U.S. patent application number 10/749706 was filed with the patent office on 2004-11-11 for methods of treating hypertension and compositions for use therein.
Invention is credited to Johnson, Richard J., Schreiner, George F..
Application Number | 20040224885 10/749706 |
Document ID | / |
Family ID | 27378887 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040224885 |
Kind Code |
A1 |
Schreiner, George F. ; et
al. |
November 11, 2004 |
Methods of treating hypertension and compositions for use
therein
Abstract
The present invention provides methods and compositions of
treating hypertension. The methods generally involve administering
a factor which increases angiogenesis and/or vascular permeability.
Compositions for use in the methods are also provided.
Inventors: |
Schreiner, George F.; (Los
Altos, CA) ; Johnson, Richard J.; (Seattle,
WA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
27378887 |
Appl. No.: |
10/749706 |
Filed: |
December 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10749706 |
Dec 31, 2003 |
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10083817 |
Feb 26, 2002 |
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10083817 |
Feb 26, 2002 |
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09392932 |
Sep 9, 1999 |
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6352975 |
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60099694 |
Sep 9, 1998 |
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60126406 |
Mar 26, 1999 |
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60126615 |
Mar 27, 1999 |
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Current U.S.
Class: |
514/8.1 ;
514/13.3; 514/15.7 |
Current CPC
Class: |
A61P 7/04 20180101; A61P
11/00 20180101; A61P 7/06 20180101; A61P 37/06 20180101; A61P 43/00
20180101; A61K 38/1866 20130101; A61P 9/12 20180101; A61P 13/12
20180101; A61K 48/00 20130101; A61P 9/00 20180101; A61P 9/10
20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/18 |
Claims
1. A method for treating impaired filtration or excretion of a
solute in the kidney, comprising administering to a patient an
effective amount of a vascular endothelial growth factor (VEGF),
wherein filtration or excretion of the solute is improved as
compared to the pre-treatment condition of the patient.
2. The method of claim 1, wherein said VEGF is selected from the
group consisting of native hVEGF121 (FIG. 6, SEQ ID NO: 1), native
hVEGF145 (FIG. 7, SEQ ID NO: 2), native hVEGF165 (FIG. 8, SEQ ID
NO: 3), native hVEGF189 (FIG. 9, SEQ ID NO: 4), and native hVEGF206
(FIG. 10, SEQ ID NO: 5).
3. The method of claim 1, wherein said VEGF lacks the ability to
bind heparin.
4. The method of claim 1, wherein said VEGF is a native hVEGF121
(FIG. 6, SEQ ID NO: 1).
5. The method of claim 1, wherein said VEGF comprises a
heparin-binding domain modified to render it incapable of binding
heparin.
6. The method of claim 1, wherein said VEGF comprises an amino acid
alteration within its heparin-binding domain.
7. The method of claim 1 comprising the administration of two or
more VEGFs.
8. The method of claim 1, wherein said VEGF is coadministered with
another angiogenic factor.
9. The method of claim 1, wherein the solute is sodium
chloride.
10. The method of claim 1, wherein the impaired filtration or
excretion of solutes comprises an aspect of hypertension.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
09/392,932 filed on Sep. 9, 1999, which claims priority to
provisional application Serial No. 60/099,694 filed on Sep. 9,
1998, provisional application Serial No. 60/126,406 filed Mar. 26,
1999, and provisional application Serial No. 60/126,615 filed Mar.
27, 1999, all disclosures are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods for treating
hypertension, using a factor that stimulates angiogenesis and/or
promotes vascular permeability.
BACKGROUND OF THE INVENTION
[0003] Systemic hypertension is the most prevalent cardiovascular
disorder in the United States, affecting over 60 million Americans.
In spite of increasing public awareness and a rapidly expanding
array of antihypertensive medications, hypertension remains one of
the leading causes of cardiovascular morbidity and mortality.
Hypertension treatments have focused on stimulating the relaxation
of the peripheral vasculature (vasodilation), depressing cardiac
function, or by stimulating salt transport by blocking epithelial
transport of sodium or chloride (diuresis). "Textbook of Medical
Physiology", Guyton and Hall, eds. p. 234 (1996) W. B. Saunders. In
addition, adverse metabolic effects have been observed with
treatment using certain classes of antihypertensive treatment in
coronary disease prevention. "Cecil Textbook of Medicine" pp.
252-269 (1992) W. B. Saunders. Therefore, there is a need to
develop improved methods of treatment of hypertension.
[0004] Essential hypertension is the pathological expression of the
inability to excrete a dietary sodium load efficiently. The causes
for and the mechanism of the development of essential hypertension
are less than clear. According to one of the several possible
theories, excreting a sodium load depends on the permeability of
the vascular/epithelial barrier in the excreting organs such as the
kidney and/or the surface area of the vascular/epithelial
structures available for solute flux.
[0005] In tissues, the basement membrane serves to separate
epithelial cells from blood vessels containing endothelial cells,
particularly in the transport or flux of solutes in solution, such
as sodium chloride, across basement membranes. Normally, the
endothelium is relatively impermeable, limiting the flux of solutes
and fluid across the basement membrane against which it is
juxtaposed. However, the function of several specialized tissues
requires permeable capillary beds to support solute flux. Such
functions include the filtration of solutes by the kidney to
regulate intravascular volume and maintain normal blood pressure,
reabsorptibn of fluid secretions in the lungs to preserve pulmonary
oxygenation, reabsorption of fluids containing solutes in the
intestines to provide nutrition, production of cerebrospinal fluid
in the choroid plexus of the brain to support and protect the
central nervous system, diffusion of nutrients toward non-vascular
tissue as occurs in certain portions of the eye or in wound
healing, and reabsorption of interstitial fluid from the
peritoneum. Impaired transport of solutes across basement membranes
contributes to or exacerbates, among other disorders, essential
hypertension, kidney disease, acute respiratory disease syndrome,
macular ischemia, intestinal inflammatory diseases, meningitis,
stroke, ascites, impaired peritoneal dialysis efficiency, and
impaired wound healing.
[0006] A number of factors may potentially affect solute flux
between the endothelial bed and epithelial tissue, including: (1)
the metabolic activity of the epithelial cells of a particular
tissue; (2) the number of the blood vessels adjacent to the
epithelium and its basement membrane; and (3) the porosity or
permeability of said blood vessels. Some of the diseases cited
above are associated with epithelial cell toxicity, such as acute
respiratory distress syndrome and kidney disease or with altered
integrity of the capillary blood vessels, as occurs in vasculitis
or ischemia. Other disease syndromes, such as essential
hypertension have no defined central mechanism. The diseases cited
above may be associated with diminished capillary number or altered
porosity of the capillary vessel.
[0007] Angiogenesis, i.e. the growth of new capillary blood
vessels, is a process which is crucial to normal tissue formation
and repair. Consequently, factors that are capable of promoting
angiogenesis are useful as wound healing agents. Angiogenesis is a
multi-step process involving capillary endothelial cell
proliferation, migration and tissue penetration. A number of known
growth factors, including basic and acidic fibroblast growth factor
transforming growth factor .alpha. and epidermal growth factor, are
broadly mitogenic for a variety of cell types as well as being
angiogenic and are, therefore, potentially useful in promoting
tissue repair. Broad spectrum mitogenicity is desirable in many
types of tissue repair applications. There are, however, specific
types of tissue repair applications in which it would be desirable
to have endothelial cell-specific mitogenic activity, since
proliferation of other cell types along with endothelial cells
could cause blockage and/or reduced blood flow.
[0008] Vascular endothelial growth factor (VEGF) is a secreted
endothelial cell mitogen that, when delivered in vivo, promotes new
blood vessel formation. The VEGF protein consists of two
polypeptide chains, linked by two disulfide bonds. Although the
protein is generally described as a homodimer, heterodimeric
species have also been reported. Through alternative splicing of
the VEGF RNA transcript, five different forms of the monomer chain
can be generated, extending 121, 145, 165, 189, and 206 amino acid
residues in length. Tischer et al. (1991) J. BioL Chem.
266:11947-11954; Houck et al. (1991) MoL Endocrinol. 5:1806-1814;
Charnock-Jones et al. (1993) Biol. Reprod. 48:1120-1128; and
Neufeld et al. (1996) Cancer Metastasis Rev. 15:153-158. The
121-residue form of VEGF (VEGF.sub.121) is unique among the five
forms in that it does not bind to heparin-like molecules associated
with the extracellular matrix. VEGF.sub.121 and the 165-residue
form, VEGF.sub.121), appear to be the most prevalent forms in
vivo.
[0009] VEGF is known to stimulate new blood vessel formation by
stimulating endothelial cell proliferation and by inducing
chemotaxis of endothelial cells. In contrast to other mitogens such
as the fibroblast growth factors, VEGF has a much more restricted
range of target cell type, and is mitogenic almost exclusively
toward endothelial cells. VEGF is also known to enhance vascular
permeability and can trigger the relaxation of blood vessels
through the release of endothelial nitric oxide. Hariawala et al
(1996) J Surgical Res. 63:77-82; and SelIke et al. (1996) Am. J.
Physiol. 271:H713-H720. In addition, VEGF has been shown to
regulate the expression of other growth factors and biological
mediators and may participate in a growth factor cascade that
promotes tissue remodeling and repair.
[0010] The activity of VEGF is mediated by interaction with
specific membrane receptors on target tissues, most notably the
vascular endothelium. Both VEGF.sub.121 and VEGF.sub.165 are known
to interact with two tyrosine kinase receptors: kinase insert
domain-containing receptor (KDR; also known as FIk-1), and fms-like
tyrosine kinase-1 (Flt-1). deVries et al. (1 992) Science
255:989-991; Termnan et al. (1 992) Biochem. Biophys. Res. Commun.
