USA, 85:
4397-4401 (1985).
Thus, the detection of a specific DNA sequence may be achieved by methods such
as hybridization, RNase
protection, chemical cleavage, direct DNA sequencing, or the use of
restriction enzymes, e.g., restriction fragment
length polymorphisms (RFLP), and Southern blotting of genomic DNA.
vii. Use to Detect PRO Polypeptide Levels
In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations can also be detected
by irz situ analysis.
Expression of nucleic acid encoding the PRO polypeptide may be linked to
vascular disease or
neovascularization associated with tumor formation. If the PRO polypeptide has
a signal sequence and the mRNA
is highly expressed in endothelial cells and to a lesser extent in smooth
muscle cells, this indicates that the PRO
polypeptide is present in serum. Accordingly, an anti-PRO polypeptide antibody
could be used to diagnose vascular
CA 02361849 2001-07-30
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disease or neovascularization associated with tumor formation, since an
altered level of this PRO polypeptide may
be indicative of such disorders.
A competition assay may be employed wherein antibodies specific to the PRO
polypeptide are attached to a
solid support and the labeled PRO polypeptide and a sample derived from the
host are passed over the solid support
and the amount of label detected attached to the solid support can be
correlated to a quantity of the PRO polypeptide
in the sample.
viii. Chromosome Mapping
The sequences 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.
1~ 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.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the
cDNA. Computer analysis for the 3'- untranslated region 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 fragment.
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 fragments 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 sitrr hybridization,
prescreening with labeled flow-sorted chromosomes, and preselection by
hybridization to construct chromosome-
specific cDNA libraries.
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 cDNA as short as 500 or
600 bases; however, clones larger than 2,000 by have a higher likelihood of
binding to a unique chromosomal
location with sufficient signal intensity for simple detection. FISH requires
use of the clones from which the gene
encoding the PR0179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 was derived, and the longer
the better. For example,
2,000 by is good. 4,000 by is better, and more than 4,000 is probably not
necessary to get good results a reasonable
percentage of the time. For a review of this technique, see. Verma er al.,
Human Chromosomes: a Manual of Basic
Technigues (Pergamon Press, New York, 1988).
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 online through Johns Hopkins
University Welch Medical Library). The
relationship between genes and diseases that have been mapped to the same
chromosomal region is then identified
through linkage analysis (coinheritance of physically adjacent genes).
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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.
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).
ix. Screening Assays for Drug Candidates
This invention encompasses methods of screening compounds to identify those
that mimic the PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 polypeptide (agonists) or prevent the effect
of the PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 polypeptide (antagonists). Screening assays for
antagonist drug candidates are
designed to identify compounds that bind or complex with the PR0179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
polypeptide encoded by the genes identified herein, or otherwise interfere
with the interaction of the encoded
polypeptides with other cellular proteins. Such screening assays will include
assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable for
identifying small molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein
binding assays, biochemical
screening assays, immunoassays, and cell-based assays, which are well
characterized in the art.
All assays for antagonists are common in that they call for contacting the
drug candidate with a PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 polypeptide encoded by a nucleic acid
identified herein under conditions
and for a time sufficient to allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the reaction
mixture. In a particular embodiment, the PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide encoded by
the gene identified herein or the drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by
covalent or non-covalent attachments. Non-covalent attachment generally is
accomplished by coating the solid
surface with a solution of the PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
and drying.
Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific
for the PRO 179. PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333. PR0840, PR0877, PR0878.
PR0879, PR0882,
PR0885 or PR0887 polypeptide to be immobilized can be used to anchor it to a
solid surface. The assay is
performed by adding the non-immobilized component, which may be labeled by a
detectable label, to the
invnobilized component, e.g., the coated surface containing the anchored
component. When the reaction is
complete, the non-reacted components are removed, e.g., by washing, and
complexes anchored on the solid surface
are detected. When the originally non-immobilized component carries a
detectable label, the detection of label
immobilized on the surface indicates that complexing occurred. Where the
originally non-immobilized component
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does not carry a label, complexing can be detected, for example, by using a
labeled antibody specifically binding
the immobilized complex.
If the candidate compound interacts with but does not bind to a particular
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 polypeptide encoded by a gene identified herein, its
interaction with that polypeptide can be
assayed by methods well known for detecting protein-protein interactions. Such
assays include traditional
approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-
purification through gradients or
chromatographic columns. In addition, protein-protein interactions can be
monitored by using a yeast-based genetic
system described by Fields and co-workers (Fields and Song, Nature (London),
340: 245-246 (1989); Chien etal.,
Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)) as disclosed by Chevray and
Nathans, Proc. Natl. Acad. Sci.
USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast
GAL4, consist of two physically
discrete modular domains, one acting as the DNA-binding domain, the other one
functioning as the transcription-
activation domain. The yeast expression system described in the foregoing
publications (generally referred to as
the "two-hybrid system" ) takes advantage of this property, and employs two
hybrid proteins, one in which the target
protein is fused to the DNA-binding domain of GAL4, and another, in which
candidate activating proteins are fused
to the activation domain. The expression of a GAL1-lacZ reporter gene under
control of a GAL4-activated
promoter depends on reconstitution of GAL4 activity via protein-protein
interaction. Colonies containing
interacting polypeptides are detected with a chromogenic substrate for ~-
galactosidase. A complete kit
(MATCHMAKERTM) for identifying protein-protein interactions between two
specific proteins using the two-
hybrid technique is commercially available from Clontech. This system can also
be extended to map protein
domains involved in specific protein interactions as well as to pinpoint amino
acid residues that are crucial for these
interactions.
Compounds that interfere with the interaction of a gene encoding a PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide identified herein and other intra- or extracellular
components can be tested as follows: usually
a reaction mixture is prepared containing the product of the gene and the
intra- or exti-acellular component under
conditions and for a time allowing for the interaction and binding of the two
products. To test the ability of a
candidate compound to inhibit binding, the reaction is run in the absence and
in the presence of the test compound.
In addition, a placebo may be added to a third reaction mixture, to serve as
positive control. The binding (complex
formation) between the test compound and the intra- or extracellular component
present in the mixture is monitored
as described hereinabove. The formation of a complex in the control reactions)
but not in the reaction mixture
containing the test compound indicates that the test compound interferes with
the interaction of the test compound
and its reaction partner.
If the PRO polypeptide has the ability to stimulate the proliferation of
endothelial cells in the presence of the
co-mitogen ConA, then one example of a screening method takes advantage of
this ability. Specifically, in the
proliferation assay, human umbilical vein endothelial cells are obtained and
cultured in 96-well flat-bottomed
culture plates (Costar, Cambridge, MA) and supplemented with a reaction
mixture appropriate for facilitating
proliferation of the cells, the mixture containing Con-A (Calbiochem, La
Jolla, CA). Con-A and the compound to
be screened are added and after incubation at 37 °C, cultures are
pulsed with 3-H-thymidine and harvested onto glass
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fiber filters (phD; Cambridge Technology, Watertown, MA). Mean'-H-thymidine
incorporation (cpm) of triplicate
cultures is determined using a liquid scintillation counter (Beckman
Instruments, Irvine, CA). Significant 3~(H)-
thymidine incorporation indicates stimulation of endothelial cell
proliferation.
To assay for antagonists, the assay described above is performed; however, in
this assay the PRO polypeptide
is added along with the compound to be screened and the ability of the
compound to inhibit 3-(H)thymidine
incorporation in the presence of the PRO polypeptide indicates that the
compound is an antagonist to the PRO
polypeptide. Alternatively, antagonists may be detected by combining the PRO
polypeptide and a potential
antagonist with membrane-bound PRO polypeptide receptors or recombinant
receptors under appropriate conditions
for a competitive inhibition assay. The PRO polypeptide can be labeled, such
as by radioactivity, such that the
number of PRO polypeptide molecules bound to the receptor can be used to
determine the effectiveness of the
potential antagonist. The gene encoding the receptor can be identified by
numerous methods known to those of skill
in the art, for example, ligand panning and FACS sorting. Coligan et al..
Current Protocols in Immun., ~:
Chapter 5 ( 1991 ). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell
responsive to the PRO polypeptide and a cDNA library created from this RNA is
divided into pools and used to
transfect COS cells or other cells that are not responsive to the PRO
polypeptide. Transfected cells that are grown
on glass slides are exposed to the labeled PRO polypeptide. The PRO
polypeptide can be labeled by a variety of
means including iodination or inclusion of a recognition site for a site-
specific protein kinase. Following fixation
and incubation, the slides are subjected to autoradiographic analysis.
Positive pools are identified and sub-pools
are prepared and re-transfected using an interactive sub-pooling and re-
screening process, eventually yielding a
single clone that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled PRO
polypeptide can be photoaffinity-
linked with cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is
resolved by PAGE and exposed to X-ray film. The labeled complex containing the
receptor can be excised,
resolved into peptide fragments, and subjected to protein micro-sequencing.
The amino acid sequence obtained
from micro-sequencing would be used to design a set of degenerate
oli';onucleotide probes to screen a cDNA library
to identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor would
be incubated with the labeled PRO polypeptide in the presence of the candidate
compound. The ability of the
compound to enhance or block this interaction could then be measured.
The compositions useful in the treatment of cardiovascular, endothelial, and
angiogenic disorders include,
without limitation. antibodies, small organic and inorganic molecules,
peptides, phosphopeptides, antisense and
ribozyme molecules, triple-helix molecules, etc., that inhibit the expression
and/or activity of the target gene.
product.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions of
immunoglobulin with a PRO polypeptide, and, in particular, antibodies
including, without limitation, poly- and
monoclonal antibodies and antibody fragments, single-chain antibodies, anti-
idiotypic antibodies, and chimeric or
humanized versions of such antibodies or fragments, as well as human
antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for
example, a mutated form of the PRO
polypeptide that recognizes the receptor but imparts no effect, thereby
competitively inhibiting the action of the
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PRO polypeptide.
Another potential PRO polypeptide antagonist or agonist is an antisense RNA or
DNA construct prepared
using antisense technology, where, e.g., an antisense RNA or DNA molecule acts
to block directly the translation
of mRNA by hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used
to control gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are
based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding
portion of the polynucleotide
sequence, which encodes the mature PRO polypeptides herein, is 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 (triple helix - see, Lee et al., Nucl.
Acids Res., 6:3073 ( 1979); Cooney et al.,
Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby
preventing transcription and the
production of the PRO polypeptide. The antisense RNA oligonucieotide
hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the PRO polypeptide (antisense - Okano,
Neurochem., 56:560 (1991);
Oli~odeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press:
Boca Raton, FL, 1988). The
oligonucleotides described above can also be delivered to cells such that the
antisense RNA or DNA may be
expressed ira vivo to inhibit production of the PRO polypeptide. When
antisense DNA is used,
oligodeoxyribonucleotides derived from the translation-initiation site, e.g.,
between about -10 and +10 positions
of the target gene nucleotide sequence, are preferred.
Antisense RNA or DNA molecules are generally at least about 5 bases in length,
about 10 bases in length,
about 15 bases in length, about 20 bases in length, about 25 bases in length,
about 30 bases in length, about 35 bases
2~ in length, about 40 bases in length, about 45 bases in length, about 50
bases in length, about 55 bases in length,
about 60 bases in length, about 65 bases in length, about 70 bases in length,
about 75 bases in length, about 80 bases
in length, about 85 bases in length, about 90 bases in length, about 95 bases
in length, about 100 bases in length,
or more.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site. or growth
factor or other relevant binding site of the PRO polypeptide, thereby blocking
the normal biological activity of the
PRO polypeptide. Examples of small molecules include, but are not limited to,
small peptides or peptide-like
molecules, preferably soluble peptides, and synthetic non-peptidyl organic or
inorganic compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA. Ribozymes
act by sequence-specific hybridization to the complementary target RNA,
followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within a potential RNA target can be
identified by known techniques. For further
details see, e.g., Rossi, Current Biolo~y, 4: 469-471 (1994), and PCT
publication No. WO 97/33551 (published
September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded and
composed of deoxynucleotides. The base composition of these oligonucleotides
is designed such that it promotes
triple-helix formation via Hoogsteen base-pairing rules, which generally
require sizeable stretches of purines or
pyrimidines on one strand of a duplex. For further details see, e.g., PCT
publication No. WO 97/33551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed hereinabove
and/or by any other screening techniques well known for those skilled in the
art.
CA 02361849 2001-07-30
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x. Types of Cardiovascular, Endothelial, and AnQiorenic Disorders to be
Treated
The PRO polypeptides, or agonists or antagonists thereto, that have activity
in the cardiovascular, angiogenic,
and endothelial assays described herein, and/or whose gene product has been
found to be localized to the
cardiovascular system, are likely to have therapeutic uses in a variety of
cardiovascular, endothelial, and angiogenic
disorders, including systemic disorders that affect vessels, such as diabetes
mellitus. Their therapeutic utility could
include diseases of the arteries, capillaries, veins, and/or lymphatics.
Examples of treatments hereunder include
treating muscle wasting disease, treating osteoporosis, aiding in implant
fixation to stimulate the growth of cells
around the implant and therefore facilitate its attachment to its intended
site, increasing IGF stability in tissues or
in serum, if applicable, and increasing binding to the IGF receptor (since IGF
has been shown in vitro to enhance
human marrow erythroid and granulocytic progenitor cell growth).
The PRO polypeptides or agonists or antagonists thereto may also be employed
to stimulate erythropoiesis
or granulopoiesis, to stimulate wound healing or tissue regeneration and
associated therapies concerned with re-
growth of tissue, such as connective tissue, skin, bone, cartilage, muscle,
lung, or kidney, to promote angiogenesis,
to stimulate or inhibit migration of endothelial cells, and to proliferate the
growth of vascular smooth muscle and
endothelial cell production. The increase in angiogenesis mediated by the PRO
polypeptide or antagonist would
be beneficial to ischemic tissues and to collateral coronary development in
the heart subsequent to coronary stenosis.
Antagonists are used to inhibit the action of such polypeptides, for example,
to limit the production of excess
connective tissue during wound healing or pulmonary fibrosis if the PRO
polypeptide promotes such production.
This would include treatment of acute myocardial infarction and heart failure.
Moreover, the present invention concerns the treatment of cardiac hypertrophy,
regardless of the underlying
cause, by administering a therapeutically effective dose of the PRO
polypeptide, or agonist or antagonist thereto.
If the objective is the treatment of human patients, the PRO polypeptide
preferably is recombinant human PRO
polypeptide (rhPRO polypeptide). The treatment for cardiac hypertrophy can be
performed at any of its various
stages, which may result from a variety of diverse pathologic conditions,
including myocardial infarction,
hypertension, hypertrophic cardiomyopathy, and valvular regurgitation. The
treatment extends to all stages of the
progression of cardiac hypertrophy, with or without structural damage of the
heart muscle, regardless of the
underlying cardiac disorder.
The decision of whether to use the molecule itself or an agonist thereof for
any particular indication, as
opposed to an antagonist to the molecule, would depend mainly on whether the
molecule herein promotes
cardiovascularization, genesis of endothelial cells, or angiogenesis or
inhibits these conditions. For example, if the
molecule promotes angiogenesis, an antagonist thereof would be useful for
treatment of disorders where it is desired
to limit or prevent angiogenesis. Examples of such disorders include vascular
tumors such as haemangioma, tumor
angiogenesis, neovascularization in the retina, choroid, or cornea, associated
with diabetic retinopathy or premature
infantretinopathyormaculardegeneration
andproliferativevitreoretinopathy,rheumatoidarthritis,Crohn'sdisease,
3S atherosclerosis, ovarian hyperstimulation, psoriasis, endometriosis
associated with neovascularization, restenosis
subsequent to balloon angioplasty, scar tissue overproduction, for example,
that seen in a keloid that forms after
surgery, fibrosis after myocardial infarction, or fibrotic lesions associated
with pulmonary fibrosis.
If, however, the molecule inhibits angiogenesis, it would be expected to be
used directly for treatment of the
above conditions.
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On the other hand, if the molecule stimulates angiogenesis it would be used
itself (or an agonist thereof) for
indications where angiogenesis is desired such as peripheral vascular disease,
hypertension, inflammatory
vasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, arterial
restenosis, thrombophlebitis,
lymphangitis, lymphedema, wound healing and tissue repair, ischemia
reperfusion injury, angina, myocardial
infarctions such as acute myocardial infarctions, chronic heart conditions,
heart failure such as congestive heart
failure, and osteoporosis.
If, however, the molecule inhibits angiogenesis, an antagonist thereof would
be used for treatment of those
conditions where angiogenesis is desired.
Specific types of diseases are described below, where the PRO polypeptide
herein or antagonists thereof may
serve as useful for vascular-related drug targeting or as therapeutic targets
for the treatment or prevention of the
disorders. Atherosclerosis is a disease characterized by accumulation of
plaques of intimal thickening in arteries,
due to accumulation of lipids, proliferation of smooth muscle cells, and
formation of fibrous tissue within the
arterial wall. The disease can affect large, medium, and small arteries in any
organ. Changes in endothelial and
vascular smooth muscle cell function are known to play an important role in
modulating the accumulation and
regression of these plaques.
Hypertension is characterized by raised vascular pressure in the systemic
arterial, pulmonary arterial, or portal
venous systems. Elevated pressure may result from or result in impaired
endothelial function and/or vascular
disease.
Inflammatory vasculitides include giant cell arteritis, Takayasu's arteritis,
polyarteritis nodosa (including the
microangiopathic form), Kawasaki's disease, microscopic polyangiitis,
Wegener's granulomatosis, and a variety of
infectious-related vascular disorders (including Henoch-Schonlein prupura).
Altered endothelial cell function has
been shown to be important in these diseases.
Reynaud's disease and Reynaud's phenomenon are characterized by intermittent
abnormal impairment of the
circulation through the extremities on exposure to cold. Altered endothelial
cell function has been shown to be
important in this disease.
Aneurysms are saccular or fusiform dilatations of the arterial or venous tree
that are associated with altered
endothelial cell andlor vascular smooth muscle cells.
Arterial restenosis (restenosis of the arterial wall) may occur following
angioplasty as a result of alteration
in the function and proliferation of endothelial and vascular smooth muscle
cells.
Thrombophlebitis and lymphangitis are inflammatory disorders of veins and
lymphatics, respectively, that may
result from, and/or in, altered endothelial cell function. Similarly,
lymphedema is a condition involving impaired
lymphatic vessels resulting from endothelial cell function.
The family of benign and malignant vascular tumors are characterized by
abnormal proliferation and growth
of cellular elements of the vascular system. For example, lymphangiomas are
benign tumors of the lymphatic system
that are congenital, often cystic, malformations of the lymphatics that
usually occur in newborns. Cystic tumors
tend to grow into the adjacent tissue. Cystic tumors usually occur in the
cervical and axillary region. They can also
occur in the soft tissue of the extremities. The main symptoms are dilated,
sometimes reticular, structured
lymphatics and lymphocysts surrounded by connective tissue. Lymphangiomas are
assumed to be caused by
improperly connected embryonic lymphatics or their deficiency. The result is
impaired local lymph drainage.