187:1579-1586; and Millauer et al. (1993) Cell 72:835-846. Both KDR
and Flt-1 consist of extracellular ligand-binding domains and
intracellular tyrosine kinase domains, the latter being
functionally activated upon engagement of VEGF. KDR is found only
on endothelial cells, while Flt-1 is found on endothelial cells and
monocytes. The angiogenic properties and other known functions of
VEGF appear to be mediated via KDR and Flt-1.
[0011] Each human kidney comprises about one million nephrons, each
capable of forming urine. Each nephron has two major components: a
glomerulus, through which large amounts of fluid are filtered from
the blood, and a long tubule, in which the filtered fluid is
converted into urine. The glomerular capillary has three major
layers: the endothelium, a basement membrane, and a layer of
epithelial cells. The capillary endothelium is perforated by
thousands of small holes called fenestrae.
[0012] VEGF can increase the permeability of blood vessels to
solutes on a long-term basis by inducing the formation of
fenestrations between endothelial cells. Roberts and Palade (995)
J. Cell Sci. 108:2369-2379; and Esser et al. (1998)J. Cell Biol.
140:947-959. In some tissues, such as the renal glomerulus, the
glomerular epithelium is known to chronically secrete VEGF,
presumably to maintain the fenestrations of the glomerular
capillary endothelium. The solute ultrafiltrate that ultimately
forms the urine produced by the kidney flows through these
fenestrations. The choroid plexus in the brain responsible for
producing cerebrospinal fluid and the distal tubule of the kidney,
where sodium and potassium are exchanged for the final control of
the urine solute concentration also contain fenestrated endothelium
adjacent to VEGF-producing epithelium. Proper sodium and potassium
exchange in the distal tubule is essential for the maintenance of
normal intravascular volume.
[0013] Other epithelia known to constitutively produce VEGF include
the epithelia of the lung, intestines, and skin. Ferrara and
Davis-Smith (1997) Endocrine Rev. 18:4-25; and Monacci et al.
(1993) Am. J. Physiol. 264:C995-C1002. Non-epithelial cells that
make VEGF are fibroblasts and vascular smooth muscle cells, which
secrete VEGF in response to tissue hypoxia, and thus stimulate the
formation of new blood vessels. Ferrara and Davis-Smith (1997)
Endocrine Rev. 18:4-25.
[0014] In contrast to the mesenchymal cells that produce VEGF,
hypoxia does not appear to be a stimulus for VEGF production in
epithelial cells. Specialized endothelia that express VEGF
receptors in the absence of hypoxia include the glomerular and
peritubular capillaries of the kidney, the capillaries of the
choroid plexus, and endothelia in the intestines, lungs, retina,
and heart valve. Little is known about modulation of VEGF secretion
by epithelia. In the kidney, it is known that hypoxia is not a
signal for VEGF secretion. Kramer et al. (1997) Kidney
International 51:444-447.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods of treating
hypertension, particularly essential hypertension. The methods
generally involve providing a stimulator of angiogenesis and/or of
blood vessel porosity to maintain or correct the transport of
solutes, including sodium chloride, and fluid across a basement
membrane separating blood vessels or other vessels containing
endothelium from epithelial cells. Such transport can be from the
blood vessel across the basement membrane to or by the epithelial
cells; or it can be from or by epithelia across the basement
membrane to blood vessels. Stimulation of vessel number or porosity
is used to increase the efficiency or extent of solute transport,
thus decreasing blood volume and the concomitant hypertension.
[0016] In one aspect, the invention concerns a method for treating
essential hypertension, comprising administering to a patient an
effective amount of an angiogenic factor, or an agonist thereof.
The angiogenic factor can be administered alone or in combination
with a further anti-hypertensive agent, such as another angiogenic
factor, and preferably is a vascular endothelial growth factor
(VEGF) molecule.
[0017] The vascular endothelial growth factor is preferably
selected from the group consisting of native hVEGF145 (FIG. 7, SEQ
ID NO: 2), native hVEGF165 (FIG. 8, SEQ ID NO: 3), native hVEGF 189
(FIG. 9, SEQ ID NO: 4), native hVEGF206 (FIG. 10, SEQ ID NO: 5),
and agonists of any one of such native VEGF proteins.
[0018] In a particularly preferred embodiment, the VEGF molecule
lacks the ability to bind heparin, and is, for example,
hVEGF121.
[0019] The hypertension preferably is salt-dependent
hypertension.
[0020] In another aspect, the invention concerns an article of
manufacture comprising:
[0021] a container;
[0022] a composition comprising an angiogenic factor or an agonist
thereof, in an amount effective in the treatment of hypertension;
and
[0023] instructions to administer the composition for the treatment
of hypertension.
[0024] Again, the composition may contain an additional
anti-hypertensive agent, e.g. a further angiogenic factor. The
angiogenic factor preferably is a VEGF molecule or an agonist
thereof.
[0025] In a further aspect, the invention concerns a method for
identifying an anti-hypertensive agonist of a VEGF molecule
comprising testing the ability of a candidate agonist to treat
hypertension in a standard animal model of hypertension, in
comparison with the VEGF molecule.
[0026] The invention further provides compositions for use is the
foregoing methods and articles of manufacture. The compositions may
contain one or more active ingredients, at least one of which is an
angiogenic factor present in an amount effective in the treatment
of hypertension. Alternatively, the compositions may comprise one
or more polynucleotides comprising nucleotide sequences which
encode an angiogenic factor, such as a VEGF, an polypeptide agonist
of an angiogenic factor, or a polypeptide factor stimulating the
production of an angiogenic factor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic representation of the various forms of
VEGF generated by alternative splicing of VEGF mRNA. The protein
sequences encoded by each of the eight exons of the VEGF gene are
represented by numbered boxes. The sequences encoded by exons 6 and
7 are rich in basic amino acid residues and confer the ability to
interact with heparin and heparin-like molecules. Asterisks
indicate N-linked glycosylation sites. Exon 1 and the first part of
exon 2 (depicted by a narrower bar) encode the secretion signal
sequence for the protein.
[0028] FIG. 2 is a graph depicting the plasma elimination kinetics
of VEGF.sub.121 and VEGF.sub.165 administered intravenously. Each
data point represents the average of the values obtained from six
rats.
[0029] FIG. 3 is a graph depicting the plasma elimination kinetics
of VEGF.sub.112, and VEGF.sub.165 administered subcutaneously. Each
data point represents the average of the values obtained from six
rats.
[0030] FIGS. 4a-c are graphs of systolic blood pressure versus days
of treatment. FIG. 4a: Rats in one group (solid diamonds) were fed
a diet containing normal levels (0.1% w/w NaCl) of salt, while rats
in a second group (open squares) were placed on a diet containing
4% (w/w) sodium chloride for three days. The vertical arrow
indicates beginning of the high-salt diet. FIG. 4b: Rats fed the
normal salt (solid diamonds) or high salt (open squares) diets, as
shown in FIG. 4a, were treated with angiotensin II from days 0 to
14. The vertial arrow indicates discontinuation of Angiotensin II
treatment. FIG. 4c: Rats on a high-salt diet were given VEGF for 14
days concurrent with a 7-day infusion of Angiotensin II (solid
triangles), Angiotensin 11 alone (solid squares), or neither (solid
diamonds). The vertical arrow indicates beginning of the VEGF
and/or Angiotensin II treatment.
[0031] FIG. 5 depicts the weight of heart and kidney from rats
treated as described in FIG. 4c.
[0032] FIG. 6 shows the amino acid sequence (SEQ ID NO: 1) and the
encoding nucleotide sequence (SEQ ID NO: 6) of native hVEGF121.
[0033] FIG. 7 shows the amino acidsequence (SEQ ID NO: 2) and the
encoding nucleotide sequence (SEQ ID NO: 7) of native hVEGF145.
[0034] FIG. 8 shows the amino acid sequence (SEQ ID NO: 3) and the
encoding nucleotide sequence (SEQ ID NO: 8) of native hVEGF165.
[0035] FIG. 9 shows the amino acid sequence (SEQ ID NO: 4) and the
encoding nucleotide sequence (SEQ ID NO: 9) of native hVEGF189.
[0036] FIG. 10 shows the amino acid sequence (SEQ ID NO: 5) and the
encoding nucleotide sequence (SEQ ID NO: 10) of native
hVEGF206.
[0037] FIG. 11 shows the amino acid sequence (SEQ ID NO: 11) of
native hVEGF110.
[0038] FIG. 12 illustrates the ability of VEGF treatment to inhibit
the development of experimental salt sensitive hypertension in
rats.
MODES OF CARRYING OUT THE INVENTION
[0039] In animals and patients with essential hypertension, in the
presence of diminished sodium chloride in the diet, the kidney is
able to maintain a normal or near-normal blood pressure. The model
system described herein creates salt-dependent hypertension in
which transient exposure to a preceding hypertensive stimulus such
as Angiotensin II (AII) or norepinephrine confers a susceptibility
to hypertension which is dependent upon the amount of sodium
chloride, or solute, in the diet, much like essential hypertension.
Johnson and Schreiner (1997) Kidney International 52:1169-1179. As
is the case with essential hypertension, kidneys with a preceding
exposure to a transient or labile hypertensive stimulus are unable
to handle the increased filtered load of solute arising from
dietary exposure, with a consequent development of elevated blood
pressure. Johnson and Schreiner (1997).
[0040] In most forms of kidney disease, renal production of VEGF is
diminished. Shulman et al. (1996) J. Am. Soc. Nephrol. 7:661-666.