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Griener etal., Lympholo~y, 4: 140-144 (1971).
Another use for the PRO polypeptides herein or antagonists thereto is in the
prevention of tumor angiogenesis,
which involves vascularization of a tumor to enable it to growth and/or
metastasize. This process is dependent on
the growth of new blood vessels. Examples of neoplasms and related conditions
that involve tumor angiogenesis
include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal
carcinomas, colorectal carcinomas, liver
carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical
carcinomas, endometrial carcinoma,
endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head
and neck cancer, nasopharyngeal
carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma,
skin carcinomas, hemangioma,
cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma,
astrocytoma, glioblastoma,
Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas,
rhabdomyosarcoma, osteogenic sarcoma,
leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor,
renal cell carcinoma, prostate
carcinoma, abnormal vascular proliferation associated with phakomatoses, edema
(such as that associated with brain
tumors), and Meigs' syndrome.
Age-related macular degeneration (AMD) is a leading cause of severe visual
loss in the elderly population.
The exudative form of AMD is characterized by choroidal neovascularization and
retinal pigment epithelial cell
detachment. Because choroidal neovascularization is associated with a dramatic
worsening in prognosis, the PRO
polypeptide or antagonist thereto is expected to be useful in reducing the
severity of AMD.
Healing of trauma such as wound healing and tissue repair is also a targeted
use for the PRO polypeptides
herein or their antagonists. Formation and regression of new blood vessels is
essential for tissue healing and repair.
This category includes bone, cartilage, tendon, ligament, and/or nerve tissue
growth or regeneration, as well as
wound healing and tissue repair and replacement, and in the treatment of
burns, incisions, and ulcers. A PRO
polypeptide or antagonist thereof that induces cartilage and/or bone growth in
circumstances where bone is not
normally formed has application in the healing of bone fractures and cartilage
damage or defects in humans and
other animals. Such a preparation employing a PRO polypeptide or antagonist
thereof may have prophylactic use
in closed as well as open fracture reduction and also in the improved fixation
of artificial joints. De rrovo bone
formation induced by an osteogenic agent contributes to the repair of
congenital, trauma-induced, or oncologic,
resection-induced craniofacial defects, and also is useful in cosmetic plastic
surgery.
PRO polypeptides or antagonists thereto may also be useful to promote better
or faster closure of non-healin;~
wounds, including without limitation pressure ulcers, ulcers associated with
vascular insufficiency, surgical and
traumatic wounds, and the like.
It is expected that a PRO polypeptide or antagonist thereto may also exhibit
activity for generation or
regeneration of other tissues, such as organs (including, for example,
pancreas, liver, intestine, kidney, skin, or
endothelium), muscle (smooth, skeletal, or cardiac), and vascular (including
vascular endothelium) tissue, or for
promoting the growth of cells comprising such tissues. Part of the desired
effects may be by inhibition or
modulation of fibrotic scarring to allow normal tissue to regenerate.
A PRO polypeptide herein or antagonist thereto may also be useful for gut
protection or regeneration and
treatment of lung or liver fibrosis, reperfusion injury in various tissues,
and conditions resulting from systemic
cytokine damage. Also, the PRO polypeptide or antagonist thereto may be useful
for promoting or inhibiting
differentiation of tissues described above from precursor tissues or cells, or
for inhibiting the growth of tissues
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described above.
A PRO polypeptide or antagonist thereto may also be used in the treatment of
periodontal diseases and in other
tooth-repair processes. Such agents may provide an environment to attract bone-
forming cells, stimulate growth
of bone-forming cells, or induce differentiation of progenitors of bone-
forming cells. A PRO polypeptide herein
or an antagonist thereto may also be useful in the treatment of osteoporosis
or osteoarthritis, such as through
stimulation of bone and/or cartilage repair or by blocking inflammation or
processes of tissue destruction
(collagenase activity, osteoclast activity, etc.) mediated by inflammatory
processes, since blood vessels play an
important role in the regulation of bone turnover and growth.
Another category of tissue regeneration activity that may be attributable to
the PRO polypeptide herein or
antagonist thereto is tendon/ligament formation. A protein that induces
tendon/ligament-like tissue or other tissue
formation in circumstances where such tissue is not normally formed has
application in the healing of tendon or
ligament tears, deformities, and other tendon or ligament defects in humans
and other animals. Such a preparation
may have prophylactic use in preventing damage to tendon or ligament tissue,
as well as use in the improved
fixation of tendon or ligament to bone or other tissues, and in repairing
defects to tendon or ligament tissue. De
novo tendon/ligament-like tissue formation induced by a composition of the PRO
polypeptide herein or antagonist
thereto contributes to the repair of congenital, trauma-induced, or other
tendon or ligament defects of other origin,
and is also useful in cosmetic plastic surgery for attachment or repair of
tendons or ligaments. The compositions
herein may provide an environment to attract tendon- or ligament-forming
cells, stimulate growth of tendon- or
ligament-forming cells, induce differentiation of progenitors of tendon- or
ligament-forming cells, or induce growth
of tendon/ligament cells or progenitors ex vivo for return iu vivo to effect
tissue repair. The compositions herein
may also be useful in the treatment of tendinitis, carpal tunnel syndrome, and
other tendon or ligament defects. The
compositions may also include an appropriate matrix and/or sequestering agent
as a carrier as is well known in the
art.
The PRO polypeptide or its antagonist may also be useful for proliferation of
neural cells and for regeneration
of nerve and brain tissue, i.e., for the treatment of central and peripheral
nervous system disease and neuropathies,
as well as mechanical and traumatic disorders, that involve degeneration,
death, or trauma to neural cells or nerve
tissue. More specifically, a PRO polypeptide or its antagonist may be used in
the treatment of diseases of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and localized neuropathies, and
central nervous system diseases, such as Alzheimer's, Parkinson's disease,
Huntington's disease, amyotrophic lateral
sclerosis, and Shy-Drager syndrome. Further conditions that may be treated in
accordance with the present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head trauma, and
cerebrovascular diseases such as stroke. Peripheral neuropathies resulting
from chemotherapy or other medical
therapies may also be treatable using a PRO polypeptide herein or antagonist
thereto.
Ischemia-reperfusion injury is another indication. Endothelial cell
dysfunction may be important in both the
initiation of, and in regulation of the sequelae of events that occur
following ischemia-reperfusion injury.
Rheumatoid arthritis is a further indication. Blood vessel growth and
targeting of inflammatory cells through
the vasculature is an important component in the pathogenesis of rheumatoid
and sera-negative forms of arthritis.
A PRO polypeptide or its antagonist may also be administered prophylactically
to patients with cardiac
hypertrophy, to prevent the progression of the condition, and avoid sudden
death, including death of asymptomatic
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patients. Such preventative therapy is particularly warranted in the case of
patients diagnosed with massive left
ventricular cardiac hypertrophy (a maximal wall thickness of 35 mm or more in
adults, or a comparable value in
children), or in instances when the hemodynamic burden on the heart is
particularly strong.
A PRO polypeptide or its antagonist may also be useful in the management of
atrial fibrillation, which
develops in a substantial portion of patients diagnosed with hypertrophic
cardiomyopathy.
Further indications include angina, myocardial infarctions such as acute
myocardial infarctions, and heart
failure such as congestive heart failure. Additional non-neoplastic conditions
include psoriasis, diabetic and other
proliferative retinopathies including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma,
thyroid hyperplasias (including Grave's disease), corneal and other tissue
transplantation, chronic inflammation,
lung inflammation, nephrotic syndrome, preeclampsia, ascites, pericardial
effusion (such as that associated with
pericarditis), and pleural effusion.
In view of the above, the PRO polypeptides or agonists or antagonists thereof
described herein, which are
shown to alter or impact endothelial cell function, proliferation, and/or
form, are likely to play an important role
in the etiology and pathogenesis of many or all of the disorders noted above,
and as such can serve as therapeutic
targets to augment or inhibit these processes or for vascular-related drug
targeting in these disorders.
xi. Administration Protocols. Schedules Doses and Formulations
The molecules herein and agonists and antagonists thereto are pharmaceutically
useful as a prophylactic and
therapeutic agent for various disorders and diseases as set forth above.
Therapeutic compositions of the PRO polypeptides or agonists or antagonists
are prepared for storage by
mixing the desired molecule having the appropriate degree of purity with
optional pharmaceutically acceptable
carriers, excipients, or stabilizers (Reminaton's Pharmaceutical Sciences,
16th edition, Osol, A. ed. ( 1980)), in the
form of lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and include buffers
such as phosphate. citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium
chloride: phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol; cyclohexanol: 3-pentanol;
and m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin.
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose, mannitol, trehalose
or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-
ionic surfactants such as TWEENT"', PLURONICSTM or polyethylene glycol (PEG).
Additional examples of such carriers include ion exchangers, alumina, aluminum
stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water,
salts, or electrolytes such as protamine
sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and
polyethylene glycol. Carriers for
topical or gel-based forms of antagonist include polysaccharides such as
sodium carboxymethylcellulose or
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methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-
polyoxypropylene-block polymers,
polyethylene glycol, and wood wax alcohols. For all administrations,
conventional depot forms are suitably used.
Such forms include, for example, microcapsules, nano-capsules, liposomes,
plasters, inhalation forms, nose sprays,
sublingual tablets, and sustained-release preparations. The PRO polypeptides
or agonists or antagonists will
typically be formulated in such vehicles at a concentration of about 0.1 mg/ml
to 100 mg/ml.
Another formulation comprises incorporating a PRO polypeptide or antagonist
thereof into formed articles.
Such articles can be used in modulating endothelial cell growth and
angiogenesis. In addition, tumor invasion and
metastasis may be modulated with these articles.
PRO polypeptide or antagonist to be used for irc vivo administration must be
sterile. This is readily
accomplished by filtration through sterile filtration membranes, prior to or
following lyophilization and
reconstitution. PRO polypeptide ordinarily will be stored in lyophilized form
or in solution if administered
systemically. If in lyophilized form, PRO polypeptide or antagonist thereto is
typically formulated in combination
with other ingredients for reconstitution with an appropriate diluent at the
time for use. An example of a liquid
formulation of PRO polypeptide or antagonist is a sterile, clear, colorless
unpreserved solution filled in a single-
dose vial for subcutaneous injection. Preserved pharmaceutical compositions
suitable for repeated use may contain,
for example, depending mainly on the indication and type of polypeptide:
a) PRO polypeptide or agonist or antagonist thereto;
b) a buffer capable of maintaining the pH in a range of maximum stability of
the polypeptide or other
molecule in solution, preferably about 4-8;
c) a detergent/surfactant primarily to stabilize the polypeptide or molecule
against agitation-induced
aggregarion;
d) an isotonifier;
e) a preservative selected from the group of phenol, benzyl alcohol and a
benzethonium halide, e.g., chloride;
and
f) water.
If the detergent employed is non-ionic, it may, for example, be polysorbates
(e.g., POLYSOR$ATET"~
(TWEENT"~) 20, 80, etc.) or poloxamers (e.g., POLOXAMERT"' 188). The use of
non-ionic surfactants permits
the formulation to be exposed to shear surface stresses without causing
denaturation of the polypeptide. Further,
such surfactant-containing formulations may be employed in aerosol devices
such as those used in a pulmonary
dosing, and needleless jet injector guns (see, e.g., EP 257,956).
An isotonifier may be present to ensure isotonicity of a liquid composition of
the PRO polypeptide or
antagonist thereto, and includes polyhydric sugar alcohols, preferably
trihydric or higher sugar alcohols, such as
glycerin, erythritol, arabitol, xylitol. sorbitol, and mannitol. These sugar
alcohols can be used alone or in
combination. Alternatively, sodium chloride or other appropriate inorganic
salts may be used to render the solutions
isotonic.
The buffer may, for example, be an acetate, citrate, succinate, or phosphate
butter depending on the pH
desired. The pH of one type of liquid formulation of this invention is
buffered in the range of about 4 to 8,
preferably about physiological pH.
The preservatives phenol, benzvl alcohol and benzethonium halides, e.g.,
chloride, are known antimicrobial
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agents that may be employed.
Therapeutic PRO polypeptide compositions generally are placed into a container
having a sterile access port,
for example, an intravenous solution bag or vial having a stopper pierceable
by a hypodermic injection needle. The
formulations are preferably administered as repeated intravenous (i.v.),
subcutaneous (s.c.), or intramuscular (i.m.)
injections, or as aerosol formulations suitable for intranasal or
intrapulmonary delivery (for intrapulmonary delivery
see, e.g., EP 257,956).
PRO polypeptide can also be administered in the form of sustained-released
preparations. Suitable examples
of sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the
protein, which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-
release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-
methacrylate) as described by Langer et
al., J. Biomed. Mater. Res., 15: 167-277 (1981 ) and Langer, Chem. Tech., 12:
98-105 ( 1982) orpoly(vinylalcohol)),
polylactides (U.S. Patent No. 3,773,919, EP 58,481 ), copolymers of L-alutamic
acid and gamma ethyl-L-glutamate
(Sidman et al., Biopolymers, 22: 547-556 (1983)), non-degradable ethylene-
vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the Lupron DepotTM
(injectable microspheres composed
of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-
3-hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
enable release of molecules for
over 100 days, certain hydrogels release proteins for shorter time periods.
When encapsulated proteins remain in
the body for a long time, they may denature or aggregate as a result of
exposure to moisture at 37°C, resulting in
a loss of biological activity and possible changes in immunogenicity. Rational
strategies can be devised for protein
stabilization depending on the mechanism involved. For example, if the
aggregation mechanism is discovered to
be intermolecular S-S bond formation through thio-disulfide interchange,
stabiiization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling
moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
Sustained-release PRO polypeptide compositions also include liposomally
entrapped PRO polypeptides.
Liposomes containing the PRO polypeptide are prepared by methods known per se:
DE 3,218, I 21; 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
patent application 83-118008; U.S.
Patent 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. % cholesterol, the selected
proportion being adjusted for the optimal therapy.
The therapeutically effective dose of PRO polypeptide or anta~~onist thereto
will, of course, vary dependin;~
on such factors as the pathological condition to be treated (including
prevention), the method of administration, the
type of compound being used for treatment, any co-therapy involved. the
patient's age, weight, general medical
condition, medical history, etc., and its determination is well within the
skill of apracticing physician. Accordingly,
it will be necessary for the therapist to titer the dosage and modify the
route of administration as required to obtain
the maximal therapeutic effect. If the PRO polypeptide has a narrow host
range, for the treatment of human patients
formulations comprising human PRO polypeptide, more preferably native-sequence
human PRO polypeptide, are
preferred. The clinician will administer PRO polypeptide until a dosa~~e is
reached that achieves the desired effect
for treatment of the condition in question. For example, if the objective is
the treatment of CHF, the amount would
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be one that inhibits the progressive cardiac hypertrophy associated with this
condition. The progress of this therapy
is easily monitored by echo cardiography. Similarly, in patients with
hypertrophic cardiomyopathy, PRO
polypeptide can be administered on an empirical basis.
With the above guidelines, the effective dose generally is within the range of
from about 0.001 to about 1.0
mg/kg, more preferably about 0.01-1.0 mg/kg, most preferably about 0.01-0.1
mg/kg.
For non-oral use in treating human adult hypertension, it is advantageous to
administer PRO polypeptide in
the form of an injection at about 0.01 to 50 mg, preferably about 0.05 to 20
mg, most preferably 1 to 20 mg, per kg
body weight, 1 to 3 times daily by intravenous injection. For oral
administration, a molecule based on the PRO
polypeptide is preferably administered at about 5 mg to I g, preferably about
10 to 100 mg, per kg body weight, 1
to 3 times daily. It should be appreciated that endotoxin contamination should
be kept minimally at a safe level,
for example, less than 0.5 ng/mg protein. Moreover, for human administration,
the formulations preferably meet
sterility, pyrogenicity, general safety, and purity as required by FDA Office
and Biologics standards.
The dosage regimen of a pharmaceutical composition containing PRO polypeptide
to be used in tissue
regeneration will be determined by the attending physician considering various
factors that modify the action of the
polypeptides, e.g., amount of tissue weight desired to be formed, the site of
damage, the condition of the damaged
tissue, the size of a wound, type of damaged tissue (e.g., bone), the
patient's age, sex, and diet, the severity of any
infection, time of administration, and other clinical factors. The dosage may
vary with the type of matrix used in
the reconstitution and with inclusion of other proteins in the pharmaceutical
composition. For example, the addition
of other known growth factors, such as IGF-I, to the final composition may
also affect the dosage. Progress can
be monitored by periodic assessment of tissue/bone growth and/or repair, for
example, X-rays, histomorphometric
determinations, and tetracycline labeling.
The route of PRO polypeptide or antagonist or agonist administration is in
accord with known methods, e.g.,
by injection or infusion by intravenous, intramuscular, intracerebral,
intraperitoneal, intracerobrospinal,
subcutaneous, intraocular, intraarticular, intrasynovial, intrathecal, oral,
topical, or inhalation routes, or by
sustained-release systems as noted below. The PRO polypeptide or antagonists
thereof also are suitably
administered by intratumoral, peritumoral, intralesional, or perilesional
routes, to exert local as well as systemic
therapeutic effects. The intraperitoneal route is expected to be particularly
useful, for example, in the treatment of
ovarian tumors.
If a peptide or small molecule is employed as an antagonist or agonist, it is
preferably administered orally or
non-orally in the form of a liquid or solid to mammals.
Examples of pharmacologically acceptable salts of molecules that form salts
and are useful hereunder include
alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal
salts (e.g., calcium salt, magnesium salt),
ammonium salts, organic base salts (e.g., pyridine salt, triethylamine salt),
inorganic acid salts (e.g., hydrochloride,
sulfate, nitrate), and salts of organic acid (e.g., acetate, oxalate, p-
toluenesulfonate).
For compositions herein that are useful for bone, cartilage, tendon, or
ligament regeneration, the therapeutic
method includes administering the composition topically, systemically, or
locally as an implant or device. When
administered, the therapeutic composition for use is in a pyrogen-free,
physiologically acceptable tOCIIl. Further,
the composition may desirably be encapsulated or injected in a viscous form
for delivery to the site of bone,
cartilage, or tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Preferably,
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for bone and/or cartilage formation, the composition would include a matrix
capable of delivering the protein-
containing composition to the site of bone and/or cartilage damage, providing
a structure for the developing bone
and cartilage and preferably capable of being resorbed into the body. Such
matrices may be formed of materials
presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic
appearance, and interface properties. The particular application of the
compositions will define the appropriate
formulation. Potential matrices for the compositions may be biodegradable and
chemically defined calcium sulfate,
tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid, and
polyanhydrides. Other potential
materials are biodegradable and biologically well-defined, such as bone or
dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components. Other potential
matrices are nonbiodegradable and
chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be
comprised of combinations of any of the above-mentioned types of material,
such as polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be
altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size, particle size,
particle shape, and biodegradability.