Both VEGF and eNOS are constitutively co-localized to collecting
ducts and medullary thick ascending limb tubular cells in the outer
medulla of the rat. With acute vasoconstriction such as that
induced by cyclosporine, an increase in both cortical hypoxia and
VEGF expression can be documented, and is consistent with the known
ability of hypoxia to induce VEGF expression. However, once chronic
tubulointerstitial disease develops, our group has noted a loss of
VEGF in the outer medulla, especially in the medullary thick
ascending limb. This finding was observed not only in rats with
cyclosporine induced nephropathy but also with the
tubulointerstitial disease that accompanies aging, hypokalemia, and
following angiotension II infusion.
[0041] Most forms of renal disease are also associated with
hypertension, raising the possibility that the impaired excretion
of solute in kidney disease is due in part to diminished endogenous
production of VEGF with consequent decrease in either the density
or permeability of the renal capillary beds.
[0042] Essential hypertension, while not linked to renal disease
per se, is associated with decreased renal natriuretic response to
salt loading or increased intravascular volume or increased
systemic blood pressure. Johnson and Schreiner (1997).
Hypertersion, as an expression of altered transport of sodium
chloride across a basement membrane in the kidney, may be a
consequence of diminished number or porosity of the capillary
plexus subserving the sodium chloride transporting epithelia of the
renal nephron.
[0043] It has now been shown that, using a model of hypertension
dependent upon increased sodium in the diet, the administration of
an angiogenic factor which increases the number and/or porosity of
capillaries adjacent to the transporting epithelium allows
increased solute load in the diet such that it can be handled in a
non-hypertensive manner.
[0044] Without being bound by any one theory, a relative deficiency
in VEGF could predispose to systemic hypertension several ways.
First, the loss of constitutive VEGF expression at sites of
tubulointerstitial damage may play a role in the capillary loss at
these sites, since VEGF is a potent angiogenic factor. Second, the
endothelium in the glomeruli and peritubular capillaries are
relatively unique in that they express fenestrations. VEGF is the
only cytokine known to induce endothelial fenestrations, and the
constitutive sites of VEGF expression in the kidney (podocytes and
collecting duct cells) suggests that they normally help maintain
this endothelial phenotype. It is possible that the local loss of
VEGF could reduce these fenestrations, and that this could impact
on glomerular permeability (kf) or the relative permeability of the
peritubular capillaries involved in pressure natriuresis (i.e., an
"interstitial"kf). Finally, since VEGF is a potent inducer of eNOS
expression and NO generation, a loss of VEGF could theoretically
result in decreased local NO concentration.
[0045] Experiments set forth in the Examples, in which VEGF.sub.121
was infused to rats with tubulointerstitial disease induced either
by angiotension II (AII) or cyclosporine (CSA), demonstrated that
VEGF infusion could prevent the development of salt-sensitive
hypertension, and in the CSA model this was shown to persist even
after VEGF administration was stopped.
[0046] An angiogenic factor, such as VEGF, may restore the proper
response to salt loading by restoring trans-basement membrane
transport of solutes such as sodium and chloride. Accordingly, the
treatment of hypertension can now be mediated by increasing either
the density of capillaries adjacent to the basement membrane, the
porosity of the capillaries, or both.
[0047] There has been no previous attempt to stimulate sodium
chloride excretion by the kidney by stimulating angiogenesis and/or
porosity of the blood vessels, with consequent blood pressure
normalization. No previous therapy has targeted the endothelium to
improve the efficiency of solute transport. Indeed, it has
heretofor been believed that administration of an angiogenic factor
in this context would be detrimental. Unlike other therapies, it
has now been found that angiogenic therapy further addresses the
mechanism by which the kidney is impaired with respect to excreting
increased salt loads.
[0048] Accordingly, the methods of the present invention treat
hypertension by administering an amount of angiogenic factors
effective to decrease hypertension. Such factors may stimulate
angiogenesis and/or promote vascular permeability.
General Techniques
[0049] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I.
[0050] Freshney, ed., 1987); "Methods in Enzymology" (Academic
Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir
& C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian
Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current
Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987);
"PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994);
and "Current Protocols in Immunology" (J. E. Coligan et al., eds.,
1991).
Definitions
[0051] The term "angiogenic factor", as used herein, refers to any
molecule (including polypeptides, peptides and small molecules),
capable of promoting the growth of new blood capillary vessels from
existing endothelium (angiogenesis), and/or increasing vascular
permeability (promoting porosity of blood vessels. Angiogenic
factors include, but are not limited to, vascular endothelial
growth factors (VEGFs) in all forms, including native sequence VEGF
molecules from any animal species, including humans, and their
functional derivatives, fibroblast growth factors (FGFs), such as
acidic and basic fibroblast growth factors (aFGFs and bFGFs) in all
forms, including native sequence FGF molecules from any animal
species, including humans, and their functional derivatives, and
VEGF-related molecules, such as PIGF, VEGF-B, and VEGF-C/VRP,
including all native sequence forms from any animal species,
including humans and other mammalian species, such as murine,
bovine, equine, porcine, ovine, canine, or feline, and their
functional derivatives. The definition specifically includes homo-
and heterodimeric forms of these and related molecules, where
dimerization is required for biological activity.
[0052] Such factors include VEGF, in any of its forms, and other
angiogenic factors, including, but not limited to, basic fibroblast
growth factor and acidic fibroblast growth factor. Additionally,
such factors include those that stimulate the production of VEGF or
other angiogenic factors or the expression of VEGF receptors or the
receptors of other angiogenic factors. Such factors include, but
are not limited to, platelet derived growth factor (PDGF),
transforming growth factor (TGF-.alpha. or -.beta.), interleukin-1
(IL-1), interleukin-6 (IL-6), insulin-like growth factor (FGF) in
all its forms, heparin-binding epidermal growth factor (HBEGF),
epidermal growth factor (EGF), adenosine, prostaglandins (PGs), and
agents that activate protein kinase C, protein kinase A, or ras
GTPase activating proteins.
[0053] Additions, substitutions or deletions of portions of any
angiogenic factor are acceptable as long as the modification allows
the factor to retain biological activity. Representative measures
of biological activity are known in the art and include, but are
not limited to, including initiation or promotion of angiogenesis
in vivo, and/or promotion of blood vessel porosity in vivo, or
stimulation of endothelial mitosis or chemotaxis, or nitric oxide
production in vitro or in vivo, or promotion of the formation and
secretion of factors that exert the aforementioned effects.
Preferably, a "biologically active angiogenic factor" is one that
increases angiogenesis and/or increases vascular/capillary
permeability. An angiogenic factor can be part of a fusion
polypeptide, i.e., one that comprises a portion that is the
angiogenic factor and at least one other portion that comprises a
different polypeptide. Examples include epitope-tagged angiogenic
factors. Covalent modifications, which are known in the art, are
also possible and included for use herein. A particularly professed
biological activity for the purpose of this invention is the
ability to reduce, most preferably, normalize hypertension.
[0054] The phrase "factor that stimulates the production of an
angiogenic factor", and grammatical equivalents thereof, are used
in the broadest sense, and include compounds (native and variant
polypeptides and peptides, small molecules, antibodies, etc.) that
stimulate the expression of angiogenic factors, or receptors of
angiogenic factors, regardless of the mechanism by which this
stimulation is achieved. As noted before, such factors include, for
example, platelet derived growth factors (PDGF) in all forms,
transforming growth factors (TGF) in all forms, interleukin-1
(IL-1), interleukin-6 (IL-6), insulin-like growth factor (IGF) in
all forms, heparin-binding epidermal growth factor, epidermal
growth factor (EGF), adenosine, prostaglandins, or agents that
activate protein kinase C, protein kinase A, or ras GTPase
activating proteins. The designations of the listed angiogenic
factors specifically include all naturally occurring forms from any
animal species, including humans and other mammalian species, such
as murine, bovine, equine, porcine, ovine, canine, or feline, and
functional derivatives thereof.
[0055] The term "vascular endothelial growth factor" or "VEGF" as
used herein refers to any naturally occurring (native) forms of a
VEGF polypeptide (also known as "vascular permeability factor" or
"VPF") from any animal species, including humans and other
mammalian species, such as murine, bovine, equine, porcine, ovine,
canine, or feline, and functional derivatives thereof. "Native
human VEGF" consists of two polypeptide chains generally occurring
as homodimers. Each monomer occurs as one of five known isoforms,
consisting of 121, 145, 165, 189, and 206 amino acid residues in
length. These isoforms will be hereinafter referred to as
hVEGF.sub.121, hVEGF.sub.145, hVEGF.sub.165, hVEGF.sub.189, and
hVEGF.sub.206, respectively. Similarly to the human VEGF, "native
murine VEGF" and "native bovine VEGF" are also known to exist in
several isoforms, 120, 164, and 188 amino acids in length, usually
occurring as homodimers. With the exception of hVEGF.sub.121, all
native human VEGF polypeptides are basic, heparin-binding
molecules. hVEGF.sub.121 is a weakly acidic polypeptide that does
not bind to heparin. These and similar native forms, whether known
or hereinafter discovered are all included in the definition of
"native VEGF" or "native sequence VEGF", regardless of their mode a
preparation, whether isolated from nature, synthesized, produced by
methods of recombinant DNA technology, or any combination of these
and other techniques. The term "vascular endothelial growth factor"
or "VEGF" includes VEGF polypeptides in monomeric, homodimeric and
heterodimeric forms. The definition of "VEGF" also includes a 110
amino acids long human VEGF species (hVEGF.sub.110), and its
homologues in other mammalian species, such as murine, bovine,
equine, porcine, ovine, canine, or feline, and functional
derivatives thereof. In addition, the term "VEGF" covers chimeric,
dimeric proteins, in which a portion of the primary amino acid
structure corresponds to a portion of either the A-chain subunit or
the B-chain subunit of platelet-derived growth factor, add a
portion of the primary amino acid structure corresponds to a
portion of vascular endothelial growth factor. In a particular
embodiment, a chimeric molecule is provided consisting of one chain
comprising at least a portion of the A- or B-chain subunit of a
platelet-derived growth factor, disulfide linked to a second chain
comprising at least a portion of a VEGF molecule. More details of
such dimers are provided, for example, in U.S. Pat. Nos. 5,194,596
and 5,219,739 and in European Patent EP-B 0 484 401, the
disclosures of which are hereby expressly incorporated by
reference. The nucleotide and amino acid sequences of hVEGF.sub.121
and bovine VEGF.sub.120 are disclosed, for example, in U.S. Pat.