One specific embodiment is a 50:50 (mole weight) copolymer of lactic acid and
glycolic acid in the form of
porous particles having diameters ranging from 150 to 800 microns. In some
applications, it will be useful to utilize
a sequestering agent, such as carboxymethyl cellulose or autologous blood
clot, to prevent the polypeptide
compositions from disassociating from the matrix.
One suitable family of sequestering agents is cellulosic materials such as
alkylcelluloses (including
hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydoxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, and carboxymethylcellulose, one preferred being
cationic salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic acid, sodium alginate,
polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer, and
polyvinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt%, based on
total formulation weight, which
represents the amount necessary to prevent desorption of the polypeptide (or
its antagonist) from the polymer matrix
and to provide appropriate handling of the composition, yet not so much that
the progenitor cells are prevented from
infiltrating the matrix, thereby providing the polypeptide (or its antagonist)
the opportunity to assist the osteogenic
activity of the progenitor cells.
xii. Combination Therapies
The effectiveness of the PRO 179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide
or an agonist or
antagonist thereof in preventing or treating the disorder in question may be
improved by administering the active
agent serially or in combination with another a~>ent that is effective for
those purposes, either in the same
composition or as separate compositions.
For example, for treatment of cardiac hypertrophy, PRO polypeptide therapy can
be combined with the
administration of inhibitors of known cardiac myocyte hypertrophy factors,
e.g., inhibitors of a-adrenergic agonists
such as phenylephrine; endothelin-I inhibitors such as BOSENTANT"' and
MOXONODINT"'~ inhibitors to CT-I
(US Pat. No. 5,679,545); inhibitors to LIF; ACE inhibitors; des-aspartate-
angiotensin I inhibitors (U.S. Pat. No.
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5,773,415), and angiotensin II inhibitors.
For treatment of cardiac hypertrophy associated with hypertension, the PRO
polypeptide can be administered
in combination with (3-adrenergic receptor blocking agents, e.g., propranolol,
timolol, tertalolol, carteolol, nadolol,
betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, or carvedilol; ACE
inhibitors, e.g., quinapril, captopril,
enalapril, ramipril, benazepril, fosinopril, or lisinopril; diuretics, e.g.,
chlorothiazide, hydrochlorothiazide,
hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide,
acetazolamide, or indapamide; and/or
calcium channel blockers, e.g., diltiazem, nifedipine, verapamil, or
nicardipine. Pharmaceutical compositions
comprising the therapeutic agents identified herein by their generic names are
commercially available, and are to
be administered following the manufacturers' instructions for dosage,
administration, adverse effects,
contraindications, etc. See, e.g., Physicians' Desk Reference (Medical
Economics Data Production Co.: Montvale,
N.J., 1997), Slth Edition.
Preferred candidates for combination therapy in the treatment of hypernophic
cardiomyopathy are (3-
adrenergic-blocking drugs (e.g., propranolol, timolol, tertalolol, carteolol,
nadolol, betaxolol, penbutolol,
acetobutolol, atenolol, metoprolol, or carvedilol), verapamil, difedipine, or
diltiazem. Treatment of hypertrophy
associated with high blood pressure may require the use of antihypertensive
drug therapy, using calcium channel
Mockers, e.g., diltiazem, nifedipine, verapamil, or nicardipine; ~3-adrenergic
blocking agents; diuretics, e.g.,
chlorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide,
benzthiazide, dichlorphenamide,
acetazolamide, or indapamide; and/or ACE-inhibitors, e.g., quinapril,
captopril, enalapril, ramipril, benazepril,
fosinopril, or lisinopril.
For other indications, PRO polypeptides or their antagonists may be combined
with other agents beneficial
to the treatment of the bone andlor cartilage defect, wound, or tissue in
question. These agents include various
growth factors such as EGF, PDGF, TGF-a or TGF-(3, IGF, FGF, and CTGF.
In addition, PRO polypeptides or their antagonists used to treat cancer may be
combined with cytotoxic,
chemotherapeutic, or growth-inhibitory agents as identified above. Also, for
cancer treatment, the PRO polypeptide
or antagonist thereof is suitably administered serially or in combination with
radiological treatments, whether
involving irradiation or administration of radioactive substances.
The effective amounts of the therapeutic agents administered in combination
with the PRO polypeptide or
antagonist thereof will be at the physician's or veterinarian's discretion.
Dosage administration and adjustment is
done to achieve maximal management of the conditions to be treated. For
example. for treating hypertension, these
amounts ideally take into account use of diuretics or digitalis, and
conditions such as hyper- or hypotension, renal
impairment, etc. The dose will additionally depend on such factors as the type
of the therapeutic agent to be used
and the specific patient being treated. Typically, the amount employed will be
the same dose as that used, if the
given therapeutic agent is administered without the PRO polypeptide.
xiii. Articles of Manufacture
An article of manufacture such as a kit containing PR0179, PR0238, PR036-1.
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR088?, PR0885 or
PR0887 polypeptide
or agonists or antagonists thereof useful for the diagnosis or treatment of
the disorders described above comprises
at least a container and a label. Suitable containers include, for example,
bottles. vials, syringes, and test tubes.
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The containers may be formed from a variety of materials such as glass or
plastic. The container holds
a composition that is effective for diagnosing or treating the condition and
may have a sterile access port (for
example, the container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic
injection needle). The active agent in the composition is the PR0179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
polypeptide or an agonist or antagonist thereto. The label on, or associated
with, the container indicates that the
composition is used for diagnosing or treating the condition of choice. The
article of manufacture may further
comprise a second container comprising a pharmaceutically-acceptable buffer,
such as phosphate-buffered saline,
Ringer's solution, and dextrose solution. It may further include other
materials desirable from a commercial and
user standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions
for use. The article of manufacture may also comprise a second or third
container with another active agent as
described above.
E. Antibodies
Some of the most promising drug candidates according to the present invention
are antibodies and antibody
fragments that may inhibit the production or the gene product of the genes
identified herein and/or reduce the
activity of the gene products.
i. Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can be
raised in a mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal
by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the PRO 179,
PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
polypeptide or a fusion protein thereof. It may be useful to conjugate the
immunizing agent to a protein known to
be immunogenic in the mammal being immunized. Examples of such immunogenic
proteins include, but are not
limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor.
Examples of adjuvants that may be employed include Freund's complete adjuvant
and MPL-TDM adjuvant
(monophosphoryl Lipid A or synthetic trehalose dicorynomycolate). The
immunization protocol may be selected
by one skilled in the art without undue experimentation.
ii. Monoclonal Antibodies
The anti-PR0179, anti-PR0238, anti-PR0364, anti-PR0844, anti-PR0846, anti-
PR01760, anti-PR0205,
anti-PR0321, anti-PR0333, anti-PR0840, anti-PR0877, anti-PR0878, anti-PR0879,
anti-PR0882, anti-PR0885
or anti-PR0887 antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared
using hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 ( 1975). In a hybridoma
method, a mouse, hamster, or other appropriate host animal is typically
immunized with an immunizing agent to
elicit lymphocytes that produce or are capable of producing antibodies that
will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in vitro.
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The immunizing agent will typically include the PRO 179, PR0238, PR0364,
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
or a fusion protein thereof. Generally, either peripheral blood lymphocytes
("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are desired. The
lymphocytes are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol,
to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and
Practice (New York: Academic Press,
1986), pp. 59-103. Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells
of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells
may be cultured in a suitable culture medium that preferably contains one or
more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the parental
cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will
include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of
HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high-level expression of
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the production
of human monoclonal antibodies. Kozbor, J. Immunol., 133:3001 ( 1984); Brodeur
et al., Monoclonal Antibody
Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987)
pp. 51-63.
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against the PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide. Preferably,
the binding specificity of monoclonal antibodies produced by the hybridoma
cells is determined by
immunoprecipitation or by an iro vitro binding assay, such as radioimmunoassay
(RIA) or enzyme-linked
immunoabsorbent assay (ELISA). Such techniques and assays are known in the
art. The binding affinity of the
monoclonal antibody can, for example, be determined by the Scatchard analysis
of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution procedures
and grown by standard methods. Goding, supra. Suitable culture media for this
purpose include, for example,
Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the
hybridoma cells may be grown
irr vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium or
ascites fluid by conventional immunoglobulin purification procedures such as,
for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described in U.S.
Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention
can be readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide probes
that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the
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invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression vectors,
which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to obtain
the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be modified, for
example, by substituting the coding
sequence for human heavy- and light-chain constant domains in place of the
homologous murine sequences (U.S.
Patent No. 4,816,567; Morrison et al., supra) or by covalently joining to the
immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide
can be substituted for the constant domains of an antibody of the invention,
or can be substituted for the variable
domains of one antigen-combining site of an antibody of the invention to
create a chimeric bivalent antibody.
1~ The antibodies may be monovalent antibodies. Methods for preparing
monovalent antibodies are well known
in the art. For example, one method involves recombinant expression of
immunoglobulin light chain and modified
heavy chain. The heavy chain is truncated generally at any point in the Fc
region so as to prevent heavy-chain
crosslinking. Alternatively, the relevant cysteine residues are substituted
with another amino acid residue or are
deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce
fragments thereof, particularly Fab fragments, can be accomplished using
routine techniques known in the art.
iii. Human and Humanized Antibodies
The anti-PR0179, anti-PR0238, anti-PR0364, anti-PR0844, anti-PR0846, anti-
PR01760, anti-PR0205,
anti-PR0321, anti-PR0333, anti-PR0840, anti-PR0877, anti-PR0878, anti-PR0879,
anti-PR0882, anti-PR0885
or anti-PR0887 antibodies may further comprise humanized antibodies or human
antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric immunoglobulins,
immunoglobulin chains. or fragments
thereof (such as Fv, Fab, Fab', F(ab')=, or other antigen-binding subsequences
of antibodies) that contain minimal
sequence derived from non-human immunoglobulin. Humanized antibodies include
human immunoglobulins
(recipient antibody) in which residues from a CDR of the recipient are
replaced by residues from a CDR of a non-
human species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity. and capacity.
In some instances, Fv framework residues of the human immunoglobulin are
replaced by correspondin '1 non-human
residues. Humanized antibodies may also comprise residues that are found
neither in the recipient antibody nor in
the imported CDR or framework sequences. In general, the humanized antibody
will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond
3~ to those of a non-human immunoglobulin, and all or substantially all of the
FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody preferably also will
comprise at least a portion of
an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Jones et al., Nature, 321: 522-
525 (1986); Riechmann etal., Nature, 332: 323-329 (1988); Presta. Curr. Op.
Struct. Biol.. 2:593-596 (1992).
Methods for humanizing non-human antibodies are well known in the art.
Generally. a humanized antibody
has one or more amino acid residues introduced into it from a source that is
non-human. These non-human amino
acid residues are often referred to as "import" residues, which are typically
taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and
co-workers (Jones et al., Nature,
321: 522-525 (1986); Riechmann etal., Nature, 332: 323-327 (1988); Verhoeyen
et al., Science. ?,9: 1534-1536
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(1988)), by substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent
No. 4,816,567), wherein
substantially less than an intact human variable domain has been substituted
by the corresponding sequence from
a non-human species. In practice, humanized antibodies are typically human
antibodies in which some CDR
residues and possibly some FR residues are substituted by residues from
analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage display
libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks etal.,
J. Mol. Biol., 222: 581 (1991).
The techniques of Cole et al. and Boerner et al. are also available for the
preparation of human monoclonal
antibodies. Cole et al., Monoclonal Antibodies and Cancer Theraoy> Alan R.
Liss, p. 77 (1985) and Boerner et al.,
1~ J. Immunol., 147(1): 86-95 (1991). Similarly, human antibodies can be made
by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody production
is observed that closely resembles
that seen in humans in all respects, including gene rearrangement, assembly,
and antibody repertoire. This approach
is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and
5,661,016, and in the following scientific publications: Marks etal.,
Bio/Technoloay, 10: 779-783 ( 1992); Lonberg
etal., Nature, 368: 856-859 ( 1994); Morrison, Nature, 368: 8l 2-813 ( 1994);
Fishwild etal., Nature Biotechnoloay,
14: 845-851 ( 1996); Neuberger, Nature Biotechnology, 14: 826 ( 1996); Lonberg
and Huszar, Intern. Rev. Immunol.,
13: 65-93 (1995).
iv. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
specificities for at least two different antigens. In the present case, one of
the binding specificities is for the
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide, the other one is for any
other antigen, and preferably
for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant production of
bispecific antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where
the two heavy chains have different specificities. Milstein and Cuello,
Nature, 305: 537-539 ( 1983). Because of
the random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a
potential mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The
3~ purification of the correct molecule is usually accomplished by affinity
chromatography steps. Similar procedures
are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al.,
EMBO J., 10: 3655-3659 ( 1991 ).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can be
fused to immunoglobulin constant-domain sequences. The fusion preferably is
with an immunoglobulin heavy-
chain constant domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first
heavy-chain constant region (CH1 ) containing the site necessary for light-
chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the immunoglobulin light
chain, are inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further
details of generating bispecific antibodies, see, for example, Suresh et al.,
Methods in EnzymoloQy, 121: 210
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WO 00/53757 PCT/US00/05004
( 1986).
v. Heteroconiu~ate Antibodies
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for
example, been proposed to target immune-system cells to unwanted cells (U.S.
Patent No. 4,676,980), and for
treatment of HIV infection. WO 91 /00360; WO 92/200373; EP 03089. It is
contemplated that the antibodies may
be prepared in vitro using known methods in synthetic protein chemistry,
including those involving crosslinking
agents. For example, immunotoxins may be constructed using a disulfide-
exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
vi. Effector Function Eneineerin~
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to enhance,
e.g., the effectiveness of the antibody in treating cancer. For example,
cysteine residues) may be introduced into
the Fc region, thereby allowing interchain disulfide bond formation in this
region. The homodimeric antibody thus
generated may have improved internalization capability and/or increased
complement-mediated cell killing and
antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al., J. Exp.
Med., 176: 1191-I 195 (1992) and
Shopes, J. Immunol., 148: 2918-2922 ( 1992). Homodimeric antibodies with
enhanced anti-tumor activity may also
be prepared using heterobifunctional cross-linkers as described in Wolff et
al., Cancer Research, 53: 2560-2565
(1993). Alternatively, an antibody can be engineered that has dual Fc regions
and may thereby have enhanced
complement lysis and ADCC capabilities. See, Stevenson et al., Anti-Cancer
Drug Design, 3: 219-230 ( 1989).
vii. Immunoconiu~ates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such
as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of
bacterial, fungal, plant, or animal origin,
or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjulates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseccdo»to»ns
aerugi»osa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleur ites fordii proteins, dianthin proteins,
Phvtolaca a»aerica»a proteins (PAPL,
PAPA, and PAP-S), momordica charantia inhibitor, curcin, croon, sapaonaria
officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for
the production of radioconjugated antibodies. Examples include -'-Bi, "'I,
"'In,'°Y, and '~~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives
of imidoesters (such as dimethyl adipimidate HCI), active esters (such as
disuccinimidyl suberate), aldehydes (such
as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyll
hexanediamine), bis-diazonium derivatives
(such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-
active fluorine compounds (such as 1,5-dilluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be
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prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-
labeled I-isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating
agent for conjugation of
radionucleotide to the antibody. See, W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such as
streptavidin) for utilization
in tumor pretargeting wherein the antibody-receptor conjugate is administered
to the patient, followed by removal
of unbound conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin)
that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
viii. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the
1~ antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA,
82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980);
and U.S. Pat. Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed in U.S.
Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter. Fab'
fragments of the antibody of the present invention can be conjugated to the
liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic agent (such as
Doxorubicin) is optionally contained within the liposome. See, Gabizon et al.,
J. National Cancer Inst., 81 (19):
1484 ( 1989).
ix. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide identified
herein, as well as other molecules identified by the screening assays
disclosed hereinbefore, can be administered
for the treatment of various disorders as noted above and below in the form of
pharmaceutical compositions.
If the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide is intracellular
and whole antibodies are
used as inhibitors, internalizing antibodies are preferred. However,
lipofections or liposomes can also be used to
deliver the antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory
fragment that specifically binds to the binding domain of the target protein
is preferred. For example, based upon
the variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the
target protein sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-
7893 ( 1993).
The formulation herein may also contain more than one active compound as
necessary for the particular
indication being treated, preferably those with complementary activities that
do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that
enhances its function, such as, for
example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-
inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the purpose
intended.
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The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-
(methylmethacylate) microcapsules, respectively, in colloidal drug delivery
systems (for example, liposomes,
albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
macroemulsions. Such techniques
are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations include
semipermeable matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of
shaped articles, e.g., films, or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels
(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919),
copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-
vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOT T"' (injectable
microspheres composed of lactic acid-
glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-
hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release of
molecules for over 100 days, certain hydrogels
release proteins for shorter time periods. When encapsulated antibodies remain
in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at 37°C,
resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be devised for
stabilization depending on the
mechanism involved. For example, if the aggregation mechanism is discovered to
be intermolecular S-S bond
formation through thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using
appropriate additives, and developing specific
polymer matrix compositions.
x. Methods of Treatment using the Antibody
It is contemplated that the antibodies to a PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR020~, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
may be used to treat various cardiovascular, endothelial, and angiogenic
conditions as noted above.
The antibodies are administered to a mammal, preferably a human, in accord
with known methods, such as
intravenous administration as a bolus or by continuous infusion over a period
of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or
inhalation routes. Intravenous administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the
antibodies of the instant invention
as noted above. For example, if the antibodies are to treat cancer, the
patient to be treated with such antibodies may
also receive radiation therapy. Alternatively. or in addition, a
chemotherapeutic agent may be administered to the
patient. Preparation and dosing schedules for such chemotherapeutic agents may
be used according to
manufacturers' instructions or as determined empirically by the skilled
practitioner. Preparation and dosing
schedules for such chemotherapy are also described in Chemotherapy Service,
Ed., M.C. Perry (Williams &
Wilkins: Baltimore, MD, 1992). The chemotherapeutic agent may precede, or
follow administration of the
antibody. or may be given simultaneously therewith. The antibody may be
combined with an anti-estrogen
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compound such as tamoxifen or EVISTAT"' or an anti-progesterone such as
onapristone (see, EP 616812) in
dosages known for such molecules.