Nos. 5,194,596 and 5,219,739, and in EP 0 484 401. hVEGF.sub.145 is
described in PCT Publication No. WO 98/10071; hVEGF.sub.165 is
described in U.S. Pat. No. 5,332,671; hVEGF.sub.189 is described in
U.S. Pat. No. 5,240,848; and hVEGF.sub.206 is described in Houck et
al. Mol. Endocrinol. 5:1806-1814 (1991). Other VEGF polypeptides
and polynucleotides have been described, including, for example,
zvegf2 (PCT Publication No. WO 98/24811), and VRP (PCT Publication
No. WO 97/09427), and are also encompassed by the term VEGF. For
the disclosure of the nucleotide and amino acid sequences of
various human VEGF isoforms see also Leung el al., Science
246:1306-1309 (1989); Kecketal., Science 246:1309-1312 (1989);
Tisheret al., J. Biol. Chem. 266:11947-11954 (1991); EP 0 370 989;
and PCT publication WO 98/10071. Forms of VEGF are shown
schematically in FIG. 1. FIGS. 2-11 (SEQ ID NOs: 1-10) show the
nucleotide and amino acid sequences of various VEGF species. For
further review, see also Klagsburn and D'Amore, Cytokine and Growth
Factor Reviews 7:259-170 (1996).
[0056] The term "VEGF" encompasses a polypeptide having an amino
acid sequence substantially homologous to one or more of the
above-mentioned native VEGF polypeptides, and which retains a
biological activity associated with VEGF. An amino acid sequence is
considered to be "substantially homologous" herein if the level of
amino acid sequence homology is at least about 50%, preferably at
least about 80%, more preferably at least about 90%, most
preferably, at least about 95%, compared with the native VEGF
protein in question.
[0057] Also included within the scope of "VEGF" herein are
biologically active fragments thereof, as well as N-terminally or
C-terminally extended versions thereof or analogs thereof
substituting and/or deleting or inserting one or more amino acid
residues which retain qualitatively the biological activities of
the protein described herein. Preferred analogs include those in
which one or more cysteine residues not required for biological
activity are substituted by a different amino acid residue,
preferably serine. Substitution of one or more cysteine residues
reduces the opportunity for intermolecular and intramolecular
disulfide bond formation, thereby rendering the molecule more
stable. For example, there are nine cysteine residues that are
present in hVEGF121 and hVEGF165. Of these, eight are conserved
with PDGF. Accordingly, a preferred analog is one in which the
ninth cysteine residue is substituted by serine. This cysteine
residue is present at position 160 of hVEGF165 and position 116 of
hVEGF121. Amino acid substitutions can be accomplished by site
specific mutagenesis of the DNA sequences described herein using
well known techniques. See, e.g., Zoller and Smith (1982) Nucleic
Acids Research 10:6487-6500.
[0058] The term "VEGF" specifically includes homodimeric and
heterodimeric forms of the VEGF molecule, in which the dimer is
formed via interchain disulfide bonds between two subunits.
Homodimers may have both of their subunits unglycosylated or
glycosylated, while in heterodimers, one subunit may be
glycosylated and the other unglycosylated. The term "VEGF"
specifically includes not only amino acid sequence variants but
also glycosylation variants of the native VEGF molecules.
[0059] In addition, the term "VEGF" covers chimeric, dimeric
proteins, in which a portion of the primary amino acid structure
corresponds to a portion of either the A-chain subunit or the
B-chain subunit of platelet-derived growth factor, add a portion of
the primary amino acid structure corresponds to a portion of
vascular endothelial growth factor. In a particular embodiment, a
chimeric molecule is provided consisting of one chain comprising at
least a portion of the A- or B-chain subunit of a platelet-derived
growth factor, disulfide linked to a second chain comprising at
least a portion of a VEGF molecule. More details of such dimers are
provided, for example, in U.S. Pat. Nos. 5,194,596 and 5,219,739
and in European Patent EP-B 0 484 401, the disclosures of which are
hereby expressly incorporated by reference.
[0060] The amino acid sequence numbering system used herein for
VEEGF is based on the mature forms of the protein, i.e. the
post-translationally processed forms. Accordingly, the residue
numbered one in the human proteins is alanine, which is the first
residue of the isolated, mature forms of these proteins.
[0061] A "polynucleotide comprising sequences encoding an
angiogenic factor" includes a polynucleotide comprising sequences
encoding any of the above-mentioned angiogenic factors. Many such
polynucleotides have been disclosed, including, for example, in the
references mentioned above, wherein VEGF polypeptides are
disclosed.
[0062] The term encompasses polynucleotide sequences which
hybridize under stringent hybridization conditions to the disclosed
sequences, as long as the polypeptide encoded thereby is
biologically active, i.e., it increases angiogenesis and/or
increases vascular permeability.
[0063] The terms "vector", "polynucleotide vector", "construct" and
"polynucleotide construct" are used interchangeably herein. A
polynucleotide vector of this invention may be in any of several
forms, including, but not limited to, RNA, DNA, DNA encapsulated in
an adenovirus coat, DNA packaged in another viral or viral-like
form (such as herpes simplex, and AAV), DNA encapsulated in
liposomes, DNA complexed with polylysine, complexed with synthetic
polycationic molecules, conjugated with transferrin, complexed with
compounds such as PEG to immunologically "mask" the molecule and/or
increase half-life, or conjugated to a non-viral protein. A
polynucleotide vector of this invention may be in the form of any
of the delivery vehicles described herein. Preferably, the
polynucleotide is DNA. As used herein, "DNA" includes not only
bases A, T, C, and G, but also includes any of their analogs or
modified forms of these bases, such as methylated nucleotides,
intemucleotide modifications such as uncharged linkages and
thioates, use of sugar analogs, and modified and/or alternative
backbone structures, such as polyamides.
[0064] "Under transcriptional control" is a term well-understood in
the art and indicates that transcription of a polynucleotide
sequence, usually a DNA sequence, depends on its being operably
(operatively) linked to an element which contributes to the
unification of, or promotes transcription.
[0065] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient of any vector of this
invention. Host cells include progeny of a single host cell, and
the progeny may not necessarily be completely identical (in
morphology or in total DNA complement) to the original parent cell
due to natural, accidental, or deliberate mutation and/or change. A
host cell includes cells transfected or infected in vivo with a
vector comprising a polynucleotide encoding an angiogenic
factor.
[0066] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm
animals, sport animals, and pets.
[0067] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of an angiogenic factor is an amount
that is sufficient to palliate, ameliorate, stabilize, reverse,
slow or delay the progression of the disease state. In particular,
the "effective amount" for the purpose of the present invention is
defined as an amount capable of reducing, and preferably
normalizing, at least transiently, high blood pressure in an
accepted animal model of hypertension, such as, for example, the
animal model of salt-dependent hypertension disclosed in Example 2
herein.
[0068] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. "Treatment" is an intervention performed with
the intention of preventing the development or altering the
pathology of a disorder. Accordingly, "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented.
Accordingly, "treatment" in the context of the present invention is
an intervention performed with the intention of preventing the
development of high blood pressure in patients at risk and/or or
reducing elevated blood pressure, preferably to a normal level. For
maintenance of acceptable levels of blood pressure, repeated
treatments may be necessary for an extended period of time.
[0069] "Palliating" a disease means that the extent and/or
undesirable clinical manifestations of a disease state are lessened
and/or time course of the progression is slowed or lengthened, as
compared to not administering a factor which increases angiogenesis
and/or vascular permeability.
[0070] Effectiveness is determined by decreased blood pressure in
response to salt loading. Methods of measuring blood pressure are
known in the art and need not be described herein.
[0071] "Sequence identity", is defined as the percentage of amino
acid residues in a candidate sequence that are identical with the
amino acid residues in a native polypeptide 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.
The % sequence identity values are generated by the NCBI BLAST2.0
software as defined by Altschul el al., (1997), "Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs",
Nucleic Acids Res., 25:3389-3402. The parameters are set to default
values, with the exception of the Penalty for mismatch, which is
set to-1.
[0072] "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 horses, sheep,
cows, pigs, dogs, cats, etc. Preferably, the mammal is human.
[0073] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0074] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0075] The term "agonist" is used in the broadest sense and
includes any molecule that mimics a biological activity of a native
angiogenic factor, such as a native VEGF polypeptide disclosed
herein. For the purpose of the methods claimed herein, the
biological activity mimicked is the ability to reduce elevated
blood pressure, regardless of the mechanism by which this effect is
achieved. Suitable molecules specifically include agonist or
antagonist antibodies or antibody fragments, fragments or amino
acid sequence variants of native polypeptides, peptides, small
organic molecules, etc.
[0076] A "small molecule" is defined herein to have a molecular
weight below about 500 daltons.
[0077] "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 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
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) 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.
[0078] 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, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0079] 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 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-deterrnining 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 (FR). The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-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. 1, 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.
[0080] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" to "CDR" (i.e.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable do Kabat el al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institute of Health, Bethesda, MD. [1991]) and/or those residues
from a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Clothia and Lesk, J. Mol BioL 196:901-917 [1987]). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0081] The term "antibody" is used herein 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.
[0082] "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, for
example, Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies;
linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062
[1995]); single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments.
[0083] 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 which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. 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
requiring 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. Pat. No. 4,816,567).
The "monoclonal antibodies" may also be isolated from phage
antibody libraries using the techniques described in Clackson et
al., Nature, 352:624-628 [1991] and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example.
[0084] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which the variable region of an antibody
heavy or light chain is derived from one mammalian species
(typically a rodent, e.g. mouse, rat or rabbit), while the constant
region is derived from a different mammalian species (typically
human), as well as fragments of such antibodies, so long as they
exhibit the desired biological activity (U.S. Pat. No.4,816,567;
Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855
[1984]).
[0085] "Humanized" forms of non-human (e.g., murine) 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 which are found neither in the recipient antibody nor in
the imported CDR or framework sequences. The humanized antibody
optimally 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); and Reichmann et al., Nature, 332:323-329 [1988].
The humanized antibody includes a PRIMATIZED.TM.antibody wherein
the antigen-binding region of the antibody is derived from an
antibody produced by immunizing macaque monkeys with the antigen of
interest.
[0086] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0087] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
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).
Angiogenic Factors
[0088] The present invention provides methods for treating
hypertension comprising administering an effective amount of an
angiogenic factor. An angiogenic factor suitable for use in the
methods of the present invention can promote (or increase, or
induce) angiogenesis and/or can promote (or increase, or induce)
vascular or capillary permeability. An angiogenic factor can be
used singly or in combination with another angiogenic factor(s) or
other therapeutic agents for use in the methods described
herein.
[0089] An angiogenic factor can be a biological or chemical
compound such as a simple or complex organic or inorganic molecule,
a peptide, or a protein. A vast array of compounds can be
synthesized, for example, oligopeptides, and synthetic inorganic
and organic-compounds based on various core structures, and these
are also included.
[0090] Whether an angiogenic factor promotes angiogenesis can be
determined by any known method. For example, a material which
serves as a matrix for invasion of new blood vessels, such as, for
example, gelatin or Matrigel, can be implanted subcutaneously in an
animal. The implanted sponges can be treated with a putatitive
angiogenic factor and, after a suitable period of time, e.g., 7 to
14 days, the implants are removed and examined morphometrically for
the quantitation of blood vessels which have invaded the implant.
Alternatively, the implants can be analyzed for the presence of an
endothelial cell marker, such as von Willebrand factor or CD34, or
the hemoglobin content of the implant can be determined.
Vascularization of an implant containing a putative angiogenic
factor is compared with an implant lacking the factor. In vitro
methods for assessing angiogenesis have also been described and can
be used to determine whether a substance is an angiogenic factor.
Magee et al. (1994) Am. J. Physiol. 267:pL433-441; Pepper et al.
(992) Biochem. Biophys. Res. Comm. 189:824-831; and Nicosia et al.
(1994) Am. J. Pathol. 145:1023-1029.
[0091] produced by any known means, including those described in
U.S. Pat. No. 5,194,596.
Amino Acid Sequence Variants of Native Angiogenic Factors
[0092] Variations in the amino acid sequence of native angiogenic
factors, such as native VEGF polypeptides, involve substitution,
deletion and/or insertion of one or more amino acids in the native
polypeptide sequence. 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 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 in any assay of high blood pressure, such as the assay
described in the Examples below.
[0093] In a preferred group of amino acid sequence variants, one
ore more cysteine residues in the VEGF structure is replaced by
another amino acid. Such substitution reduced the opportunity for
intermolecular and intramolecular disulfide bond formation, thereby
rendering the molecule more stable. There are nine cysteine
residues present in hVEGF120, hVEGF165, and in the respective
bovine homologues. Of these, eight are conserved with PDGF.
Accordingly, the most preferred analog is in which the ninth
cysteine residue is subtituted by serine. This cysteine residue is
presented at position 160 of hVEGF165 and position 116 of hVEGF121,
and the corresponding positions of the bovine forms. Some
additional information about variant forms of VEGF molecules is
provided in U.S. Pat. No. 5,332,671. Specifically included herein
are the variant VEGF molecules described in PCT Publication WO
98/36075, the disclosure of which is expressly incorporated by
reference. Such VEGF molecules contain modifications in the
C-terminal heparin binding domain that are described to result in
functional modification of the pharmacokinetic profile, and yield
molecules having a reduced clearance rate compared with the
corresponding heparin-binding native VEGF molecule. Preferred
embodiments include the replacement of positively charged amino
acids with negatively charged or neutral amino acids within the
heparin-binding domain of a heparin-binding VEGF species. In
addition, VEGF variants in which portions of the C-terminal
heparin-binding domain are deleted are included within the scope of
the present invention.
[0094] 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 et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., 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 DNA encoding a VEGF variant.
[0095] 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 One indication
that a factor promotes angiogenesis is its ability to induce
mitogenesis of vascular endothelial cells. Mitogenic activity for
vascular endothelial cells can be determined by an assay which
uses, as target cells, adrenal cortex-derived capillary endothelial
cells (ACE cells). This assay is carried out essentially as
described in Gospodarowicz et al. ((1986) J. Cell Physiol.
127:121-136), the disclosure of which is incorporated herein by
reference. Generally, stock cultures of ACE cells are maintained in
the presence of Dulbecco's modified Eagle's medium (DMEM-21)
supplemented with 10% calf serum. The antibiotics penicillin (50
IU/ml), streptomycin (50 mu g/ml), gentamycin (50 mu g/ml), and
Fungizone (0.25 mu g/ml) and 2 mM L-glutamine can also be added to
the medium. Cells are passaged weekly on tissue culture dishes at a
split ratio of between 1:40 and 1:200 (the preferred split ratio is
that which gives 2.5.times.10.sup.5 cells in 15 ml of medium in T75
flasks). For the mitogenic assay, cells are seeded in 12 well
cluster plates at a density of 5.times.10.sup.3 cells per well in 1
ml Dulbecco's modified Eagle's medium supplemented with 10% calf
serum and antibiotics as described in Gospodarowicz et al. (1988)
Eur. J. Cell. Biol. 46:144-151. Alternatively, the ACE cells are
plated in 35 mm dishes or 6 well cluster plates at a density of
5-10.times.10.sup.3 cells per dish or well in 2 ml of medium as
described in Gospodarowicz et al. (1986). Ten-microliter aliquots
of appropriate dilutions of each sample are then added to duplicate
or triplicate wells in the dishes on days 0 and 2. After 4 or 5
days in culture, the plates are trypsinized and cell densities
determined in a Coulter counter. For purposes of description
herein, a factor is considered to have mitogenic activity for
vascular endothelial cells if the cell density at the end of this
assay is at least 1.5 times and preferably at least 3 times the
cell density of control wells receiving no factor additions.
[0096] Determination of whether an angiogenic factor induces
capillary permeability can be determined by any known method. The
method used may depend upon the tissue being examined. For example,
for determination of renal peritubular capillary permeability, one
can administer to an animal proteins of various molecular weights
which are labelled with a dye detectable by electron microscopy.
Venkatachalam and Kamovsky (1972) J. Lab. Invest. 27:435-444.
Alternatively, the proteins can be enzymes which act on a substrate
to produce a signal. Another method to measure peritubular
capillary permeability is by renal micropuncture. See, for example,
Baer et al. (1978) Kidney Int. 13:452-466. Methods to assess
capillary permeability in the lung have been described (Lull et al.
(1983) Semin. Nucl. Med. 13:223-237), as have methods to measure
cerebral vascular permeability (Terada et al. (1992) Neuroradiol.
34:290-296).
[0097] Capillary density can be measured immunohistochemically
using commercially available antibodies which bind specifically to
endothelial cell-specific cell surface markers, such as von
Willebrand factor or CD34.
[0098] In determining whether an angiogenic factor is effective in
treating hypertension, an animal model such as the one described
herein can be used. Hypertension is induced, as described in
Example 1, a putative angiogenic factor administered, and blood
pressure monitored. Decreased blood pressure in response to
treatment with the factor, as compared with a hypertensive animal
not treated with the factor, is an indication that the factor is
effective in treating hypertension. Other animal models of
hypertension are known in the art, and can be used to determine
whether an angiogenic factor is effective in treating hypertension.
These include the Dahl rat. Roman et al. (1986) Am. J Physiol.
251:F57-F65.
Production of Angiogenic Factors
[0099] Methods of producing known angiogenic factors are well known
in the art and can be used to produce an angiogenic factor. These
methods include synthetic and recombinant methods, as well as
methods for isolating angiogenic factors from natural sources, from
tissue culture supernatants, and the like. An example of an
angiogenic factor which can be used in the methods of the present
invention is VEGF, which can be 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.
Delivery Vehicles Comprising Polynucleotides Encoding an Angiogenic
Factor
[0100] The present invention also provides delivery vehicles
suitable for delivery of a polynucleotide encoding an angiogenic
factor into cells (whether in vivo, ex vivo, or in vitro).
Generally, a polynucleotide encoding an angiogenic factor will be
operably linked to a promoter and a heterologous polynucleotide. A
polynucleotide encoding an angiogenic factor can be contained
within a cloning or expression vector, using methods well known in
the art, or within a viral vector. These vectors (especially
expression vectors) can in turn be manipulated to assume any of a
number of forms which may, for example, facilitate delivery to
and/or entry into a target cell. Delivery of the polynucleotide
constructs of the invention to eukaryotic cells, particularly to
mammalian cells, more particularly to distal tubule cells of the
kidney, can be accomplished by any suitable art-known method.
Delivery can be accomplished in vivo, ex vivo, or in vitro.
[0101] The invention provides methods and compositions for
transferring such expression constructs into cells, especially in
vivo for treatment of hypertension. It is also an object of the
invention to provide compositions for the therapy of
hypertension.