If the antibodies are used for treating cancer, it may be desirable also to
administer antibodies against other
tumor-associated antigens, such as antibodies that bind to one or more of the
ErbB2, EGFR, ErbB3, ErbB4, or
VEGF receptor(s). These also include the agents set forth above. Also, the
antibody is suitably administered
serially or in combination with radiological treatments, whether involving
irradiation or administration of
radioactive substances. Alternatively, or in addition, two or more antibodies
binding the same or two or more
different antigens disclosed herein may be co-administered to the patient.
Sometimes, it may be beneficial also to
administer one or more cytokines to the patient. In a preferred embodiment,
the antibodies herein are co-
1~ administered with a growth-inhibitory agent. For example, the growth-
inhibitory agent may be administered first,
followed by an antibody of the present invention. However, simultaneous
administration or administration of the
antibody of the present invention first is also contemplated. Suitable dosages
for the growth-inhibitory agent are
those presently used and may be lowered due to the combined action (synergy)
of the growth-inhibitory agent and
the antibody herein.
In one embodiment, vascularization of tumors is attacked in combination
therapy. The anti-PRO polypeptide
antibody and another antibody (e.g., anti-VEGF) are administered to tumor-
bearing patients at therapeutically
effective doses as determined, for example, by observing necrosis of the tumor
or its metastatic foci, if any. This
therapy is continued until such time as no further beneficial effect is
observed or clinical examination shows no
trace of the tumor or any metastatic foci. Then TNF is administered, alone or
in combination with an auxiliary agent
such as alpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin,
anti-heregulin antibody, D-factor,
interleukin-I (IL-1 ), interleukin-2 (IL-2), granulocyte-macrophage colony
stimulating factor (GM-CSF), or agents
that promote microvascular coagulation in tumors, such as anti-protein C
antibody, anti-protein S antibody, or C4b
binding protein (see, WO 91/01753, published 21 February 1991 ), or heat or
radiation.
Since the auxiliary agents will vary in their effectiveness, it is desirable
to compare their impact on the tumor
by matrix screening in conventional fashion. The administration of anti-PRO
polypeptide antibody and TNF is
repeated until the desired clinical effect is achieved. Alternatively, the
anti-PRO polypeptide antibody is
administered together with TNF and, optionally, auxiliary agent(s). In
instances where solid tumors are found in
the limbs or in other locations susceptible to isolation from the general
circulation, the therapeutic agents described
herein are administered to the isolated tumor or organ. In other embodiments,
a FGF or PDGF antagonist, such as
an anti-FGF or an anti-PDGF neutralizing antibody, is administered to the
patient in conjunction with the anti-PRO
polypeptide antibody. Treatment with anti-PRO polypeptide antibodies
preferably may be suspended during periods
of wound healing or desirable neovascularization.
For the prevention or treatment of cardiovascular, endothelial, and
angio~~enic disorder. the appropriate dosage
of an antibody herein will depend on the type of disorder to be treated, as
defined above. the severity and course
of the disease, whether the antibody is administered for preventive or
therapeutic purposes, previous therapy, the
patient's clinical history and response to the antibody, and the discretion of
the attendin~~ physician. The antibody
is suitably administered to the patient at one time or over a series of
treatments.
For example, depending on the type and severity of the disorder, about 1 ~g/k~
to 50 mg/kg (e.g., 0.1-20
mg/kg) of antibody is an initial candidate dosage for administration to the
patient, whether, for example, by one or
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more separate administrations, or by continuous infusion. A typical daily or
weekly dosage might range from about
I ~cg/kg to 100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over
several days or longer, depending on the condition, the treatment is repeated
or sustained until a desired suppression
of disorder symptoms occurs. However, other dosage regimens may be useful. The
progress of this therapy is
easily monitored by conventional techniques and assays, including, for
example, radiographic tumor imaging.
xi. Articles of Manufacture with Antibodies
An article of manufacture containing a container with the antibody and a label
is also provided. Such articles
are described above, wherein the active agent is an anti-PR0179, anti-PR0238,
anti-PR0364, anti-PR0844, anti-
PR0846, anti-PR01760, anti-PR0205, anti-PR0321, anti-PR0333, anti-PR0840, anti-
PR0877, anti-PR0878,
anti-PR0879, anti-PR0882, anti-PR0885 or anti-PR0887 antibody.
xii. Diagnosis and Prognosis of Tumors using Antibodies
If the indication for which the antibodies are used is cancer, while cell-
surface proteins, such as growth
receptors over expressed in certain tumors, are excellent targets for drug
candidates or tumor (e.g., cancer)
treatment, the same proteins along with PRO polypeptides find additional use
in the diagnosis and prognosis of
tumors. For example, antibodies directed against the PRO polypeptides may be
used as tumor diagnostics or
prognostics.
For example, antibodies, including antibody fragments, can be used
qualitatively or quantitatively to detect
the expression of genes including the gene encoding the PRO polypeptide. The
antibody preferably is equipped
with a detectable, e.g., fluorescent label, and binding can be monitored by
light microscopy, flow cytometry,
fluorimetry, or other techniques known in the art. Such binding assays are
performed essentially as described
above.
In situ detection of antibody binding to the marker gene products can be
performed, for example, by
immunofluorescence or immunoelectron microscopy. For this purpose, a
histological specimen is removed from
the patient, and a labeled antibody is applied to it, preferably by overlaying
the antibody on a biological sample.
This procedure also allows for determining the distribution of the marker gene
product in the tissue examined. It
will be apparent to those skilled in the art that a wide variety of
histological methods are readily available for iv situ
detection.
The following Examples are offered for illustrative purposes only, and are not
intended to limit the scope of
the present invention in any way.
The disclosures of all patent and literature references cited in the present
specification are hereby incorporated
by reference in their entirety.
EXAMPLES
Commercially available reagents referred to in the Examples were used
according to manufacturer's
instructions unless otherwise indicated. The source of those cells identified
in the following Examples, and
throughout the specification, by ATCC accession numbers is the American Type
Culture Collection, Manassas, VA.
Unless otherwise noted, the present invention uses standard procedures of
recombinant DNA technology, such as
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those described hereinabove and in the following textbooks: Sambrook et al.,
supra; Ausubel et al., Current
Protocols in Molecular BioloQy (Green Publishing Associates and Wiley
Interscience, N.Y., 1989); Innis etal., PCR
Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y.,
1990); Harlow et al., Antibodies:
A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988);
Gait, OliQonucleotide Synthesis (IRL
Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan etal.,
Current Protocols in Immunology, 1991.
EXAMPLE I
Extracellular Domain Homology Screening to Identify Novel Polypeptides and
cDNA Encoding Therefor
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about 950
known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public databases (e.g., GenBank), and proprietary databases
(e.g., LIFESEQ°, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST-2
[Altschul etal., Methods in Enzymology> 266:460-480 (1996)] as a comparison of
the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons with a BLAST
score of 70 (or in some cases, 90)
or greater that did not encode known proteins were clustered and assembled
into consensus DNA sequences with
the program "phrap" (Phil Green, University of Washington. Seattle,
Washington).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative to other
identified EST sequences using phrap. In addition, the consensus DNA sequences
obtained were often (but not
always) extended using repeated cycles of BLAST and phrap to extend the
consensus sequence as far as possible
using the sources of EST sequences discussed above.
Based upon the consensus sequences obtained as described above,
oligonucleotides were then synthesized and
used to identify by PCR a cDNA library that contained the sequence of interest
and for use as probes to isolate a
clone of the full-length coding sequence for a PRO polypeptide. Forward and
reverse PCR primers generally range
from 20 to 30 nucleotides and are often designed to give a PCR product of
about 100-1000 by in length. The probe
sequences are typically 40-55 by in length. In some cases, additional
oligonucleotides are synthesized when the
consensus sequence is greater than about 1-1.5 kbp. In order to screen several
libraries for a full-length clone, DNA
from the libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular
Biology, supra, with the PCR primer pair. A positive library was then used to
isolate clones encoding the gene of
interest using the probe oligonucleotide and one of the primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by
standard methods using
commercially available reagents such as those from Invitrogen, San Diego, CA.
The eDNA was primed with oligo
dT containing a Notl site, linked with blunt to SaII hemikinased adaptors,
cleaved with NotI, sized appropriately
by gel electrophoresis, and cloned in a defined orientation into a suitable
clonin;~ vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD that does not contain the SfiI site; sc:e,
Holmes et al., Science, 253:1278-1280
(1991)) in the unique XhoI and NotI sites.
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EXAMPLE 2
Isolation of cDNA clones by Amylase Screening
1. Preparation of olio dT primed cDNA library
mRNA was isolated from a human tissue of interest using reagents and protocols
from Invitrogen, San Diego,
CA (Fast Track 2). This RNA was used to generate an oligo dT primed cDNA
library in the vector pRKSD using
reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script
Plasmid System). In this
procedure, the double stranded cDNA was sized to greater than 1000 by and the
SaII/NotI Tinkered cDNA was
cloned into XhoIlNotI cleaved vector. pRKSD is a cloning vector that has an
sp6 transcription initiation site
followed by an SfiI restriction enzyme site preceding the XhoI/NotI cDNA
cloning sites.
1~ 2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the 5' ends of the primary cDNA
clones. Sp6 RNA was generated from the primary library (described above), and
this RNA was used to generate
a random primed cDNA library in the vector pSST-AMY.O using reagents and
protocols from Life Technologies
(Super Script Plasmid System, referenced above). In this procedure the double
stranded cDNA was sized to 500-
1000 bp, Tinkered with blunt to NotI adaptors, cleaved with SfiI, and cloned
into SfiI/NotI cleaved vector. pSST-
AMY.O is a cloning vector that has a yeast alcohol dehydrogenase promoter
preceding the cDNA cloning sites and
the mouse amylase sequence (the mature sequence without the secretion signal)
followed by the yeast alcohol
dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into
this vector that are fused in frame with
amylase sequence will lead to the secretion of amylase from appropriately
transfected yeast colonies.
3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to
which was added electrocompetent
DHlOB bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was
then electroporated as
recommended by the manufacturer. Subsequently, SOC media (Life Technologies, 1
ml) was added and the mixture
was incubated at 37°C for 30 minutes. The transformants were then
plated onto 20 standard 150 mm LB plates
containing ampicillin and incubated for 16 hours (37°C). Positive
colonies were scraped off the plates and the
DNA was isolated from the bacterial pellet using standard protocols, e.g.,
CsCI-gradient. The purified DNA was
then carried on to the yeast protocols below.
The yeast methods were divided into three categories: (1) Transformation of
yeast with the plasmid/cDNA
combined vector; (2) Detection and isolation of yeast clones secreting
amylase; and ( 3) PCR amplification of the
insert directly from the yeast colony and purification of the DNA for
sequencing and further analysis.
The yeast strain used was HD56-SA (ATCC-90785). This strain has the following
genotype: MAT alpha,
ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL', SUC+, GAL'. Preferably,
yeast mutants can be employed that
have deficient post-translational pathways. Such mutants may have
translocation deficient alleles in sec71, sec72,
sec62. with truncated sec71 being most preferred. Alternatively, antagonists
(including antisense nucleotides and/or
3S ligandsl which interfere with the normal operation of these genes, other
proteins implicated in this post translation
pathway (e.g., SEC6lp, SEC72p, SEC62p, SEC63p, TDJIp or SSAIp-4p) or the
complex formation of these
proteins may also be preferably employed in combination with the amylase-
expressing yeast.
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Transformation was performed based on the protocol outlined by Gietz et al.,
Nucl. Acid. Res., 20:1425
(1992). Transformed cells were then inoculated from agar into YEPD complex
media broth (100 ml) and grown
overnight at 30°C. The YEPD broth was prepared as described in Kaiser
et al., Methods in Yeast Genetics, Cold
Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The overnight
culture was then diluted to about 2
x 10~ cells/ml (approx. OD~~,=0.1 ) into fresh YEPD broth (500 ml) and regrown
to 1 x 10' cells/ml (approx.
OD~~,=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor bottles in a Sorval
GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then
resuspended into sterile water, and
centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant was
discarded and the cells were subsequently washed with LiAc/'TE (10 ml, 10 mM
Tris-HCI, 1 mM EDTA pH 7.5,
100 mM Li~00CCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100,u1) with freshly
denatured single stranded salmon
testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (I ,ug,
vol. < 10 ul) in microfuge tubes.
The mixture was mixed briefly by vortexing, then 40% PEGfTE (600,u1, 40%
polyethylene glycol-4000, 10 mM
Tris-HCI, 1 mM EDTA, 100 mM Li=OOCCH3, pH 7.5) was added. This mixture was
gently mixed and incubated
at 30°C while agitating for 30 minutes. The cells were then heat
shocked at 42°C for 15 minutes, and the reaction
vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and
resuspended into TE (500,u1, 10
mM Tris-HCI, 1 mM EDTA pH 7.5) followed by recentrifugation. The cells were
then diluted into TE (I ml) and
aliquots (200 ~cl) were spread onto the selective media previously prepared in
150 mm growth plates (VWR).
2~ Alternatively, instead of multiple small reactions, the transformation was
performed using a single, large scale
reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil
(SCD-Ura) prepared as
described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, NY, p. 208-
210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was performed by including red
starch in the selective growth
media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as per the
procedure described by Biely et
al., Anal. Biochem., 172:176-179 ( 1988). The coupled starch was incorporated
into the SCD-Ura agar plates at a
final concentration of 0.15% (w/v), and was buffered with potassium phosphate
to a pH of 7.0 (50-100 mM final
concentration).
3~ The positive colonies were picked and streaked across fresh selective media
(onto 150 mm plates) in order
to obtain well isolated and identifiable single colonies. Well isolated single
colonies positive for amylase secretion
were detected by direct incorporation of red starch into buffered SCD-Ura
agar. Positive colonies were determined
by their ability to break down starch resulting in a clear halo around the
positive colony visualized directly.
4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile water
(30 /cl) in a 96 well plate. At this time, the positive colonies were either
frozen and stored for subsequent analysis
or immediately amplified. An aliquot of cells (5 ul) was used as a template
for the PCR reaction in a 25 ul volume
containing: 0.5 ~1 Klentaq (Clontech. Palo Alto, CA); 4.Opl 10 mM dNTP's
(Perkin Elmer-Cetus); 2.5,u1 Klentaq
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buffer (Clontech); 0.25 ,ul forward oligo 1; 0.25 ,ul reverse oligo 2; 12.5
/cl distilled water.
The sequence of the forward oliQonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID N0:33)
The sequence of the reverse olieonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID N0:34)
PCR was then performed as follows:
a. Denature 92°C, 5 minutes
b. 3 cycles of: Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds
Extend 72°C, 60 seconds
c. 3 cycles of: Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds
d. 25 cycles of: Denature 92°C, 30 seconds
Anneal 55°C, 30 seconds
Extend 72°C, 60 seconds
e. Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter
region and the amylase region,
respectively, and amplified a 307 by region from vector pSST-AMY.O when no
insert was present. Typically, the
first 18 nucleotides of the 5' end of these oligonucleotides contained
annealing sites for the sequencing primers.
Thus, the total product of the PCR reaction from an empty vector was 343 bp.
However, signal sequence-fused
cDNA resulted in considerably longer nucleotide sequences.
Following the PCR, an aliquot of the reaction (5 /rl) was examined by agarose
gel electrophoresis in a l X70
agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described by
Sambrook et al., supra. Clones
resulting in a single strong PCR product larger than 400 by were further
analyzed by DNA sequencing after
purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth,
CA).
EXAMPLE 3
Isolation of cDNA Clones Using Signal Algorithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a proprietary signal
sequence finding algorithm developed by Genentech, Inc., (South San Francisco,
CA) upon ESTs as well as
clustered and assembled EST fragments from public (e.g., GenBank) and/or
private (LIFESEQ°, Incyte
Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm
computes a secretion signal score
based on the character of the DNA nucleotides surrounding the first and
optionally the second methionine codon(s)
(ATG) at the 5'-end of the sequence or sequence fragment under consideration.
The nucleotides following the first
ATG must code for at least 35 unambiguous amino acids without any stop codons.
If the first ATG has the required
amino acids, the second is not examined. If neither meets the requirement, the
candidate sequence is not scored.
In order to determine whether the EST sequence contains an authentic signal
sequence, the DNA and correspondin~.T
amino acid sequences surrounding the ATG codon are scored usin~T a set of
seven sensors (evaluation parameters)
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known to be associated with secretion signals. Use of this algorithm resulted
in the identification of numerous
polypeptide-encoding nucleic acid sequences.
EXAMPLE 4
Isolation of cDNA Clones Encoding Human PR0179
A cDNA sequence was isolated in a screen as described in Example 2 above. The
cDNAsequence isolated
in the above screen was found, by BLAST and FastA sequence alignment, to have
sequence homology to a
nucleotide sequence encoding various angiopoietin proteins. This cDNA sequence
is herein designated DNA I 0028
and/or DNA25250.
Based on the sequence homology, oligonucleotide probes were then generated
from the sequence of the
DNA10028 molecule and used to screen a human fetal liver (LIB6) library
prepared as described in paragraph 1
of Example 2 above. The cloning vector was pRKSB (pRKSB is a precursor of
pRKSD that does not contain the
SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991 )) in the unique
XhoI and NotI sites, and the cDNA size
cut was less than 2800 bp.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for PR0179
and the derived protein sequence for PR0179. The entire nucleotide sequence of
DNA16451-1388 is shown in
Figure 1 (SEQ ID NO:1). Clone DNA16451-1388 contains a single open reading
frame with an apparent
translational initiation site at nucleotide positions 37-39 and ending at the
stop codon at nucleotide positions 1417
1419 (Figure I ). The predicted polypeptide precursor is 460 amino acids long
(Figure 2, SEQ ID N0:2). The full
length PR0179 protein shown in Figure 2 has an estimated molecular weight of
about 53,637 daltons and a pI of
about 6.61.
Analysis of the full-length PR0179 sequence shown in Figure 2 (SEQ ID N0:2)
evidences the presence of
a variety of important polypeptide domains as shown in Figure 2, wherein the
locations given for those important
polypeptide domains are approximate as described above. Analysis of the full-
length PR0179 sequence (Figure
2; SEQ ID N0:2), evidences the presence of the following: a signal peptide
from about amino acid 1 to about amino
acid 16; leucine zipper patterns from about amino acid 120 to about amino acid
142 and from about amino acid 127
to about amino acid 149; N-glycosylation sites from about amino acid 23 to
about amino acid 27, from about amino
acid 115 to about amino acid l 19, from about amino acid 296 to about amino
acid 300, and from about amino acid
357 to about amino acid 361; and fibrinogen beta and gamma chains C-terminal
domains from about amino acid
271 to about amino acid 310, from about amino acid 312 to about amino acid
322, from about amino acid 331 to
about amino acid 369, and from about amino acid 393 to about amino acid 424.
Clone DNA16451-1388 has been deposited with the ATCC on April 14, 1998 and is
assi'_ned ATCC deposit
no. 209776. Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and
the sequences provided herein are based on known sequencing techniques.