[0102] Delivery vehicles suitable for incorporation of a
polynucleotide encoding an angiogenic factor of the present
invention for introduction into a host cell include non-viral
vehicles and viral vectors. Verma and Somia (1997) Nature
389:239-242.
[0103] A wide variety of non-viral vehicles for delivery of a
polynucleotide encoding an angiogenic factor are known in the art
and are encompassed in the present invention. A polynucleotide
encoding an angiogenic factor can be delivered to a cell as naked
DNA (U.S. Pat. No. 5,692,622; WO 97/40163). Alternatively, a
polynucleotide encoding an angiogenic factor can be delivered to a
cell associated in a variety of ways with a variety of substances
(forms of delivery) including, but not limited to cationic lipids;
biocompatible polymers, including natural polymers and synthetic
polymers; lipoproteins; polypeptides; polysaccharides;
lipopolysaccharides; artificial viral envelopes; metal particles;
and bacteria. A delivery vehicle can be a microparticle. Mixtures
or conjugates of these various substances can also be used as
delivery vehicles. A polynucleotide encoding an angiogenic factor
can be associated non-covalently or covalently with these various
forms of delivery. Liposomes can be targeted to a particular cell
type, e.g., to a glomerular epithelial cell.
[0104] Viral vectors include, but are not limited to, DNA viral
vectors such as those based on adenoviruses, herpes simplex virus,
poxviruses such as vaccinia virus, and parvoviruses, including
adeno-associated virus; and RNA viral vectors, including, but not
limited to, the retroviral vectors. Retroviral vectors include
murine leukemia virus, and lentiviruses such as human
immunodeficiency virus. Naldini et al. (1996) Science
272:263-267.
[0105] Non-viral delivery vehicles comprising a polynucleotide
encoding an angiogenic factor can be introduced into host cells
and/or target cells by any method known in the art, such as
transfection by the calcium phosphate coprecipitation technique;
electroporation; electropermeabilization; liposome-mediated
transfection; ballistic transfection; biolistic processes including
microparticle bombardment, jet injection, and needle and syringe
injection; or by microinjection. Numerous methods of transfection
are known to the skilled worker in the field.
[0106] Viral delivery vehicles can be introduced into cells by
infection. Alternatively, viral vehicles can be incorporated into
any of the non-viral delivery vehicles described above for delivery
into cells. For example, viral vectors can be mixed with cationic
lipids (Hodgson and Solaiman (1996) Nature Biotechnol. 14:339-342);
or lamellar liposomes (Wilson et al. (1977) Proc. Nall. Acad. Sci.
USA 74:3471; and Faller et al. (1984) J. Virol. 49:269).
[0107] For in vivo delivery, the delivery vehicle(s) can be
introduced into an individual by any of a number of methods, each
of which is familiar in the art.
Pharmaceutical Compositions
[0108] Pharmaceutical compositions for use in the methods of the
present invention can comprise a polynucleotide encoding an
angiogenic, or, alternatively, pharmaceutical compositions can
comprise an angiogenic factor itself.
[0109] Suitable forms, in part, depend upon the use or the route of
entry, for example oral, transdermal, or by injection. Such forms
should allow the agent or composition to reach a target cell
whether the target cell is present in a multicellular host or in
culture. For example, pharmacological agents or compositions
injected into the blood stream should be soluble. Other factors are
known in the art, and include considerations such as toxicity and
forms which prevent the agent or composition from exerting its
effect.
[0110] Compositions comprising an angiogenic factor or an
angiogenic factor-encoding polynucleotide can also be formulated as
pharmaceutically acceptable salts (e.g., acid addition salts)
and/or complexes thereof. Pharmaceutically acceptable salts are
non-toxic at the concentration at which they are administered.
[0111] Pharmaceutically acceptable salts include acid addition
salts such as those containing sulfate, hydrochloride, phosphate,
sulfonate, sulfamate, sulfate, acetate, citrate, lactate, tartrate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, cyclolexylsulfonate, cyclohexylsulfamate and
quinate. Pharmaceutically acceptable salts can be obtained
from-acids such as hydrochloric acid, sulfuric acid, phosphoric
acid, sulfonic acid, sulfamic acid, acetic acid, citric acid,
lactic acid, tartaric acid, malonic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclohexylsulfonic acid, cyclohexylsulfamic acid, and quinic acid.
Such salts may be prepared by, for example, reacting the free acid
or base forms of the product with one or more equivalents of the
appropriate base or acid in a solvent or medium in which the salt
is insoluble, or in a solvent such as water which is then removed
in vacuo or by freeze-drying or by exchanging the ions of an
existing salt for another ion on a suitable ion exchange resin.
[0112] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, vegetable oils, polyethylene glycols and
physiologically compatible solvents. The compositions or
pharmaceutical composition can be administered by different routes
including, but not limited to, intravenous, intraperitoneal,
subcutaneous, and intramuscular, oral, topical, or
transmucosal.
[0113] The desired isotonicity of the compositions can be
accomplished using sodium chloride or other pharmaceutically
acceptable agents such as dextrose, boric acid, sodium tartrate,
propylene glycol, polyols (such as mannitol and sorbitol), or other
inorganic or organic solutes.
[0114] Pharmaceutical compositions comprising an angiogenic factor
or a polynucleotide encoding an angiogenic factor can be formulated
for a variety of modes of administration, including systemic and
topical or localized administration. Techniques and formulations
generally may be found in Remington's Pharmaceutical Sciences, 18th
Edition, Mack Publishing Co., Easton, Pa. 1990. See, also, Wang and
Hanson "Parenteral Formulations of Proteins and Peptides: Stability
and Stabilizers", Journal of Parenteral Science and Technology,
Technical Report No. 10, Supp. 42-2S (1988). A suitable
administration format can best be determined by a medical
practitioner for each patient individually.
[0115] For systemic administration, injection is preferred, e.g.,
intramuscular, intravenous, intraperitoneal, subcutaneous,
intrathecal, or intracerebrovascular. For injection, the compounds
of the invention are formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution.
[0116] Alternatively, the compounds of the invention are formulated
in one or more excipients (e.g., propylene glycol) that are
generally accepted as safe as defined by USP standards. They can,
for example, be suspended in an inert oil, suitably a vegetable oil
such as sesame, peanut, olive oil, or other acceptable carrier.
Preferably, they are suspended in an aqueous carrier, for example,
in an isotonic buffer solution at pH of about 5.6 to 7.4. These
compositions can be sterilized by conventional sterilization
techniques, or can be sterile filtered. The compositions can
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
buffering agents. Useful buffers include for example, sodium
acetate/acetic acid buffers. A form of repository or "depot" slow
release preparation can be used so that therapeutically effective
amounts of the preparation are delivered into the bloodstream over
many hours or days following transdermal injection or delivery. In
addition, the compounds can be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included.
[0117] Alternatively, the compounds can be administered orally. For
oral administration, the compounds are formulated into conventional
oral dosage forms such as capsules, tablets and tonics.
[0118] Systemic administration can also be transmucosal or
transdermal means, or the molecules can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration, bile salts and fusidic
acid derivatives. In addition, detergents can be used to facilitate
permeation. Transmucosal administration can be, for example,
through nasal sprays or using suppositories. For oral
administration, the molecules are formulated into conventional oral
administration dosage forms such as capsules, tablets, and liquid
preparations.
[0119] For topical administration, the compounds of the invention
are formulated into ointments, salves, gels, or creams, as is
generally known in the art.
[0120] If desired, solutions of the above compositions can be
thickened with a thickening agent such as methyl cellulose. They
can be prepared in emulsified form, either water in oil or oil in
water. Any of a wide variety of pharmaceutically acceptable
emulsifying agents can be employed including, for example, acacia
powder, a non-ionic surfactant (such as a Tween), or an ionic
surfactant (such as alkali polyether alcohol sulfates or
sulfonates, e.g., a Triton).
[0121] Compositions useful in the invention are prepared by mixing
the ingredients following generally accepted procedures. For
example, the selected components can be mixed simply in a blender
or other standard device to produce a concentrated mixture which
can then be adjusted to the final concentration and viscosity by
the addition of water or thickening agent and possibly a buffer to
control pH or an additional solute to control tonicity.
[0122] The amounts of various compounds for use in the methods of
the invention to be administered can be determined by standard
procedures. Generally, a therapeutically effective amount is
between about 1 nrnole and 3 .mu.mole of the molecule, preferably
between about 10 nmole and 1 .mu.mole depending on the age and size
of the patient, and the disease or disorder associated with the
patient. Generally, it is an amount between about 0.05 and 50
mg/kg, preferably 1 and 20 mg/kg of the individual to be
treated.
[0123] For use by the physician, the compositions are provided in
dosage unit form containing an amount of an angiogenic factor.
Antibodies
[0124] Some drug candidates according to the present invention are
agonist antibodies which mimic the anti-hypertensive properties of
an angiogenic factor, preferably a VEGF.
[0125] Methods of preparing polyclonal antibodies are known in the
art. 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. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized, such as serum albumin, or soybean trypsin
inhibitor. Examples of adjuvants which may be employed include
Freund's complete adjuvant and MPL-TDM.
[0126] According to one approach, 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. 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, 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. 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.
[0127] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the particular angiogenic factor used, such as
VEGF. Preferably, the binding specificity of monoclonal antibodies
produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RJA) 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).
[0128] 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 in vivo as ascites in a mammal.
[0129] 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.
[0130] Alternatively, monoclonal antibodies may be made by
recombinant DNA methods, such as those described in U.S. Pat. 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 discussed above 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 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.
[0131] The antibodies, including antibody fragments, such as Fv,
Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of
antibodies, may be humanized. Humanized antibodies contain minimal
sequence derived from a non-human immunoglobulin. More
specifically, in humanized antibodies residues from a complementary
determining region (CDR) of a human inmunoglobulin (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 also replaced by
corresponding non-human residues. Humanized antibodies may
additionally comprise residues that are found neither in the
recipient antibody nor in the imported CDR or framework sequences
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,
Nature, 332:323-329 (1988)].