Analysis of the amino acid sequence of the full-length PR0179 polypeptide
suggests that it possesses
significant similarity to the angiopoietin family of proteins, thereby
indicating that PR0179 may be a novel
angiopoietin family member. More specifically, an analysis of the Dayhoff
database (version 35.45 SwissProt 35),
evidenced significant homology between the PR0179 amino acid sequence and the
following Dayhoff sequences:
AF004326_1, P 894605, HSU83508_l, P 894603, P 894317. AF025818_I, HSY16132_1,
P R65760.I37391
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and HUMRSC 192 I .
EXAMPLE 5
Isolation of cDNA Clones Encoding Human PR0238
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described in
Example I above. This consensus sequence is herein designated DNA30908. Based
on the DNA30908 consensus
sequence, oligonucleotides were synthesized: 1 ) to identify by PCR a cDNA
library that contained the sequence
of interest, and 2) for use as probes to isolate a clone of the full-length
coding sequence for PR0238. PCR primers
(forward and reverse) were synthesized based upon the DNA30908 sequence.
Additionally, a synthetic
oligonucleotide hybridization probe was constructed from the consensus
DNA30908 sequence.
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification, as per Ausubel et al., Current Protocols in Molecular
Bioloey, supra, with the PCR primer
pair. A positive library was then used to isolate clones encoding the PR0238
gene using the probe oligonucleotide
and one of the PCR primers.
The oligonucleotide sequences used in the above procedure were the following:
forward PCRprimer 1:
5'-GGTGCTAAACTGGTGCTCTGTGGC-3' (SEQ ID N0:35)
forward PCR primer 2:
5'-CAGGGCAAGATGAGCATTCC-3' (SEQ ID N0:36)
reverse PCR primer:
5'-TCATACTGTTCCATCTCGGCACGC-3' (SEQ ID N0:37)
hybridization probe:
5'-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCTTCTCATGC-3' (SEQ ID N0:38)
RNA for construction of the cDNA libraries was isolated from human fetal liver
tissue. The cDNA libraries
used to isolate the cDNA clones were constructed by standard methods using
commercially available reagents such
as those from Invitrogen, San Diego, CA. The cDNA was primed with NotI site,
linked with blunt to SaII
hemikinased adaptors, cleaved with NotI, sized appropriately by gel
electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a
precursor of pRKSD that does not
contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991 )) in
the unique XhoI and NotI sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for PR0238
[herein designated as DNA35600-1162] (Figure 3, SEQ ID N0:3) and the derived
protein sequence for PR0238.
The entire nucleotide sequence of DNA35600-1 162 is shown in Fi~~ure 3 (SEQ ID
N0:3). Clone DNA35600-
1162 contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 134-
136 and ending at the stop codon at nucleotide positions I 064-1066 (Figure
3). The predicted polypeptide precursor
is 310 amino acids long (Figure 4; SEQ ID N0:4), and has an estimated
molecular weight of about 33,524 daltons
and a pI of about 9.55.
Analysis of the full-length PR0238 sequence shown in Figure 4 (SEQ ID N0:4)
evidenced the presence of
a variety of important polypeptide domains as shown in Figure 4, wherein the
locations given for those important
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polypeptide domains are approximate as described above. Analysis of the full-
length PR0238 sequence (Figure
4; SEQ ID N0:4) evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino
acid 21; transmembrane domains from about amino acid 102 to about amino acid I
19 and from about amino acid
278 to about amino acid 292; an N-glycosylation site from about amino acid 228
to about amino acid 232; a
glycosaminoglycan attachment site from about amino acid 47 to about amino acid
51; a tyrosine kinase
phosphorylation site from about amino acid 145 to about amino acid 153; N-
myristoylation sites from about amino
acid 44 to about amino acid 50, from about amino acid 105 to about amino acid
1 I 1, from about amino acid 238
to about amino acid 244, from about amino acid 242 to about amino acid 248,
and from about amino acid 291 to
about amino acid 297; an amidation site from about amino acid 265 to about
amino acid 269; and a prokaryotic
membrane lipoprotein lipid attachment site from about amino acid 6 to about
amino acid 17. Clone DNA35600-
1162 has been deposited with ATCC on October 16, 1997 and is assigned ATCC
deposit no. 209370.
Analysis of the amino acid sequence of the full-length PR0238 polypeptide
suggests that portions of it possess
significant homology to reductase, particularly oxidoreductase, thereby
indicating that PR0238 may be a novel
reductase.
EXAMPLE 6
Isolation of cDNA Clones Encoding Human PR0364
An expressed sequence tag (EST) DNA database (LIFESEQ°, Incyte
Pharmaceuticals, Palo Alto, CA) was
searched and an Incyte EST sequence (Incyte EST No. 3003460) was identified
that showed homology to members
of the tumor necrosis factor receptor (TNFR) family of polypeptides.
2~ A consensus DNA sequence was then assembled relative to the Incyte 3003460
EST and other EST sequences
using repeated cycles of BLAST (Altshul etal., Methods in EnzvmoloQy, 266:460-
480 (1996)) and "phrap" (Phil
Green, University of Washington, Seattle Washington). The consensus sequence
is herein designated
"<consen0l >" and is also herein designated as DNA44825.
Oligonucleotide probes based upon the DNA44825 and "<consen0l>" consensus
sequences were then
synthesized: 1 ) to identify by PCR a cDNA library that contained the sequence
of interest, and 2) for use as probes
to isolate a clone of the full-length coding sequence for PR0364. Forward and
reverse PCR primers general ly range
from 20-30 nucleotides and are often designed to give a PCR product of about
100-1000 by in length. The probe
sequences are typically 40-55 by in length. In order to screen several
libraries for a full-length clone, DNA from
the libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular Biolo~y,
3~ supra, with the PCR primer pair. A positive library was then used to
isolate clones encoding the gene of interest
using the probe oligonucleotide and one of the primer pairs.
Pairs of PCR primers (forward and reverse) were synthesized:
forward PCR primer (44825.f1 ):
5'-CACAGCACGGGGCGATGGG-3' (SEQ ID N0:39)
forward PCR primer (44825.f2):
5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID N0:40)
forward PCR primer (44825.GITR.f):
5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3' (SEQ ID N0:41 )
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reverse PCR primer (44825.r1 ):
5'-CTGGTCACTGCCACCTTCCTGCAC-3' (SEQ ID N0:42)
reverse PCR primer (44825. r2):
5'-CGCTGACCCAGGCTGAG-3' (SEQ ID N0:43)
reverse PCR primer (44825.GITR.r):
5'-GAAGGTCCCCGAGGCACAGTCGATACA-3' (SEQ ID N0:44)
Additonally, synthetic oligonucleotide hybridization probes were constructed
from the consensus DNA44825
sequence which had the following nucleotide sequences:
hybridization probe (44825.p1):
5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3' (SEQ ID N0:45)
hybridization probe (44825.GITR.p):
5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID N0:46)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pairs identified above. A positive
library was then used to isolate clones
encoding the PR0364 gene using the probe oligonucleotides and one of the PCR
primers.
RNA for construction of the cDNA libraries was isolated from human bone marrow
tissue. The eDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a NotI
site, sized appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector
2~ (such as pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain
the SfiI site; see, Holmes et al.,
Science, 253:1278-1280 ( 1991 )) in the unique XhoI and NotI sites.
A cDNA clone was identified and sequenced in entirety. The entire nucleotide
sequence of DNA47365-1206
is shown in Figure 5 (SEQ ID N0:5). Clone DNA47365-1206 contains a single open
reading frame with an
apparent translational initiation site at nucleotide positions 121-123, and a
stop codon at nucleotide positions 8-I4-
846 (Figure 5; SEQ ID N0:5). The predicted polypeptide precursor is 241 amino
acids long and has a predicted
molecular weight of approximately 26,000 daltons and an estimated pI of about
6.34. The full-length PR0364
protein is shown in Figure 6 (SEQ ID N0:6).
Analysis of the full-length PR0364 sequence shown in Figure 6 (SEQ ID N0:6)
evidences the presence of
important polypeptide domains as shown in Figure 6, wherein the locations
given for those important polypeptide
domains are approximate as described above. Analysis of the full-length PR0364
sequence (Figure 6; SEQ ID
N0:6) evidences the presence of the following: a signal peptide from about
amino acid 1 to about amino acid 25;
a potential transmembrane domain from about amino acid 162 to about amino acid
180; an N-glycosylation site from
about amino acid 146 to about amino acid 150; N-myristoylation sites from
about amino acid 5 to about amino acid
1 I , from about amino acid 8 to about amino acid 14, from about amino acid 25
to about amino acid 31, from about
amino acid 30 to about amino acid 36, from about amino acid 33 to about amino
acid 39, from about amino acid
118 to about amino acid 124, from about amino acid 122 to about amino acid
128, and from about amino acid 156
to about amino acid 162; a prokaryotic membrane lipoprotein lipid attachment
site from about amino acid 166 to
about amino acid 177; and a leucine zipper pattern from about amino acid 171
to about amino acid 19 ~.
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Clone DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is
assigned ATCC deposit
no. 209436. It is understood that the deposited clone has the actual correct
sequence rather than the representations
provided herein.
Analysis of the amino acid sequence of the full-length PR0364 polypeptide
suggests that portions of it possess
homology to members of the tumor necrosis factor receptor family, thereby
indicating that PR0364 may be a novel
member of the tumor necrosis factor receptor family. The intracellular domain
of PR0364 contains a motif (in the
region of amino acids 207-214) similar to the minimal domain within CD30
receptor shown to be required for
TRAF2 binding and which is also present within TNFR2. There are three apparent
extracellular cysteine-rich
domains characteristic of the TNFR family (see, Naismith and Sprang, Trends
Biochem. Sci., 23:74-79 (1998)),
of which the third CRD has 3 rather than the more typical 4 or 6 cysteines of
the TNFR family. As compared to
the mouse GITR (described below) the PR0364 amino acid sequence has 8
cysteines in the CRD1 relative to 5
cysteines in CRD1 of mouse GITR, and the presence of one potential N-linked
glycosylation site in the ECD as
compared to 4 potential N-linked glycosylation sites in mouse GITR.
A detailed review of the putative amino acid sequence of the full-length
native PR0364 polypeptide and the
nucleotide sequence that encodes it evidences sequence homology with the mouse
GITR (mGITR) protein reported
by Nocentini et al., Proc. Natl. Acad. Sci. USA, 94:6216-6221 (1997). It is
possible, therefore, that PR0364
represents the human counterpart or ortholog to the mouse GITR protein
reported by Nocentini et al.
EXAMPLE 7
Isolation of cDNA Clones Encoding Human PR0844
An expressed sequence tag (EST) DNA database (LIFESEQ°, Incyte
Pharmaceuticals, Palo Alto, CA) was
searched and an EST was identified that showed sequence identity with a LP.
Based on the information and
discoveries provided herein, the clone for this EST, Incyte clone 2657496 from
a cancerous lung library (309-
LUNGTUT09) was further examined.
A cDNA clone was identified and sequenced in entirety. The entire nucleotide
sequence of DNA59838-1462
is shown in Figure 7 (SEQ ID N0:7). Clone DNA59838-1462 contains a single open
reading frame with an
apparent translational initiation site at nucleotide positions 5-7, and a stop
codon at nucleotide positions 338-340
(Figure 7; SEQ ID N0:7). The predicted polypeptide precursor is 11 1 amino
acids long and has a predicted
molecular weight of approximately 12,050 daltons and an estimated pI of about
5.45. The full-length PR0844
protein is shown in Figure 8 (SEQ ID N0:8).
Analysis of the full-length PR0844 sequence shown in Figure 8 (SEQ ID N0:8)
evidences the presence of
important polypeptide domains as shown in Figure 8, wherein the locations
given for those important polypeptide
domains are approximate as described above. Analysis of the full-length PR0844
sequence (Figure 8; SEQ ID
N0:8) evidences the presence of the following: a signal peptide from about
amino acid 1 to about amino acid 19;
N-myristoylation sites from about amino acid 23 to about amino acid 29, from
about amino acid ?7 to about amino
acid 33. from about amino acid 32 to about amino acid 38, and from about amino
acid 102 to about amino acid 108;
and a WAP-type 'four-disulfide core' domain signature from about amino acid 49
to about amino acid 63.
Clone DNA59838-1462 has been deposited with ATCC on June 16, 1998 and is
assigned ATCC deposit no.
209976. It is understood that the deposited clone has the actual correct
sequence rather than the representations
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provided herein.
Analysis of the amino acid sequence of the full-length PR0844 polypeptide
suggests that it possesses
significant similarity to serine protease inhibitors, thereby indicating that
PR0844 may be a novel proteinase
inhibitor. More specifically, an analysis of the Dayhoff database (version
35.45 SwissProt 35) evidenced significant
homology between the PR0844 amino acid sequence and at least the following
Dayhoff sequences:
ALKI HUMAN, P P82403, P P82402, ELAF HUMAN and P P60950.
EXAMPLE 8
Isolation of cDNA Clones Encoding Human PR0846
l~ A consensus DNA sequence was assembled relative to other EST sequences
using phrap as described in
Example 1 above. This consensus sequence is herein designated DNA39949 and
"<consen1322>". Based on the
DNA39949 consensus sequence and the "<consen1322>" sequnece, oligonucleotides
were synthesized: 1 ) to
identify by PCR a cDNA library that contained the sequence of interest, and 2)
for use as probes to isolate a clone
of the full-length coding sequence for PR0846. PCR primers (forward and
reverse) were synthesized based upon
the DNA39949 and "<consen 1322>" consensus sequences. Additionally, a
synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30908 sequence.
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification, as per Ausubel et al., Current Protocols in Molecular
Biology, supra, with the PCR primer
pair. A positive library was then used to isolate clones encoding the PR0846
gene using the probe oligonucleotide
and one of the PCR primers.
The oligonucleotide sequences used in the above procedure were the following:
forward PCR~rimer (39949.f1):
5'-CCCTGCAGTGCACCTACAGGGAAG-3' (SEQ ID N0:47)
reverse PCR primer (39949.r1 ):
5'-CTGTCTTCCCCTGCTTGGCTGTGG-3' (SEQ ID N0:48)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA39949
sequence which had the following nucleotide sequence:
hybridization probe (39949.p1):
5'-GGTGCAGGAAGGGTGGGATCCTCTTCTCTCGCTGCTCTGGCCACATC-3' (SEQ ID N0:49)
3~ RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227). The
cDNA libraries used to isolate the cDNA clones were constructed by standard
methods using commercially
available reagents such as those from Invitrogen, San Diego, CA. The cDNA was
primed with NotI site, linked
with blunt to SaII hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned
in a defined orientation into a suitable cloning vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD
that does not contain the SfiI site; see, Holmes etal., Science, 253:1278-1280
(1991 )) in the unique XhoI and Notl
sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for PR0846
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[herein designated as DNA44196-1353) (Figure 9, SEQ ID N0:9) and the derived
protein sequence for PR0846
The entire nucleotide sequence of DNA44196-1353 is shown in Figure 9 (SEQ ID
N0:9). Clone DNA44196-
1353 contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 25-27
and ending at the stop codon at nucleotide positions 1021-1023 (Figure 9). The
predicted polypeptide precursor
is 332 amino acids long and has an estimated molecular weight of approximately
36,143 daltons and pI of about
5.89 (Figure 10; SEQ ID NO:10).
Analysis of the full-length PR0846 sequence shown in Figure 10 (SEQ ID NO:10)
evidenced the presence
of a variety of important polypeptide domains as shown in Figure 10, wherein
the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0846 sequence
(Figure 10; SEQ ID NO:10) evidences the presence of the following: a signal
peptide from about amino acid 1 to
about amino acid 17; a transmembrane domain from about amino acid 248 to about
amino acid 269; an N
glycosylation site from about amino acid 96 to about amino acid 100; a
fibrinogen beta and gamma chains C
terminal domain from about amino acid 104 to about amino acid 114; and a Ig
like V-type domain from about amino
acid 13 to about amino acid 128. Clone DNA44196-1353 has been deposited with
ATCC on May 6, 1998 and is
assigned ATCC deposit no. 209847.
EXAMPLE 9
Isolation of cDNA Clones Encoding Human PR01760
Use of the signal sequence algorithm described in Example 3 above allowed
identification of an EST cluster
sequence from the Incyte database. This EST cluster sequence was then compared
to a variety of expressed
sequence tag (EST) databases which included public EST databases (e.g.,
GenBank) and a proprietary EST DNA
database (LIFESEQ°, Incyte Pharmaceuticals, Palo Alto, CA) to identify
existing homologies. One or more of the
ESTs was derived from a prostate tumor library. The homology search was
performed using the computer program
BLAST or BLAST2 (Altshul et al., Methods in Enzymoloay, 266:460-480 (1996)).
Those comparisons resulting
in a BLAST score of 70 (or in some cases, 90) or greater that did not encode
known proteins were clustered and
assembled into a consensus DNA sequence with the program "phrap" (Phil Green,
University of Washington,
Seattle, Washington). The consensus sequence obtained therefrom is herein
designated DNA58798.
In light of an observed sequence homology between the DNA58798 consensus
sequence and the Incyte EST
3358745, the clone including this EST was purchased and the cDNA insert was
obtained and sequenced. It was
found herein that that insert encoded a full-length protein. The sequence of
this cDNA insert is shown in Figure
11 (SEQ ID NO:11 ) and is herein designated DNA76532-1702.
Clone DNA76532-1702 contains a single open reading frame with an apparent
translational initiation site at
nucleotide positions 60-62 and ending at the stop codon at nucleotide
positions 624-626 (Figure 11 ). The predicted
polypeptide precursor is 188 amino acids long (Figure 12; SEQ ID N0:12). The
full-length PRO1760 protein
shown in Figure 12 has an estimated molecular weight of about 21,042 daltons
and a pI of about 5.36.
3S Analysis of the full-length PRO 1760 sequence shown in Figure 12 (SEQ ID
N0:12) evidences the presence
of a variety of important polypeptide domains as shown in Figure 12, wherein
the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PRO 1760 sequence
evidences the presence of the follov~in~; features: a signal peptide from
about amino acid 1 to about amino acid 20;
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N-glycosylation sites from about amino acid 121 to about amino acid 125 and
from about amino acid 171 to about
amino acid 175; a tyrosine kinase phosphorylation site from about amino acid
25 to about amino acid 32; and N-
myristoylation sites from about amino acid 54 to about amino acid 60 and from
about amino acid 160 to about
amino acid 166. Clone DNA76532-l 702 has been deposited with ATCC on November
17, 1998 and is assigned
ATCC deposit no. 203473.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence alignment
analysis of the full-length sequence shown in Figure 12 (SEQ ID NO: I 2),
evidenced sequence identity between the
PR01760 amino acid sequence and the following Dayhoff sequences: CELT07F12_2,
T22J18_16, ATF1C12_3,
APE3 YEAST, P W22471, SAU56908_ 1, SCPA_STRPY, ATAC00423817, SAPURCLUS 2 and
AF041468_9.
EXAMPLE 10
Stimulation of Heart Neonatal Hypertrophy (Assay I )
This assay is designed to measure the ability of PRO polypeptides to stimulate
hypertrophy of neonatal heart.
PRO polypeptides testing positive in this assay are expected to be useful for
the therapeutic treatment of various
cardiac insufficiency disorders.
Cardiac myocytes from I-day old Harlan Sprague Dawley rats were obtained.
Cells (180 ~1 at 7.5 x 10''/ml,
serum <0. I %, freshly isolated) are added on day 1 to 96-well plates
previously coated with DMEM/F12 + 4% FCS.
Test samples containing the test PRO polypeptide or growth medium only
(negative control) (20 ~1/well) are added
directly to the wells on day l . PGF (20,u1/well) is then added on day 2 at a
final concentration of 10-~ M. The cells
are then stained on day 4 and visually scored on day 5, wherein cells showing
no increase in size (as compared to
negative controls) are scored 0.0, cells showing a small to moderate increase
in size (as compared to negative
controls) are scored 1.0 and cells showing a large increase in size (as
compared to negative controls) are scored 2Ø
A positive result in the assay is a score of 1.0 or greater.
PR0882 tested positive in this assay as shown in TABLE 4 below:
TABLE 4
PRO # Concentration/Dilution Relative Increase in Size
(Compared to Negative Control)
PR0882 0.01 % 3
PR0882 0.01 % 3
PR0882 0.10% 4
PR0882 O.10% 4
PR0882 I .00% 6
PR0882 1.00% 6
PR0882 0.01 % 3
PR0882 0.10% 4.5
PR0882 I.00% 6
PR0882 I .00% 5.5
PR0882 2.6 nM
5.75
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EXAMPLE 1 1
Inhibition of Vascular Endothelial Growth Factor (VEGF) Stimulated
Proliferation of Endothelial Cell Growth
Assa 9
The ability of various PRO polypeptides to inhibit VEGF stimulated
proliferation of endothelial cells was
tested. Polypeptides testing positive in this assay are useful for inhibiting
endothelial cell growth in mammals where
such an effect would be beneficial, e.g., for inhibiting tumor growth.
Specifically, bovine adrenal cortical capillary endothelial cells (ACE) (from
primary culture, maximum of 12-
14 passages) were plated in 96-well plates at 500 cells/well per 100
microliter. Assay media included low glucose
DMEM, 10% calf serum, 2 mM glutamine, and 1X
penicillin/streptomycin/fungizone. Control wells included the
following: (I) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5
ng/ml FGF; (4) ACE cells plus 3
ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE
cells plus 3 ng/ml VEGF plus
5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in 100
microliter volumes), were then added to
the wells (at dilutions of 1 %, 0.1 % and 0.01 %, respectively). The cell
cultures were incubated for 6-7 days at
37°C/5% CO~. After the incubation, the media in the wells was
aspirated, and the cells were washed 1X with PBS.
An acid phosphatase reaction mixture (100 microliter; O.1M sodium acetate, pH
5.5, 0.1 % Triton X-100. 10 mM
p-nitrophenyl phosphate) was then added to each well. After a 2 hour
incubation at 37 °C, the reaction was stopped
by addition of 10 microliters I N NaOH. Optical density (OD) was measured on a
microplate reader at 405 nm.
The activity of PRO polypeptides was calculated as the percent inhibition of
VEGF (3 ng/ml) stimulated
proliferation (as determined by measuring acid phosphatase activity at OD 405
nm) relative to the cells without
stimulation. TGF-beta was employed as an activity reference at I ng/ml, since
TGF-beta blocks 70-90% of VEGF
stimulated ACE cell proliferation. The results, as shown in TABLE 5 below, are
indicative of the utility of the PRO
polypeptides in cancer therapy and specifically in inhibiting tumor
angiogenesis. The numerical values (relative
inhibition) shown in TABLE 5 are determined by calculating the percent
inhibition of VEGF stimulated
proliferation by the PRO polypeptides relative to cells without stimulation
and then dividing that percentage into
the percent inhibition obtained by TGF-(3 at 1 ng/ml which is known to block
70-90% of VEGF stimulated cell
proliferation. The results are considered positive if the PRO ploypeptide
exhibits 30% or greater inhibition of
VEGF stimulation of endothelial cell growth (relative inhibition 30% or
greater).
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TABLE 5
Inhibition of VEGF Stimilated Endothelial Cell Growth
PRO Name PRO Concentration Relative % Inhibition
PR0333 0.01 % 97.0
PR0333 0.10% 90.0
PR0333 1.00% 63.0
PR0877 0.01 % 101.0
PR0877 0.01 % 108.0
PR0877 0.10% 86.0
10PR0877 0.10% 99.0
PR0877 1.00% 46.0
PR0877 1.00% 46.0
PR0879 0.01 % 100.0
PR0879 0.01 % 105.0
15PR0879 0.10% 97.0
PR0879 0.10% I O 1.0
PR0879 I .00% 55.0
PR0879 1.00% 66.0
PR0882 0.01 % 96.0
20PR0882 0.10% 86.0
PR0882 I .00% 70.0
PR0885 0.01 % 100.0
PR0885 0.10% 93.0
PR0885 1.00% 64.0
25 EXAMPLE 12
Induction of c-fos in Endothelial Cells (Assay 34)
This assay is designed to determine whether PRO polypeptides show the ability
to induce c-fos in endothelial
cells. PRO polypeptides testing positive in this assay would be expected to be
useful for the therapeutic treatment
of conditions or disorders where angiogenesis would be beneficial including,
for example, wound healing, and the
30 like (as would agonists of these PRO polypeptides). Antagonists of the PRO
polypeptides testing positive in this
assay would be expected to be useful for the therapeutic treatment of
cancerous tumors.
Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in growth
media (50% Ham's F12
w/o GHT: low glucose, and 50% DMEM without glycine: with NaHC03, 1 %
glutamine, 10 mM HEPES, 10%
FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at a cell density
of 1 x 10~ cells/well. The day after
35 plating, the cells were starved by removing the growth media and treating
the cells with 100 ~l/well test samples
and controls (positive control: growth media; negative control: 10 mM HEPES,
140 mM NaCI, 4% (w/v) mannitol,
pH 6.8). The cells were incubated for 30 minutes at 37°C, in 5% CO=.
The samples were removed, and the first
part of the bDNA kit protocol (Chiron Dia_nostics, cat. #6005-037) was
followed, where each capitalized
reagenbbuffer listed below was available from the kit.
40 Briefly, the amounts of the TM Lysis Buffer and Probes needed for the tests
were calculated based on
information provided by the manufacturer. The appropriate amounts of thawed
Probes were added to the TM Lysis
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Buffer. The Capture Hybridization Buffer was warmed to room temperature. The
bDNA strips were set up in the
metal strip holders, and 100 ~cl of Capture Hybridization Buffer was added to
each b-DNA well needed, followed
by incubation for at least 30 minutes. The test plates with the cells were
removed from the incubator, and the media
was gently removed using the vacuum manifold. 100 ~1 of Lysis Hybridization
Buffer with Probes were quickly
pipetted into each well of the microtiter plates. The plates were then
incubated at 55 °C for I S minutes. Upon
removal from the incubator, the plates were placed on the vortex mixer with
the microtiter adapter head and
vortexed on the #2 setting for one minute. 80 ~l of the lysate was removed and
added to the bDNA wells containing
the Capture Hybridization Buffer, and pipetted up and down to mix. The plates
were incubated at 53 °C for at least
16 hours.
On the next day, the second part of the bDNA kit protocol was followed.
Specifically, the plates were
removed from the incubator and placed on the bench to cool for 10 minutes. The
volumes of additions needed were
calculated based upon information provided by the manufacturer. An Amplifier
Working Solution was prepared
by making a 1:100 dilution of the Amplifier Concentrate (20 fm/,ul) in AL
Hybridization Buffer. The hybridization
mixture was removed from the plates and washed twice with Wash A. 50 ,ul of
Amplifier Working Solution was
added to each well and the wells were incubated at 53 °C for 30
minutes. The plates were then removed from the
incubator and allowed to cool for 10 minutes. The Label Probe Working Solution
was prepared by making a 1:100
dilution of Label Concentrate (40 pmoles/~1) in AL Hybridization Buffer. After
the 10-minute cool-down period,
the amplifier hybridization mixture was removed and the plates were washed
twice with Wash A. 50 ~1 of Label
Probe Working Solution was added to each well and the wells were incubated at
53 °C for I S minutes. After
cooling for 10 minutes, the Substrate was warmed to room temperature. Upon
addition of 3 ~I of Substrate
Enhancer to each ml of Substrate needed for the assay, the plates were allowed
to cool for 10 minutes, the label
hybridization mixture was removed, and the plates were washed twice with Wash
A and three times with Wash D.
50 ~l of the Substrate Solution with Enhancer was added to each well. The
plates were incubated for 30 minutes
at 37°C and RLU was read in an appropriate luminometer.
The replicates were averaged and the coefficient of variation was determined.
The measure of activity of
the fold increase over the negative control (HEPES buffer described above)
value was indicated by
chemiluminescence units (RLU). The results are shown in TABLE 6 below, and are
considered positive if the PRO
polypeptide exhibits at least a two-fold value over the negative control.
Negative control = 1.00 RLU at 1.00%
dilution. Positive control = 8.39 RLU at 1.00% dilution.
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TABLE 6
Induction of c-fos in Endothelial
Cells
PRO Name PRO Concentration RLU values
PR0321 O.OIl nM 1.51
PR0321 O.ll nM 1.07
PR0321 1.1 nM 2.11
PR0321 0.011 nM 2.13
PR0321 0.11 nM 2.27
PR0321 1.10 nM 2.65
10PR0840 2.44 nM 1.85
PR0840 24.4 nM 2.21
PR0840 244 nM 3.04
PR0840 2.44 nM 2.82
PR0840 24.4 nM 2.90
15PR0840 244 nM 1.01
PR0878 0.01 % 2.43
PR0878 0.10% 2.71
PR0878 1.00% 1.39
PR0878 0.01 % 2.48
20PR0878 0.10% 2.45
PR0878 1.00% I .89
PR0879 0.01 % 1.23
PR0879 0.10% 1.33
PR0879 1.00% 2.54
25PR0879 0.01 % 2.06
PR0879 0. I 0% 1.65
PR0879 1.00% 2.25
EXAMPLE 13
Enhancement of Heart Neonatal Hypertrophy Induced by F2a (Assay 37)
30 This assay is designed to measure the ability of PRO polypeptides to
stimulate hypertrophy of neonatal heart.
PRO polypeptides testing positive in this assay are expected to be useful for
the therapeutic treatment of various
cardiac insufficiency disorders.
Cardiac myocytes from 1-day old Harlan Sprague Dawley rats were obtained.
Cells (l80 ~cl at 7.5 x 10~/ml,
serum <0.1 %, freshly isolated) are added on day 1 to 96-well plates
previously coated with DMEM/Fl 2 + 4% FCS.
35 Test samples containing the test PRO polypeptide (20 fcl/well) are added
directly to the wells on day l . PGF (20
~cl/well) is then added on day 2 at a final concentration of 10-h M. The cells
are then stained on day 4 and visually
scored on day 5. Visual scores are based on cell size, wherein cells showing
no increase in size as compared to
negative controls are scored 0.0, cells showing a small to moderate increase
in size as compared to negative controls
are scored 1.0 and cells showing a lar~~e increase in size as compared to
negative controls are scored 2Ø A score
40 of 1.0 or greater is considered positive.
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No PBS is included, since calcium concentration is critical for assay
response. Plates are coated with
DMEM/F12 plus 4% FCS (200 ~1/well). Assay media included: DMEM/F12 (with 2.44
gm bicarbonate), 10
~g/ml transferrin, 1 ~g/ml insulin, 1 ~g/ml aprotinin, 2 mmol/L glutamine, 100
U/ml penicillin G, 100 ~g/ml
streptomycin. Protein buffer containing mannitol (4%) gave a positive signal
(score 3.5) at 1/10 (0.4%) and 1/100
(0.04%), but not at 1/1000 (0.004%). Therefore the test sample buffer
containing mannitol is not run.
PR0205, PR0882 and PR0887 polypeptides tested positive in this assay:
EXAMPLE 14
Inhibition of Heart Adult Hypertrophy (Assay 42)
This assay is designed to measure the inhibition of heart adult hypertrophy.
PRO polypeptides testing positive
in this assay may find use in the therapeutic treatment of cardiac disorders
associated with cardiac hypertrophy.
Ventricular myocytes are freshly isolated from adult (250g) Harlan Sprague
Dawley rats and the cells are
plated at 2000/well in 180 ~cl volume. On day two, test samples (20 ~l)
containing the test PRO polypeptide are
added. On day five, the cells are fixed and then stained. An increase in ANP
message can also be measured by
PCR from cells after a few hours. Results are based on a visual score of cell
size: 0 = no inhibition, -1 =small
inhibition, -2 = large inhibition. A score of less than 0 is considered
positive. Activity reference corresponds to
phenylephrin (PE) at 0.1 mM, as a positive control. Assay media included: M199
(modified)-glutamine free,
NaHC03, phenol red, supplemented with 100 nM insulin, 0.2% BSA, 5 mM creatine,
2 mM L-carnitine, 5 mM
taurine, 100 U/ml penicillin G, 100 ~g/ml streptomycin (CCT medium). Only
inner 60 wells are used in 96 well
plates. Of these, 6 wells are reserved for negative and positive (PE)
controls.
PR0878 polypeptide provided a score of less than 0 in the above assay:
EXAMPLE 1 _5
Induction of Endothelial Cell Apoptosis (Assay 73)
The ability of PRO polypeptides to induce apoptosis in endothelial cells was
tested in human venous umbilical
vein endothelial cells (HUVEC, Cell Systems). A positive test in the assay is
indicative of the usefulness of the
polypeptide in therapeutically treating tumors as well as vascular disorders
where inducing apoptosis of endothelial
cells would be beneficial.
The ability of PRO polypeptides to induce apoptosis in endothelial cells was
tested in human venous umbilical
vein endothelial cells (HUVEC, Cell Systems), using a 96-well format, in 0%
serum media supplemented with 100
ng/ml VEGF. (As HUVEC cells are easily dislodged from the plating surface, all
pipetting in the wells must be
done as gently as practicable.)
The medium was aspirated and the cells washed once with PBS. 5 ml of I x
trypsin was added to the cells
in a T-175 flask, and the cells were allowed to stand until they were released
from the plate (about 5-10 minutes).
Trypsinization was stopped by adding 5 ml of growth media. The cells were spun
at 1000 rpm for ~ minutes at 4 °C.
The media was aspirated and the cells were resuspended in 10 ml of 10% serum
complemented medium (Cell
Systems), 1 x penicillin/streptomycin.
The cells were plated on 96-well microtiter plates (Amersham Life Science,
cytostar-T scintillating microplate,
RPNQ160, sterile. tissue-culture treated, individually wrapped). in 10% serum
(CSG-medium, Cell Systems), at
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a density of 2 x 10' cells per well in a total volume of 100 ~1. Test PRO
polypeptide samples were added in
triplicate at dilutions of I %, 0.33% and 0.1 I %. Wells without cells were
used as a blank and wells with cells only
were used as a negative control. As a positive control I :3 serial dilutions
of 50 ~I of a 3x stock of staurosporine
were used. The ability of the test PRO polypeptide to induce apoptosis was
determined using Annexin-V, a member
of the calcium and phospholipid binding proteins, to detect apoptosis.
0.2 ml Annexin V - Biotin stock solution ( 100 /eg/ml) were diluted in 4.6 ml
2 x Cav+ binding buffer and 2.5%
BSA (1:25 dilution). 50 GIs of the diluted Annexin V - Biotin solution were
added to each well (except controls)
to a final concentration of 1.0 ~g/ml. The samples were incubated for 10-15
minutes with Annexin-Biotin prior
to direct addition of 35S-Streptavidin. 35S-Streptavidin was diluted in 2x
Caz+Binding buffer, 2.5% BSA and was
added to all wells at a final concentration of 3 x 104 cpm/well. The plates
were then sealed, centrifuged at 1000
rpm for 15 minutes and placed on orbital shaker for 2 hours. The analysis was
performed on 1450 Microbeta Trilux
(Wallac). The results are shown in TABLE 7 below where percent above
background represents the percentage
amount of counts per minute above the negative controls. Percents greater than
or equal to 30% above background
are considered positive.
PR0333, PR0364 and PR0879 scored positive results in the above described
assay.
TABLE 7
Induction of Endothelial Cell Apoptosis
PRO Name PRO Concentration Percent Above Background
PR0333 0.11 % 61.7 %
PR0333 0.33% 37.6 %
PR0364 2.99 nM 19.3 %
PR0364 8.99 nM 6.9 %
PR0364 27.23 nM 31.5 %
PR0879 1.00% 64.2 %
PR0879 0.11 % 65.5%
PR0879 0.33% 14.7%
EXAMPLE 16
Inhibition of Heart Neonatal Hypertrophy Induced by LIFplus Endothelin-I (ET-1
) (Assay 74)
This assay is designed to determine whether PRO polypeptides of the present
invention show the ability to
inhibit neonatal heart hypertrophy induced by LIF and endothelin-1 (ET-7 ). A
test compound that provides a
positive response in the present assay would be useful for the therapeutic
treatment of cardiac insufficiency diseases
or disorders characterized or associated with an undesired hypertrophy of the
cardiac muscle.
Cardiac myocytes from 1-day old Harlan Sprague Dawley rats (180 ~l at 7.5 x
10'/ml, serum <0.1, freshly
isolated) are introduced on day 1 to 96-well plates previously coated with
DMEM/F12 + 4%nFCS. Test PRO
polypeptide samples or growth medium alone (negative control) are then added
directly to the wells on day 2 in 20
~I volume. LIF + ET-I are then added to the wells on day 3. The cells are
stained after an additional 2 days in
culture and are then scored visually the next day. A positive in the assay
occurs when the PRO polypeptide treated
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myocytes are visually smaller on the average or less numerous than the
untreated myocytes.
PR0238 and PR01760 polypeptides tested positive in this assay.
EXAMPLE 17
Stimulation of Endothelial Tube Formation -gout formation (Assay 86)
This assay is designed to determine whether PRO polypeptides show the ability
to promote endothelial vacuole
and lumen formation in the absence of exogenous growth factors. PRO
polypeptides testing positive in this assay
would be expected to be useful for the therapeutic treatment of disorders
where endothelial vacuole and/or lumen
formation would be beneficial including, for example, where the stimulation of
pinocytosis, ion pumping, vascular
permeability and/or functional formation would be beneficial.