[0132] 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 non-human source. 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); Rie chmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human
antibody.
[0133] In addition, human antibodies can be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Moi. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boemer et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of 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, which 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. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0134] The antibodies may be bispecific, in which one specificity
is for an angiogenic factor, and the other specificity for another
protein, such as, a second angiogenic factor, or a different
epitope of the same angiogenic factor.
Screening Assays for Drug Candidates
[0135] Screening assays for drug candidates are designed to
identify agonists, such as small molecule agonists, of the
angiogenic factors (e.g. VEGF) used in the methods and compositions
of the present invention. Such screening assays will include assays
amenable to high-throughput screening of chemical libraries, making
them particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds, including peptides, preferably soluble
peptides, (poly)peptide-immunoglobulin fusions, and 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.
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.
[0136] In addition, drug candidates can be tested or their initial
activity can be confirmed in various animal models of hypertension
(e.g. salt-sensitive hypertension).
[0137] Microvascular injury has been identified as a possible
mechanism for tubuinterstitial (TI) injury and associated
salt-dependent hypertension. For example, it has been reported that
short term infusion of angiotensin II (AII) results in focal TI
injury, and predisposes rats to the subsequent development of
salt-sensitive hypertension (Lombardi et al., Hypertension
33:1013-1019,1999). Similarly, aging has been strongly associated
with salt-sensitive hypertension (Weinberger and Fineberg,
Hypertension 18:67-71, 1991), and it was shown that
tubulointerstitial damage in the aging rat is also susceptible to
salt-sensitive hypertension (Masilamani et al., Am. J. Kid. Des.
32:605-610. 1998).
Methods of Treatment Using an Angiogenic Factor
[0138] The invention provides methods for treating hypertension.
The methods generally involve administering to an individual an
amount of an angiogenic factor effective to decrease hypertension.
An effective amount is as described above. Effectiveness of the
treatment is determined by decreased blood pressure particularly in
response to salt loading.
[0139] The methods of the present invention can also be useful in
treating disorders-relating to abnormal transport of solutes across
endothelial cells. Such disorders include (1) the treatment or
prevention of kidney disease associated with impaired filtration or
excretion of solutes; (2) the treatment or prevention of diseases
of the central nervous system associated with alterations in
cerebrospinal fluid synthesis, composition, or circulation,
including stroke, meningitis, tumor, infections, and disorders of
bone growth;
[0140] (3) the treatment or prevention of hypoxia or hypercapnia or
fibrosis arising from accumulation of fluid secretions in the lungs
or impediments to their removal, including but not restricted to
acute respiratory distress syndrome, toxic alveolar injury, as
occurs in smoke inhalation, pneumonia, including viral and
bacterial infections, surgical intervention, cystic fibrosis, and
other inherited or acquired disease of the lung associated with
fluid accumulation in the pulmonary air space; (4) the treatment or
prevention of pulmonary dysfunction arising from injury to the
pulmonary endothelium, including disorders arising from birth
prematurity, and primary and secondary causes of pulmonary
hypertension; (5) the treatment or prevention of disease arising
from disordered transport of fluid and solutes across the
intestinal epithelium, including but not restricted to inflammatory
bowel disease, infections diarrhea, surgical intervention; (6) the
treatment or prevention of ascites accumulation in the peritoneum
as occurs in failure of the heart, liver, or kidney, or in
infectious or tumor states; (7) the enhancement of efficacy of
solute flux as it can be needed for peritoneal dialysis in the
treatment of kidney failure or installation of therapeutics or
nutrition into the peritoneum; (8) the preservation or enhancement
of function of organ allografis, including but not restricted to
transplants of kidney, heart, liver, lung, pancreas, skin, bone,
intestine, and xenografts; and (9) the treatment of cardiac valve
disease.
[0141] The following examples are provided to illustrate but not
limit the invention.
EXAMPLE 1
Plasma Elimination Kinetics of VEGF.sub.121 and VEGF.sub.165
[0142] Studies were conducted to determine plasma elimination
kinetics of VEGF.sub.121 or VEGF.sub.165 when administered
intravenously or subcutaneously. Recombinant VEGF.sub.121 or
VEGF.sub.65 was injected as a 10 .mu.g/kg bolus into the tail veins
of rats. Plasma samples were taken over the following two hours,
and the concentration of VEGF in the samples was determined using a
sandwich ELISA assay. The results are shown in FIG. 2. Recombinant
VEGF.sub.121 or VEGF.sub.165 was injected into rats as a
subcutaneous 100 .mu./kg bolus. Plasma samples were taken over the
following eight hours, and the concentration of VEGF in the samples
was determined as before. The results are shown in FIG. 3. These
data indicate that VEGF.sub.121, exhibits the characteristics of
being rapidly absorbed into the circulation following subcutaneous
injection, remaining in circulation for a significant time period,
and then being eliminated.
EXAMPLE 2
Animal Model of Salt-Dependent Hypertension
[0143] Laboratory rats (e.g., Sprague-Dawley) can be rendered
hypertensive in a salt-dependent manner to emulate the human
example of essential hypertension. Indeed, recently published data
(Lombardi et al., Hypertension 33:1013-1019, 1999) confirm that
short term infusion of angiotensin II (AII) results in focal
tubulointerstitial (TI) injury and predisposes rats to the
subsequent development of salt-sensitive hypertension.
[0144] The model used in the present experiment consists of placing
normal laboratory rats on a diet with an increased amount of sodium
chloride (4% w/w) for three days. Rats were placed on this diet
alone for up to two weeks. These rats showed no hypertension
compared to rats on a normal lab chow diet, which comprises 0.1%
NaCl (FIG. 4a).
[0145] After three days, the rats were then subjected to continuous
intravenous infusion of angiotensin II (AII) at 200 ng/kg/min for
1-2 weeks. As shown in FIG. 4b, all AII-treated animals developed
systolic hypertension. If the rats were on a low salt diet
throughout, the blood pressure rapidly normalized. If the animals
were placed on a high salt diet, the blood pressure remained
elevated after the cessation of All infusion, demonstrating a form
of salt-dependent hypertension. As shown in FIG. 4c, if rats
received VEGF.sub.121 at 20 .mu.g/kg/day for 14 days concurrent
with a 7 day infusion of AII, both groups were comparable with
respect to initial elevation in blood pressure. Both sets of rats
were fed a high-salt diet. Upon cessation of All however, systolic
blood pressure in the animals additionally treated with VEGF drops
to normal or below-normal levels, while the systolic blood pressure
of the animals receiving a transient exposure to AII remained
elevated. Comparison of organ within these animal groups
demonstrated that the kidneys of animals treated with VEGF
demonstrate marked hypertrophy, while those exposed to high salt or
high salt plus AII infusions did not (FIG. 5). This unexpected
result demonstrates that angiogenic stimulation facilitates the
hypertrophy of the kidney to adapt to the filtration demands
imposed by high salt diet and a preceding or concurrent state of
hypertension.
EXAMPLE 3
[0146] Treatment of Salt-Sensitive Hypertension Induced by
Transient Angiotensin II (AII) Generation, with hVEGF.sub.121
[0147] Laboratory rats (Sprague-Dawley males, weighing 300 grams)
on a high salt diet (4% NaCl) received a continuous infusion of
angiotension II (AII) (444 ng/kg/min, s.c.) for 7 days. The high
salt diet started 3 days prior to the infusion of AII, and was
continued throughout the experiment (an additional 7 days).
VEGF.sub.121 (20 .mu.g/kg/day, i.v.) or vehicle (phosphate bufered
saline) was administered during the 14 days period. The VEGF
treatment did not lower the blood pressure in rats of normal salt
diet or reduce the initial hypertensive response to acute AII
infusion. However, blood pressure during the period following AII
administration was significantly lower than in vehicle treated rats
and similar to control rats on a high salt diet that had never been
exposed to AII (day 14: AII/vehicle: 167.+-.5; AIIVEGF: 136.+-.3,
high salt control: 138.+-.5, p<0.05). VEGF infusion was also
associated with increased urinary nitrite excretion (VEGF vs.
vehicle, 150 vs 35 nmol/day, p<0.0 1), and with hypertrophy of
the renal medulla.
[0148] All infusion results in dysregulated VEGF expression with
alterations in interstitial capillary structure and nitric oxide
generation that predisposes rats to salt sensitive hypertension.
VEGF infusion corrects the nitric oxide abnormalities and prevents
the development of post AII induced hypertension.
EXAMPLE 4
VEGF.sub.121 Reduced Blood Pressure Response to High Salt Diet in
Rats with Chronic Cyclosporin Nephropathy
[0149] Cyclosporin (CSA) has been associated with tubulo
interstitial disease and the development of salt-sensitive
hypertension. Previous study suggests that the renal
vasoconstriction and injury might be mediated by a decrease in
local nitric oxide concentration (Kidney Int. 53:897, 1998). We
decided to test whether VEGF is able to prevent the development of
salt-sensitive hypertension in rats with established chronic CSA
nephropathy. The model was induced in laboratory rats
(Sprague-Dawley males, weighing 300 grams) by subcutaneous
injection of CSA (15 mg/kg/day) for 45 days, while the rats were
kept on a low salt diet (0.125% NaCl). After 5 days of washout
period (day 50), the diet was switched to high salt (4% NaCl) and
the rats received subcutaneous injection of either VEGF.sub.121
(100 .mu.g/kg/day) or vehicle for 14 days. Then all treatment was
discontinued for an additional 5 days and rats were sacrificed.