HUVEC cells (passage <8 from primary) are mixed with type I rat tail collagen
(final concentration 2.6 mg/ml)
at a density of 6x105 cells per ml and plated at 50 ~l per well of M199
culture media supplemented with 1 % FBS
and I ~M 6-FAM-FITC dye to stain the vacuoles while they are forming and in
the presence of the PRO
polypeptide. The cells are then incubated at 37°C/5% CO~ for 48 hours,
fixed with 3.7% formalin at room
temperature for 10 minutes, washed 5 times with M199 medium and then stained
with Rh-Phalloidin at 4°C
overnight followed by nuclear staining with 4 ~M DAPI. A positive result in
the assay is equal to or less than 2
[I = cells are all round, 2 = cells are elongated, 3 cells are forming tubes
with some connections, 4 = cells are
forming complex tubular networks].
PR0179 polypeptide tested positive in this assay.
EXAMPLE 18
Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)
The ability of PRO polypeptides to induce apoptosis in endothelial cells was
tested in human venous umbilical
vein endothelial cells (HUVEC, Cell Systems) using a 96-well format, in 0%
serum media supplemented with 100
ng/ml VEGF, 0.1 % BSA, IX penn/strep. A positive result in this assay
indicates the usefulness of the polypeptide
for therapeutically treating any of a variety of conditions associated with
undesired endothelial cell growth
including, for example, the inhibition of tumor growth. The 96-well plates
used were manufactured by Falcon (No.
3072). Coating of 96 well plates were prepared by allowing gelatinization to
occur for >30 minutes with l00 ~.cl
of 0.2% gelatin in PBS solution. The gelatin mix was aspirated thoroughly
before plating HUVEC cells at a final
concentration of 2 x 10'' cells/ml in 10% serum containing medium - 100 ~cl
volume per well. The cells were grown
for 24 hours before adding test samples containing the PRO polypeptide of
interest.
To all wells, 100 ~I of 0% serum media (Cell Systems) complemented with l 00
ng/ml VEGF, 0.1 % BSA, 1 X
penn/strep was added. Test samples containing PRO polypeptides were added in
triplicate at dilutions of I %,
0.33% and 0.11 %. Wells without cells were used as a blank and wells with
cells only were used as a negative
control. As a positive control, 1:3 serial dilutions of 50 ~cl of a 3x stock
of staurosporine were used. The cells were
incubated for 24 to 35 hours prior to ELISA.
ELISA was used to determine levels of apoptosis preparing solutions accordinn
to the Boehringer Manual
[Boehringer, Cell Death Detection ELISA plus, Cat No. 1 920 685]. Sample
preparations: 96 well plates were spun
down at 1 krpm for 10 minutes (200g); the supernatant was removed by fast
inversion, placing the plate upside
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down on a paper towel to remove residual liquid. To each well, 200 pl of IX
Lysis buffer was added and
incubation allowed at room temperature for 30 minutes without shaking. The
plates were spun down for I 0 minutes
at 1 krpm, and 20 pl of the lysate (cytoplasmic fraction) was transferred into
streptavidin coated MTP. 80 ~l of
immunoreagent mix was added to the 20 ~l lystate in each well. The MTP was
covered with adhesive foil and
incubated at room tempearature for 2 hours by placing it on an orbital shaker
(200 rpm). After two hours, the
supernatant was removed by suction and the wells rinsed three times with 250
pl of 1 X incubation buffer per well
(removed by suction). Substrate solution was added (100 pl) into each well and
incubated on an orbital shaker at
room temperature at 250 rpm until color development was sufficient for a
photometric analysis (approx. after 10-20
minutes). A 96 well reader was used to read the plates at 405 nm, reference
wavelength, 492 nm. The levels
obtained for PIN 32 (control buffer) was set to 100%. Samples with levels
>130% were considered positive for
induction of apoptosis.
PR0846, and PR0844 polypeptides tested positive in this assay.
EXAMPLE 19
Ira situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection
and localization of nucleic acid
sequences within cell or tissue preparations. It may be useful, for example,
to identify sites of gene expression,
analyze the tissue distribution of transcription, identify and localize viral
infection, follow changes in specific
mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the
protocol by Lu and Gillett, Cell
Vision, 1: 169-176(1994), usingPCR-generated3~P-labeledriboprobes.
Briefly,formalin-fixed, paraffin-embedded
human tissues were sectioned, deparaffinized, deproteinated in proteinase K
(20 g/ml) for 15 minutes at 37 °C, and
further processed for irr- situ hybridization as described by Lu and Gillett,
suprn. A (i3-P)UTP-labeled antisense
riboprobe was generated from a PCR product and hybridized at 55 °C
overnight. The slides were dipped in Kodak
NTB2TM nuclear track emulsion and exposed for 4 weeks.
33P-Riboprobe synthesis
6.0 ,ul (125 mCi) of ~'P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed-
vacuum dried. To each
tube containing dried 3'P-UTP, the following ingredients were added:
2.0 pl 5x transcription buffer
1.0 ~1 DTT (100 mM)
2.0 ~1 NTP mix (2.5 mM: 10 pl each of 10 mM GTP, CTP & ATP + 10 p.l H=O)
1.0 ~l UTP (50 p.M)
1.0 ~I RNAsin
1.0 ~cl DNA template ( 1 fig)
I .0 ~l H,O
1.0 ,~I RNA polymerase (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37°C for one hour. A total of 1.0 rrl RQI
DNase was added, followed by
incubation at 37°C for 15 minutes. A total of 90 ~cl TE (70 mM Tris pH
7.6/1 mM EDTA pH 8.0) was added, and
the mixture was pipetted onto DE81 paper. The remaining solution was loaded in
a MICROCON-50Th'
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ultrafiltration unit, and spun using program 10 (6 minutes). The filtration
unit was inverted over a second tube and
spun using program 2 (3 minutes). After the final recovery spin, a total of
100 ~l TE was added, then 1 ul of the
final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR IITM.
The probe was run on a TBE/urea gel. A total of 1-3 ~l of the probe or 5 ~cl
of RNA Mrk III was added to
3 ~l of loading buffer. After heating on a 95 °C heat block for three
minutes, the gel was immediately placed on
ice. The wells of gel were flushed, and the sample was loaded and run at 180-
250 volts for 45 minutes. The gel
was wrapped in plastic wrap (SARANTM brand) and exposed to XAR film with an
intensifying screen in a -70°C
freezer one hour to overnight.
33P-Hybridization
A. Pretreatment of frozerz sectiozas
The slides were removed from the freezer, placed on aluminum trays, and thawed
at room temperature for 5
minutes. The trays were placed in a 55 °C incubator for five minutes to
reduce condensation. The slides were fixed
for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in
0.5 x SSC for 5 minutes, at room
temperature (25 ml 20 x SSC + 975 ml SQ H=O). After deproteination in 0.5
fzg/ml proteinase K for 10 minutes
at 37°C (12.5 ~zl of 10 mg/ml stock in 250 ml prewarmed RNAse-free
RNAse buffer), the sections were washed
in 0.5 x SSC for 10 minutes at room temperature. The sections were dehydrated
in 70%, 95%, and 100% ethanol,
2 minutes each.
B. Pretreatment of para~zz-embedded sections
The slides were deparaffinized, placed in SQ HZO, and rinsed twice in 2 x SSC
at room temperature, for 5
2~ minutes each time. The sections were deproteinated in 20 ~g/ml proteinase K
(500 ~1 of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 °C, 15 minutes) for human embryo tissue, or
8 x proteinase K ( 100 ~I in 250 ml Rnase
buffer, 37°C, 30 minutes) for formalin tissues. Subsequent rinsing in
0.5 x SSC and dehydration were performed
as described above.
C. Prehybridizatiozt
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50%
formamide) - saturated filter
paper. The tissue was covered with 50 ~1 of hybridization buffer (3.75 g
dextran sulfate + 6 ml SQ H_O), vortexed,
and heated in the microwave for 2 minutes with the cap loosened. After cooling
on ice, 18.75 ml formamide, 3.75
ml 20 x SSC, and 9 ml SQ HZO were added, and the tissue was vortexed well and
incubated at 42°C for 1-4 hours.
D. Hybridization
3~ 1.0 x 10'' cpm probe and l .0 ~zl tRNA (50 mghnl stock) per slide were
heated at 95°C for 3 minutes. The
slides were cooled on ice, and 48 ~1 hybridization buffer was added per slide.
After vortexing, 50 e< 1 "P mix was
added to 50 ~1 prehybridization on the slide. The slides were incubated
overnight at 55°C.
E. Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml
20 x SSC + 16 ml 0.25
M EDTA, V,=4L), followed by RNAseA treatment at 37°C for 30 minutes
(500 ~.l of 10 mg/ml in ?50 ml Rnase
buffer = 20 ~.g/ml), The slides were washed 2 x10 minutes with 2 x SSC, EDTA
at room temperature. The
stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x
SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
V,=4L 1.
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F. Oligo~aucleotides
If2 situ analysis was performed on three of the DNA sequences disclosed
herein. The oligonucleotides
employed for these analyses are as follows:
( 1 ) DNA47365-1206 (PR0364) (TNF receptor homology
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC AAC CCG AGC ATG GCA CAG CAC-3' (SEQ ID
N0:50)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TCT CCC AGC CGC CCC TTC TC-3' (SEQ ID
N0:51 )
(2) DNA30868 (PR0205) (follistatin homoloa)
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC AGA GAC AGG GCA AGC AGA ATG-3' (SEQ ID
N0:52)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GAA GGG GAT GAC TGG AGG AAC-3' (SEQ ID
N0:53)
(3) DNA41374 (PR0333) (CD33 homolor)
pI:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CTC CAC AGA ACC TCG CCA TCA-3' (SEQ ID
N0:54)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TGG GGC AAG ACT CAC AAG CAG -3' (SEQ ID
N0:55)
G. Results
In. situ analysis was performed on the above three DNA sequences disclosed
herein. The results from these
analyses are as follows:
(1 ) DNA47365-1206 (PR0364) (TNF receptor homology
In the fetus, there was expression in the fascia lining the anterior surface
of the vertebral body. There was
also expression over the fetal retina. However, there was low level expression
over the fetal neurones. All other
tissues were negative.
(2) DNA30868 (PR0205) (follistatin homology
In fetal tissue, there was expression in the spinal cord, autonomic ganglia,
enteric nerves, sacral plexus,
peripheral and cranial nerves. All other fetal and adult tissues were
negative.
Fetal tissues (12-16 weeks) examined included: placenta, umbilical cord,
liver. kidney, adrenals, thyroid,
lungs, heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord,
body wall, pelvis and lower limb.
Adult tissues included: liver, kidney, adrenal, myocardium, aorta, spleen,
lymph node, pancreas, lung and
skin.
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(3) DNA41374 (PR0333) (CD33 homoloa)
This molecule has been shown to be immunostimulatory (enhances T lymphocyte
proliferation in the one-way ,
mixed lymphocyte reaction in T lymphocyte co-stimulation assays). The
distribution pattern of this molecule was
evaluated with a limited tissue screen comprising tissues available at the
initiation of this study.
In numerous tissues evaluated, a weak diffuse expression was detected in
thymic T lymphocytes (spleen and
lymph node were not evaluated). This result was confirmed in the subsequent in
situ hybridization study. The
results of that study showed similar low level expression in non-human primate
thymus and in human tonsil in T
lymphocyte specific regions. The limited distribution pattern suggests
expression by T lymphocytes or cells closely
associated with T lymphocytes such as antigen presenting cells (dendritic cell
populations, etc). In inflamed human
1 ~ tissue with significant lymphocytic inflammation and presence of reactive
follicle formation (in inflammatory bowel
disease and chronic lymphocytic interstitial pneumonia/bronchitis) there was
no detectable expression in areas
which contained significant numbers of T lymphocytes.
The distribution discrepancy (i.e. expression in thymic and tonsillar T
lymphocyte areas but not in areas with
T lymphocytic inflammation) suggests the following possibilities:
1. That there is selectivelrestricted expression in a T lymphocyte subset
population present in thymus and
tonsillar lymph node but not inflamed tissue. Immature, non-committed T
lymphocytes are present in both
tonsil and thymus but likely would not be a major population in chronically
inflamed tissues.
2. That expression in T lymphocytes is weak and differences in detection in
tissues with T lymphocytes is a
reflection of RNA quality in those tissues sections rather than a reflection
of different T lymphocyte cell
population types.
That expression in thymus and tonsil is not in lymphocytes but rather in a
specific cell population closely
associated with T lymphocytes, that is not present in the inflamed lung and
bowel evaluated. One such
possibility is a dendritic cell subpopulation.
In a non-human primate there was weak diffuse expression in thymic
lymphocytes.
In a subsequent study, the following results were reported:
Inflammed lung: (chronic lymphocytic and granulomatous pneumonitis): a weak to
negative signal was observed
in the interstitium compared to the control sense probe; there was weak
expression in the normal chimp thymus
(human thymus not available) and in the human tonsil. In the later, the
expression was predominantly in T
lymphocyte areas of this structure including the perifollicular marginal zone
and in the paracortex.
3~ There was no detectable expression in the following human tissues:
inflammatory bowel disease (8 patient
specimens), chronically inflamed and normal lung (6 patient specimens),
chronic sclerosing nephritis ( 1 patient
specimen), chronically and acutely inflammed and cirrhotic liver (10 specimen
multiblock), normal and psoriatic
skin, and peripheral lymph node (non-reactive).
EXAMPLE 20
Use of PR0179, PR0238, PR0364, PR0844 PR0846 PR01760 PR0205 PR0321 PR0333
PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 as a Hybridization Probe
The following method describes use of a nucleotide sequence encoding PRO 179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878. PR0879,
PR0882, PR0885 or
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PR0887 as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PR0179, PR0238,
PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 (as shown in Figures 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, and 31, respectively, SEQ ID NOS:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31, respectively)
or a fragment thereof is employed as a probe
to screen for homologous DNAs (such as those encoding naturally-occurring
variants of PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887) in human tissue cDNA libraries or human tissue
genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following high-
1~ stringency conditions. Hybridization of radiolabeled probe derived from the
gene encoding PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 polypeptide to the filters is performed in a solution
of 50% formamide, 5x SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x
Denhardt's solution, and 10%
dextran sulfate at 42°C for 20 hours. Washing of the filters is
performed in an aqueous solution of O.lx SSC and
0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence can then be
identified using standard techniques known in the art.
EXAMPLE 21
Expression of Nucleic Acid Encoding PR0179 PR0238 PR0364 PR0844 PR0846 PR01760
PR0205
2~ PR0321, PR0333, PR0840 PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887 in E
coli
This Example illustrates preparation of an unglycosylated form of PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 by recombinant expression in E. coli.
The DNA sequence encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PRO878, PR0879, PR0882, PR0885 or PR0887 (SEQ ID NOS:
I, 3, 5, 7, 9, 1 l,
13, 15, 17, 19, 21, 23, 25, 27, 29, or 31, respectively) is initially
amplified using selected PCR primers. The primers
should contain restriction enzyme sites that correspond to the restriction
enzyme sites on the selected expression
vector. A variety of expression vectors may be employed. An example of a
suitable vector is pBR322 (derived
from E. coli; see Bolivar etal., Gene, 2: 95 (1977)), which contains genes for
ampicillin and tetracycline resistance.
The vector is digested with restriction enzyme and dephosphorylated. The PCR-
amplified sequences are then
ligated into the vector. The vector will preferably include sequences that
encode an antibiotic-resistance gene, a
trp promoter, a poly-His leader (including the first six STII codons, poly-His
sequence, and enterokinase cleavage
site), the region encoding PRO179, PR0238, PR0364, PR0844, PR0846, PRO 1760,
PR0205, PR0321. PRO333,
PR0840, PR0877. PR0878, PR0879, PR0882, PR0885 or PR0887, lambda
transcriptional terminator, and an
argU gene.
The ligation mixture is then used to transform a selected E. coli strain
usin~T the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic-resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed by
restriction analysis and DNA
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WO 00/53757 PCT/US00/05004
sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture may subsequently be used to inoculate a
larger-scale culture. The cells are then
grown to a desired optical density, during which the expression promoter is
turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell pellet
obtained by the centrifugation can be solubilized using various agents known
in the art, and the solubilized PRO 179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887 polypeptide can then be purified using a
metal-chelating column under
conditions that allow tight binding of the polypeptide.
PR0238, PR0364 and PRO 1760 were successfully expressed in E. coli in a poly-
His tagged form by the
above procedure.
EXAMPLE 22
Expression of Nucleic Acid Encoding PR0179 PR0238 PR0364 PR0844 PR0846 PR01760
PR0205
PR0321, PR0333, PR0840, PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887 in
Mammalian Cells
This Example illustrates preparation of a potentially glycosylated form of
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 by recombinant expression in mammalian cells.
The vector, pRKS (see, EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205,
PR0321, PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 DNA is ligated into ARKS with
selected restriction
enzymes to allow insertion of the DNA encoding PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 using ligation
methods such as described in Sambrook et al., supra. The resulting vector is
called pRKS-(DNA encoding
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760. PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887).
In one embodiment, the selected host cells are 293 cells. Human 293 cells
(ATCC CCL 1573) are grown to
confluence in tissue culture plates in medium such as DMEM supplemented with
fetal calf serum and optionally,
nutrient components and/or antibiotics. About 10 ~g DNA of ARKS-(DNA encoding
PRO 179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PRO878,
PR0879, PR0882,
PR0885 or PR0887) is mixed with about I ,ug DNA encoding the VA RNA gene
(Thimmappaya et al., Cell, 31:
543 ( 1982)) and dissolved in 500 ~l of I mM Tris-HCI, 0.1 mM EDTA, 0.227 M
CaCh. To this mixture is added,
dropwise, 500 ~l of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO~, and a
precipitate is allowed to form
for 10 minutes at 25°C. The precipitate is suspended and added to the
293 cells and allowed to settle for about four
hours at 37°C. The culture medium is aspirated off and 2 ml of 20 is
glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum-free medium, fresh medium is added, and
the cells are incubated for about
5 days.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with culture
medium (alone) or culture medium containing 200 ~rCi/ml''S-cvsteine and 200
~.Ci/ml'SS-methionine. After a 12-
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WO 00/53757
hour incubation, the conditioned medium is collected, concentrated on a spin
filter, and loaded onto a 15°~o SDS
gel. The processed gel may be dried and exposed to film for a selected period
of time to reveal the presence of the
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 polypeptide. The cultures containing
transfected cells may
undergo further incubation (in serum-free medium) and the medium is tested in
selected bioassays.
In an alternative technique, the gene encoding PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 may be
introduced into 293 cells transiently using the dextran sulfate method
described by Somparyrac et al., Proc. Natl.
Acad. Sci., 12: 7575 (1981 ). 293 cells are grown to maximal density in a
spinner flask and 700 ~g pRKS-(DNA
encoding PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887) is added. The cells are
first concentrated from the
spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell pellet
for four hours. The cells are treated with 20% glycerol for 90 seconds, washed
with tissue culture medium, and re-
introduced into the spinner flask containing tissue culture medium, 5 ~g/ml
bovine insulin, and 0.1 ~g/ml bovine
transferrin. After about four days, the conditioned media is centrifuged and
filtered to remove cells and debris.
The sample containing the expressed gene encoding the PR0179, PR0238, PR0364,
PR0844, PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 polypeptide
can then be concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
In another embodiment, the gene encoding PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 can be
expressed in CHO cells. The pRKS-(DNA encoding PR0179, PR0238, PR0364, PR0844,
PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887) nucleic acid
can be transfected into CHO cells using known reagents such as CaPO~ or DEAE-
dextran. As described above,
the cell cultures can be incubated, and the medium replaced with culture
medium (alone) or medium containing a
radiolabel such as 35S-methionine. After determining the presence of PROl79,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 polypeptide, the culture medium may be replaced with serum-free medium.
Preferably. the cultures are
incubated for about 6 days, and then the conditioned medium is harvested. The
medium containing the expressed
PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR03 33,
PR0840, PR0877,
PR0878, PR0879, PR0882, PR0885 or PR0887 can then be concentrated and purified
by any selected method.
Epitope-tagged gene encoding the PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205,
PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887
polypeptide may also
be expressed in host CHO cells. The gene encoding PR0179, PR0238, PR0364, PR08-
I-1. PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 may be
subcloned out of the ARKS vector. The subclone insert can undergo PCR
amplification to fuse in frame with a
selected epitope tag such as a poly-His tag into a baculovirus expression
vector. The gene insert encoding the poly-
His-tagged-[PR0179, PR0238, PR0364, PR0844, PR0846, PRO1760, PR0205, PRO321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887] can then be subcloned into a
SV40- driven vector
containing a selection marker such as DHFR for selection of stable clones.
Finally, the CHO cells can be
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transfected (as described above) with the SV40-driven vector. Labeling may be
performed, as described above, to
verify expression. The culture medium containing the expressed gene encoding
the poly-His-tagged-[PR0179,
PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840,
PR0877, PR0878,
PR0879, PR0882, PR0885 or PR0887] can then be concentrated and purified by any
selected method, such as
by Ni'+-chelate affinity chromatography.
PR0179, PR0364, PR0840, PR0844, PR0846 and PR0205 were stably expressed in CHO
cells by the
above described method. In addition, PR0364 and PR0846 were expressed in CHO
cells by a transient procedure.
EXAMPLE 23
Expression of Nucleic Acid Encoding PR0179 PR0238 PR0364 PR0844 PR0846 PR01760
PR0205
PR0321, PR0333, PR0840, PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887 in Yeast
The following method describes recombinant expression of the gene encoding
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 from the ADH2/GAPDH promoter. DNA encoding PR0179,
PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 and the promoter is inserted into suitable restriction enzyme
sites in the selected plasmid to
direct intracellular expression of the gene encoding PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887. For secretion,
DNA encoding PRO 179, PR0238, PR0364, PR0844, PR0846, PRO 1760, PR0205,
PR0321, PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 can be cloned into the
selected plasmid, together with
DNA encoding the ADH2/GAPDH promoter, a native PR0179, PR0238, PR0364, PR0844,
PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 signal peptide
or other mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signal/leader
sequence, and linker sequences (if needed) for expression of the gene encoding
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described above
and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation
with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining
of the gels with Coomassie Blue
stain.
Recombinant PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333,
PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 can subsequently be
isolated and purified
by removing the yeast cells from the fermentation medium by centrifugation and
then concentrating the medium
using selected cartridge filters. The concentrate containing PR0179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887
may further be purified using selected column-chromatography resins.
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EXAMPLE 24
Expression of Nucleic Acid Encoding PR0179 PR0238. PR0364 PR0844 PR0846
PR01760 PR0205
PR0321, PR0333. PR0840 PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887 in
Baculovirus-
Infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected
insect cells.
The sequence coding for PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 is fused
upstream of an epitope
tag contained within a baculovirus expression vector. Such epitope tags
include poly-His tags and immunoglobulin
tags (like Fc regions of IgG). A variety of plasmids may be employed,
including plasmids derived from
commercially available plasmids such as pVL1393 (Novagen). Briefly, the
sequence encoding PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887 or the desired portion of the coding sequence of
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887 [such as the sequence encoding the extracellular domain of a
transmembrane protein or the
sequence encoding the mature protein if the protein is extracellular] is
amplified by PCR with primers
complementary to the 5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites.
The product is then digested with those selected restriction enzymes and
subcloned into the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoldTM virus DNA
(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711 ) using
lipofectin (commercially available
from GIBCO-BRL). After 4 - 5 days of incubation at 28 °C, the released
viruses are harvested and used for further
amplifications. Viral infection and protein expression are performed as
described by O'Reilley et al., Baculovirus
Expression Vectors: A Laboratory Manual (Oxford: Oxford University Press
(1994)).
Expressed poly-His tagged-[PR0179, PR0238, PR0364, PR0844, PR0846, PR01760,
PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR088~ or PR0887] can then be
purified, for example,
by Ni '-'-chelate affinity chromatography as follows. Extracts are prepared
from recombinant virus-infected Sf9
cells as described by Rupert et al., Nature, 362:175-179 (1993). Briet7y, Sf9
cells are washed, resuspended in
sonication buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCI=; 0.1 mM EDTA; 10%
glycerol; 0. I % NP-40; 0.4 M KCl),
and sonicated twice for 20 seconds on ice. The sonicates are cleared by
centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol,
pH 7.8) and filtered through a
0.45 ~m filter. A Ni '+-NTA agarose column (commercially available from
Qiagen) is prepared with a bed volume
of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of loading
buffer. The filtered cell extract is
loaded onto the column at 0.5 ml per minute. The column is washed to baseline
A=~" with loading buffer, at which
point fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate:
300 mM NaCl, 10% glycerol, pH 6.0), which elutes non-specifically- bound
protein. After reaching A~h~, baseline
again, the column is developed with a 0 to 500 mM imidazole gradient in the
secondary wash buffer. One ml
fractions are collected and analyzed by SDS-PAGE and silver stainin'_ or
Western blot with Ni''-NTA-conjugated
to alkaline phosphatase (Qiagen). Fractions containing the eluted His",-tagged-
[PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333. PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887], respectively, are pooled and dialyzed against loading
buffer.
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Alternatively, purification of the IgG-tagged (or Fc tagged)-[PRO 179, PR0238,
PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882,
PR0885 or PR0887]
can be performed using known chromatography techniques, including for
instance, Protein A or protein G column
chromatography.
While expression was actually performed in a 0.5-2 L scale, it can be readily
scaled up for larger (e.g., 8 L)
preparations. The proteins were expressed as an IgG construct (immunoadhesin),
in which the protein extracellular
region was fused to an IgGI constant region sequence containing the hinge, CH2
and CH3 domains and/or in
poly-His tagged forms.
Following PCR amplification, the respective coding sequences were subcloned
into a baculovirus expression
vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged
proteins), and the vector and Baculogold~
baculovirus DNA (Pharmingen) were co-transfected into 105 Spodoptera
frugiperda ("Sf9") cells (ATCC CRL
1711 ), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are
modifications of the commercially available
baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker
regions to include the His or Fc
tag sequences. The cells were grown in Hink's TNM-FH medium supplemented with
10% FBS (Hyclone). Cells
were incubated for 5 days at 28°C. The supernatant was harvested and
subsequently used for the first viral
amplification by infecting Sf9 cells in Hink's TNM-FH medium supplemented with
10% FBS at an approximate
multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28
°C. The supernatant was harvested and
the expression of the constructs in the baculovirus expression vector was
determined by batch binding of 1 ml of
supernatant to 25 ml of Ni 2+-NTA beads (QIAGEN) for histidine tagged proteins
or Protein-A Sepharose CL-4B
beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis
comparing to a known concentration
of protein standard by Coomassie blue staining.
The first viral amplification supernatant was used to infect a spinner culture
(500 ml) of Sf9 cells grown in
ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells
were incubated for 3 days at
28°C. The supernatant was harvested and filtered. Batch binding and SDS-
PAGE analysis were repeated, as
necessary. until expression of the spinner culture was confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) was harvested
by centrifugation to remove the
cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein construct was purified
using a Ni ''-NTA column (Qiagen). Before purification, imidazole was added to
the conditioned media to a
concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni'+-NTA
column equilibrated in 20 mM
Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow rate
of 4-5 ml/min. at 4°C. After
loading, the column was washed with additional equilibration buffer and the
protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly purified protein was subsequently
desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25
Superfine (Pharmacial column
and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the
conditioned media as follows.
The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia)
which had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column was washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein was
immediately neutralized by collecting I ml
fractions into tubes containing 275 ml of 1 M Tris buffer. pH 9. The highly
purified protein was subsequently
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desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity of the proteins
was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal
amino acid sequencing by Edman
degradation.
PR0205, PR0321, PR0840, PR0846, PR0885 and PR0887 were successfully expressed
in Baculovirus-
infected insect Sf9 cells by the above procedure.
Alternatively, a modified baculovirus procedure may be used incorporating high-
5 cells. In this procedure,
the DNA encoding the desired sequence was amplified with suitable systems,
such as Pfu (Stratagene), or fused
upstream (5'-of) of an epitope tag contained with a baculovirus expression
vector. Such epitope tags include
poly-His tags and immunoglobulin tags (like Fc regions of IgG). A variety of
plasmids may be employed, including
1~ plasmids derived from commercially available plasmids such as pIEI-I
(Novagen). The pIEl-1 and pIEl-2 vectors
are designed for constitutive expression of recombinant proteins from the
baculovirus iel promoter in
stably-transformed insect cells. The plasmids differ only in the orientation
of the multiple cloning sites and contain
all promoter sequences known to be important for iel-mediated gene expression
in uninfected insect cells as well
as the hr5 enhancer element. pIEl-1 and pIEl-2 include the translation
initiation site and can be used to produce
fusion proteins. Briefly, the desired sequence or the desired portion of the
sequence (such as the sequence encoding
the extracellular domain of a transmembrane protein) was amplified by PCR with
primers complementary to the
5' and 3' regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product was then
digested with those selected restriction enzymes and subcloned into the
expression vector. For example, derivatives
of pIEI -1 can include the Fc region of human IgG (pb.PH.IgG) or an 8
histidine (pb.PH.His) tag downstream (3'-of)
the desired sequence. Preferably, the vector construct is sequenced for
confirmation.
High-5 cells are grown to a confluency of 50% under the conditions of, 27
°C, no CO~, NO penlstrep. For each
150 mm plate, 30 ~g of pIE based vector containing the sequence was mixed with
1 ml Ex-Cell medium (Media:
Ex-Cell 401 + l/100 L-Glu JRH Biosciences #14401-78P (note: this media is
light sensitive)), and in a separate
tube, 100 ~l of CellFectin (CelIFECTIN (GibcoBRL #10362-O10) (vortexed to
mix)) was mixed with 1 ml of
Ex-Cell medium. The two solutions were combined and allowed to incubate. at
room temperature for 15 minutes.
8 ml of Ex-Cell media was added to the 2 ml of DNA/CeIIFECTIN mix and this is
layered on high-5 cells that have
been washed once with Ex-Cell media. The plate is then incubated in darkness
for 1 hour at room temperature. The
DNA/CeIIFECTIN mix is then aspirated, and the cells are washed once with Ex-
Cell to remove excess
CeIIFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are incubated
for 3 days at 28 °C. The
supernatant was harvested and the expression of the sequence in the
baculovirus expression vector was determined
by batch binding of 1 ml of supernatant to 25 ml of Ni ~~-NTA beads (QIAGEN)
for histidine tagged proteins or
Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed
by SDS-PAGE analysis comparing
to a known concentration of protein standard by Coomassie blue staining.
The conditioned media from the transfected cells (0.5 to 3 L) was harvested by
centrifugation to remove the
cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein comprising the
sequence is purified using a Ni -+-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned
media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml
Ni ~+-NTA column equilibrated
in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a
flow rate of 4-5 ml/min. at 48 °C.
After loading, the column was washed with additional equilibration buffer and
the protein eluted with equilibration
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WO 00/53757 PCT/US00/05004
buffer containing 0.25 M imidazole. The highly purified protein was then
subsequently desalted into a storage
buffer containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25
ml G25 Superfine (Pharmacia)
column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the
conditioned media as follows.
The conditioned media was pumped onto a 5 ml Protein A column (Pharmacia)
which had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column was washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein was
immediately neutralized by collecting 1 ml
fractions into tubes containing 275 ml of 1 M Tris buffer, pH 9. The highly
purified protein was subsequently
desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity of the sequence
was assessed by SDS polyacrylamide gels and by N-terminal amino acid
sequencing by Edman degradation and
other analytical procedures as desired or necessary.
PR0179, PR0205, PR0321, PR0333, PR0364, PR0844, PR0846, PR0877, PR0879,
PR0882, PR0885
and PR01760 were expressed in high 5 cells by the above described method.
EXAMPLE 25
Preparation of Antibodies that Bind PR0179 PR0238 PR0364 PR0844 PR0846 PR01760
PR0205
PR0321, PR0333, PR0840 PR0877 PR0878 PR0879 PR0882 PR0885 or PR0887
This Example illustrates preparation of monoclonal antibodies that can
specifically bind PR0179, PR0238,
PR0364, PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877,
PR0878, PR0879,
PR0882, PR0885 or PR0887.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for instance, in
Goding, supra. Immunogens that may be employed include purified PR0179,
PR0238, PR0364, PR0844,
PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879,
PR0882, PR0885 or
PR0887 fusion proteins containing PRO 179, PR0238, PR0364, PR0844, PR0846> PRO
1760, PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887, and cells
expressing the gene
encoding PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205> PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 on the cell surface.
Selection of the immunogen can
be made by the skilled artisan without undue experimentation.
Mice, such as Balblc, are immunized with the PR0179, PR0238, PR0364. PR0844,
PR0846, PRO 1760,
PR0205, PR0321, PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or
PR0887 immunogen
emulsified in complete Freund's adjuvant and injected subcutaneously or
intraperitoneally in an amount from 1 to
100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adiuvant
(Ribi Immunochemical
Research, Hamilton, MT) and injected into the animal's hind foot pads. The
immunized mice are then boosted 10
to 12 days later with additional immunogen emulsified in the selected
adjuvant. Thereafter, for several weeks, the
mice may also be boosted with additional immunization injections. Serum
samples may be periodically obtained
from the mice by retro-orbital bleeding for testing in ELISA assays to detect
anti-PR0179, anti-PR0238, anti-
PR0364, anti-PR0844, anti-PR0846, anti-PR01760, anti-PR0205, anti-PR0321. anti-
PR0333, anti-PR0840,
anti-PR0877, anti-PR0878, anti-PR0879, anti-PR0882, anti-PR0885 or anti-PR0887
antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with a
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CA 02361849 2001-07-30
WO 00/53757 PCT/US00/05004
final intravenous injection of PR0179, PR0238, PR0364, PR0844, PR0846,
PR01760, PR0205, PR0321,
PR0333, PR0840, PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887. Three to
four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells are then
fused (using 35% polyethylene glycol)
to a selected murine myeloma cell line such as P3X63AgU.l, available from
ATCC, No. CRL 1597. The fusions
generate hybridoma cells that can then be plated in 96-well tissue culture
plates containing HAT medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against
PR0179, PR0238, PR0364,
PR0844, PR0846, PR01760, PR0205, PR0321, PR0333, PR0840, PR0877, PR0878,
PR0879, PR0882,
PR0885 or PR0887. Determination of "positive" hybridoma cells secreting the
desired monoclonal antibodies
against PR0179, PR0238, PR0364, PR0844, PR0846, PR01760, PR0205, PR0321,
PR0333, PR0840,
PR0877, PR0878, PR0879, PR0882, PR0885 or PR0887 is within the skill in the
art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce ascites
containing the anti-PR0179, anti-PR0238, anti-PR0364, anti-PR0844, anti-
PR0846, anti-PR01760, anti-PR0205,
anti-PR0321, anti-PR0333, anti-PR0840, anti-PR0877, anti-PR0878, anti-PR0879,
anti-PR0882, anti-PR0885
or anti-PR0887 monoclonal antibodies. Alternatively, the hybridoma cells can
be grown in tissue-culture flasks
or roller bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using
ammonium-sulfate precipitation, followed by gel-exclusion chromatography.
Alternatively, affinity chromatography
based upon binding of antibody to protein A or protein G can be employed.
Deposit of Material
The following materials) has/have been deposited with the American Type
Culture Collection, 10801
University Blvd., Manassas, VA 20110-2209, USA (ATCC):
Material ATCC Dep. No. Deposit Date
DNA16451- 1388 209776 April 14, 1998
DNA35600- l 162 209370 October 16,
1997
DNA47365-1206 209436 November 7,
1997
DNA59838- 1462 209976 June 16, 1998
DNA44196- 1353 209847 May 6, 1998
DNA76532- 1702 203473 November 17,
1998
DNA34433 209719 March 31, 1998
DNA53987 209858 May 12, 1998
This deposit was made under the provisions of the Budapest Treaty on the
International Recognition of the
Deposit of Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty).
This assures maintenance of a viable culture of the deposit for 30 years from
the date of deposit. The deposit will
be made available by ATCC under the terms of the Budapest Treaty, and subject
to an agreement between
3S Genentech, Inc., and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture
of the deposit to the public upon issuance of the pertinent U.S. patent or
upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures availability
of the progeny to one determined by
136
CA 02361849 2001-07-30
WO 00/53757 PCT/IJS00/05004
the U.S. Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC ~122 and the
Commissioner's rules pursuant thereto (including 37 CFR ~1.14 with particular
reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the
materials) on deposit should die or
be lost or destroyed when cultivated under suitable conditions, the materials)
will be promptly replaced on
notification with another of the same. Availability of the deposited
materials) is not to be construed as a license
to practice the invention in contravention of the rights granted under the
authority of any government in accordance
with its patent laws.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to practice
the invention. The present invention is not to be limited in scope by the
constructs) deposited, since the deposited
1~ embodiment(s) is/are intended as single illustrations) of certain aspects
of the invention and any constructs that
are functionally equivalent are within the scope of this invention. The
deposit of materials) herein does not
constitute an admission that the written description herein contained is
inadequate to enable the practice of any
aspect of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims
to the specific illustrations that it represents. Indeed, various
modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in the art
from the foregoing description and fall
within the scope of the appended claims.
137