[0150] The VEGF-treated rats had lower blood pressure in response
to a high salt diet during the post-CSA injection period, and this
effect persisted even after stopping the VEGF.sub.121 injections
for 5 days. Interestingly, there was no difference in histology
(light microscopy) or renal function (BUN) in the VEGF and vehicle
treated rats at the end of the study. Urinary nitrates/nitrites
(nitric oxide metabolites) were undetectable in the urine of VEGF
and vehicle treated rats for 7 days, and were not different at
sacrifice (2932.+-.738 vs. 2516.+-.564 nmol/day, p=NS). These data
indicate that VEGF reduces the blood pressure response to high salt
in rats exposed to CSA, and the effect is persistent even after
VEGF administration is stopped.
EXAMPLE 5
Inhibition of Salt Sensitive Hypertension in Rats by VEGF
Treatment
[0151] Laboratory rats (Sprague-Dawley males, 3-months of age) were
divided into three treatment groups, and one control group, each
group including 10 animals. Group I was kept on a high salt diet
(4% NaCl) and received a continuous infusion (via Alzet pump) of
angiotension II (AII) (440 ng/kg/min, s.c.) for 7 days. Group 2 was
kept on a low salt diet (0.01% NaCl) and received a continuous
infusion (via Alzet pump) of angiotension 11 (AII) (440 ng/kg/min,
s.c.) for 7 days. Group 3 was kept on a high salt diet, just as
Group 1, but also received rhVEGF.sub.121 (100 .mu.kg/kg/day, s.q.)
starting at day 0, and continuing through day 48, then stopping for
observation. Group 4 (control group) received no treatment. The
blood pressure of animals in all groups was measured twice weekly,
at days 1, 2, 4, 7, 11,14, 21, 25, 28, 32, 35, 41, and 48. Urine
was collected at week 2 from Group 1, Group 3, and Group 4 animals
(6 animals from each group) and specimen centrifuged and kept
frozen for future protein, electrolytes, creatinine and urine
intrates measurements. As illustrated in FIG. 12, animals in all
three treatment groups showed a hypertensive response to acute AII
infusion. When the AII treatment was discontinued, the blood
pressure normalized but then increased again in the group kept on
high salt diet in the absence of VEGF treatment (Group 1). In
contrast, VEGF treatment kept the blood pressure of animals kept on
high salt diet (Group 3) at normal level, demonstrating that VEGF
treatment is capable of preventing the development of hypertension
in this experimental model of salt sensitive hypertension.
[0152] All publications and patent applications mentioned in this
specification are herein incorporated be reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0153] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
11 1 147 PRT Homo sapiens 1 Met Asn Phe Leu Leu Ser Trp Val His Trp
Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser
Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His
Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys
His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro
Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85
90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro
His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His
Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln
Glu Lys Cys Asp Lys 130 135 140 Pro Arg Arg 145 2 145 PRT Homo
sapiens 2 Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val
Val Lys 1 5 10 15 Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro
Ile Glu Thr Leu 20 25 30 Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu
Ile Glu Tyr Ile Phe Lys 35 40 45 Pro Ser Cys Val Pro Leu Met Arg
Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60 Gly Leu Glu Cys Val Pro
Thr Glu Glu Ser Asn Ile Thr Met Gln Ile 65 70 75 80 Met Arg Ile Lys
Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Leu Gln
His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg 100 105 110
Gln Glu Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys 115
120 125 Arg Lys Lys Ser Arg Tyr Lys Ser Trp Ser Val Cys Asp Lys Pro
Arg 130 135 140 Arg 145 3 191 PRT Homo sapiens 3 Met Asn Phe Leu
Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu
His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30
Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35
40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln
Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys
Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly
Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln
Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu
Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys
Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser
Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr 145 150 155 160
Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165
170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg
180 185 190 4 108 PRT Homo sapiens 4 Met Phe Leu Trp His Ser Ala
Leu Tyr His Ala Trp Gln Ala Met Glu 1 5 10 15 Gly Gln His Glu Val
Phe Asp Tyr Arg Tyr His Ile Thr Val Ile Gln 20 25 30 Tyr Asp Ile
Tyr Phe Pro Cys Pro Met Cys Gly Cys Asp Gly Glu Val 35 40 45 Thr
Glu Asn Thr Gln Met Ile Pro Gln Gln Ile Glu Ser Leu His Lys 50 55
60 Glu Arg Lys Asp Ala Gln Lys Ser Arg Lys Lys Gln Arg Arg Lys Arg
65 70 75 80 Lys Trp Val Cys Pro Ser Arg Lys Leu Val Asp Gln Cys Cys
Cys Asn 85 90 95 Asp Arg Lys Arg Leu Leu Glu Thr Arg Asp Pro Arg
100 105 5 116 PRT Homo sapiens 5 Met Phe Leu Trp His Ser Ala Leu
Tyr His Ala Trp Gln Ala Met Glu 1 5 10 15 Gly Gln His Glu Val Phe
Asp Tyr Arg Tyr His Ile Thr Val Ile Gln 20 25 30 Tyr Asp Ile Tyr
Phe Pro Cys Pro Met Cys Gly Cys Asp Gly Glu Val 35 40 45 Thr Glu
Asn Thr Gln Met Ile Pro Gln Gln Ile Glu Ser Leu His Lys 50 55 60
Glu Arg Lys Asp Ala Gln Lys Ser Arg Lys Lys Gln Arg Arg Lys Arg 65
70 75 80 Lys Trp Val Val Ala Cys Leu Pro Ser Pro Pro Pro Gly Cys
Glu Arg 85 90 95 His Phe Gln Pro Thr Lys Ser Lys Thr Ser Cys Ala
Gln Glu Asn Arg 100 105 110 Cys Cys Lys Arg 115 6 444 DNA Homo
sapiens 6 atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta
cctccaccat 60 gccaagtggt cccaggctgc acccatggca gaaggaggag
ggcagaatca tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagc
tactgccatc caatcgagac cctggtggac 180 atcttccagg agtaccctga
tgagatcgag tacatcttca agccatcctg tgtgcccctg 240 atgcgatgcg
ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 300
aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg
360 agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc
aagacaagaa 420 aaatgtgaca agccgaggcg gtga 444 7 516 DNA Homo
sapiens 7 atgaactttc tgctgtcttg ggtggattgg agccttgcct tgctgctcta
cctccaccat 60 gccaagtggt cccaggctgc acccatggca gaaggaggag
ggcagaatca tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagc
tactgccatc caatcgagac cctggtggac 180 atcttccagg agtaccctga
tgagatcgag tacatcttca agccatcctg tgtgcccctg 240 atgcgatgcg
ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 300
aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg
360 agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc
aagacaagaa 420 aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaa
agcgcaagaa atcccggtat 480 aagtcctgga gcgtatgtga caagccgagg cggtga
516 8 576 DNA Homo sapiens 8 atgaactttc tgctgtcttg ggtgcattgg
agcctcgcct tgctgctcta cctccaccat 60 gccaagtggt cccaggctgc
acccatggca gaaggaggag ggcagaatca tcacgaagtg 120 gtgaaattca
tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 180
atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg
240 atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac
tgaggagtcc 300 aacatcacca tgcagattat gcggatcaaa cctcaccaag
gccagcacat aggagagatg 360 agcttcctac agcacaacaa atgtgaatgc
agaccaaaga aagatagagc aagacaagaa 420 aatccctgtg ggccttgctc
agagcggaga aagcatttgt ttgtacaaga tccgcagacg 480 tgtaaatgtt
cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac 540
gaacgtactt gcagatgtga caagccgagg cggtga 576 9 642 DNA Homo sapiens
9 atgaactttc tgctgtcttg ggtgcattgg agcctcgcct tgctgctcta cctccaccat
60 gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca
tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagc tactgccatc
caatcgagac cctggtggac 180 atcttccagg agtaccctga tgagatcgag
tacatcttca agccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctg
caatgacgag ggcctggagt gtgtgcccac tgaggagtcc 300 aacatcacca
tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg 360
agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa
420 aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaa agcgcaagaa
atcccggtat 480 aagtcctgga gcgtggggcc ttgctcagag cggagaaagc
atttgtttgt acaagatccg 540 cagacgtgta aatgttcctg caaaaacaca
gactcgcgtt gcaaggcgag gcagcttgag 600 ttaaacgaac gtacttgcag
atgtgacaag ccgaggcggt ga 642 10 699 DNA Homo sapiens 10 atgaactttc
tgctgtcttg ggtgcattgg agcctcgcct tgctgctcta cctccaccat 60
gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg
120 gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac
cctggtggac 180 atcttccagg agtaccctga tgagatcgag tacatcttca
agccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctg caatgacgag
ggcctggagt gtgtgcccac tgaggagtcc 300 aacatcacca tgcagattat
gcggatcaaa cctcaccaag gccagcacat aggagagatg 360 agcttcctac
agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa 420
aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaa agcgcaagaa atcccggtat
480 aagtcctgga gcgtgtacgt tggtgcccgc tgctgtctaa tgccctggag
cctccctggc 540 ccccatccct gtgggccttg ctcagagcgg agaaagcatt
tgtttgtaca agatccgcag 600 acgtgtaaat gttcctgcaa aaacacagac
tcgcgttgca aggcgaggca gcttgagtta 660 aacgaacgta cttgcagatg
tgacaagccg aggcggtga 699 11 110 PRT Homo sapiens 11 Ala Pro Met Ala
Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys 1 5 10 15 Phe Met
Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30
Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35
40 45 Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu 50 55 60 Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr
Met Gln Ile 65 70 75 80 Met Arg Ile Lys Pro His Gln Gly Gln His Ile
Gly Glu Met Ser Phe 85 90 95 Leu Gln His Asn Lys Cys Glu Cys Arg
Pro Lys Lys Asp Arg 100 105 110
* * * * *