U.S. patent application number 13/685892 was filed with the patent office on 2013-04-04 for variants of vegfr and their use in the diagnosis and treatment of pregnancy associated medical conditions.
This patent application is currently assigned to Hadasit Medical Research Services and Development Ltd.. The applicant listed for this patent is Hadasit Medical Research Services and Developm, Yissum Research Development Company of the H. Invention is credited to AHUVA ITIN, ELI KESHET, SHAY SELA, SIMCHA YAGEL.
Application Number | 20130084583 13/685892 |
Document ID | / |
Family ID | 39271517 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130084583 |
Kind Code |
A1 |
KESHET; ELI ; et
al. |
April 4, 2013 |
VARIANTS OF VEGFR AND THEIR USE IN THE DIAGNOSIS AND TREATMENT OF
PREGNANCY ASSOCIATED MEDICAL CONDITIONS
Abstract
An isolated polypeptide comprising an amino acid sequence at
least 70% homologous to SEQ ID NO: 4 and an isolated polynucleotide
encoding same are disclosed. A polynucleotide comprising a nucleic
acid sequence capable of specifically hybridizing to the isolated
polynucleotide and an isolated antibody comprising an antigen
recognition domain which specifically binds the isolated
polypeptide are also disclosed. Pharmaceutical compositions,
methods of diagnosing and treating comprising same are also
disclosed.
Inventors: |
KESHET; ELI; (MOSHAV
AMINADAV, IL) ; ITIN; AHUVA; (JERUSALEM, IL) ;
SELA; SHAY; (HAIFA, IL) ; YAGEL; SIMCHA;
(JERUSALEM, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yissum Research Development Company of the H;
Hadasit Medical Research Services and Developm; |
Jerusalem
Jerusalem |
|
IL
IL |
|
|
Assignee: |
Hadasit Medical Research Services
and Development Ltd.
Jerusalem
IL
Yissum Research Development Company of the Hebrew University of
Jerusalem Ltd.
Jerusalem
|
Family ID: |
39271517 |
Appl. No.: |
13/685892 |
Filed: |
November 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12448404 |
Jan 13, 2010 |
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PCT/IL2007/001589 |
Dec 20, 2007 |
|
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13685892 |
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60875822 |
Dec 20, 2006 |
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Current U.S.
Class: |
435/7.8 ;
436/501 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 35/00 20180101; G01N 33/689 20130101; A61P 9/00 20180101; A61P
27/02 20180101; A61P 3/10 20180101; A61P 43/00 20180101; C07K 14/71
20130101; A61P 9/12 20180101 |
Class at
Publication: |
435/7.8 ;
436/501 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method of diagnosing a pregnancy-associated medical condition
associated with maternal or fetal stress in a subject in need
thereof, the method comprising: (a) detecting expression level of
sFlt-14 (SEQ ID NO: 2 or 4) in a biological sample of the subject
using an agent capable of recognizing sFlt-14 (SEQ ID NO: 2 or 4)
and not sFlt-1 (SEQ ID NO: 10); and (b) diagnosing said subject as
having a pregnancy-associated medical condition associated with
maternal or fetal stress based on the presence of increased level
of said sFlt-14 (SEQ ID NO: 2 or 4) in said biological sample as
compared to a sample of a normal pregnant subject.
2. A method of diagnosing preeclampsia in a subject in need
thereof, the method comprising: (a) detecting expression level of
sFlt-14 (SEQ ID NO: 2 or 4) in a biological sample of the subject
using an agent capable of recognizing sFlt-14 (SEQ ID NO: 2 or 4)
and not sFlt-1 (SEQ ID NO: 10); and (b) diagnosing said subject as
having a preeclampsia based on the presence of increased level of
said sFlt-14 (SEQ ID NO: 2 or 4) in said biological sample as
compared to a sample of a normal pregnant subject.
3. A method of diagnosing preeclampsia in a subject in need
thereof, the method comprising: (a) detecting expression level of
sFlt-14 (SEQ ID NO: 2 or 4) in a serum sample or a placenta tissue
sample of the subject using an antibody capable of recognizing
sFlt-14 (SEQ ID NO: 2 or 4) and not sFlt-1 (SEQ ID NO: 10); and (b)
diagnosing said subject as having a preeclampsia based on the
presence of increased level of said sFlt-14 (SEQ ID NO: 2 or 4) in
said serum sample or said placenta tissue sample as compared to a
sample of a normal pregnant subject.
4. The method of claim 1, wherein said condition is selected from
the group consisting of preeclampsia, gestational diabetes,
gestational hypertension, fetal growth restriction (FGR), and fetal
alcohol syndrome (FAS).
5. The method of claim 1, wherein said agent is an antibody.
6. The method of claim 2, wherein said agent is an antibody.
7. The method of claim 1, wherein said biological sample is
selected from the group consisting of a urine sample, a blood
sample, a serum sample, a placenta biopsy, a chorionic villus
sample, and an amniotic fluid sample.
8. The method of claim 2, wherein said biological sample is
selected from the group consisting of a urine sample, a blood
sample, a serum sample, a placenta biopsy, a chorionic villus
sample, and an amniotic fluid sample.
9. The method of claim 1, being effected in vitro or ex vivo.
10. The method of claim 2, being effected in vitro or ex vivo.
11. The method of claim 3, being effected in vitro or ex vivo.
12. The method of claim 1, wherein said biological sample is of a
gestation week 13 and on.
13. The method of claim 2, wherein said biological sample is of a
gestation week 13 and on.
14. The method of claim 3, wherein said serum sample or said
placenta tissue sample is of a gestation week 13 and on.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/448,404 filed on Jan. 13, 2010, which is a National
Phase of PCT Patent Application No. PCT/IL2007/001589 filed on Dec.
20, 2007, which claims the benefit of priority under 35 USC
.sctn.119(e) of U.S. Provisional Patent Application No. 60/875,822
filed on Dec. 20, 2006. The contents of the above applications are
all incorporated by reference as if fully set forth herein in their
entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to isolated polypeptides and polynucleotides encoding same for the
diagnosis and treatment of VEGF-associated medical conditions.
[0003] Vascular endothelial growth factor (VEGF), an endothelial
specific mitogen, plays a key role in promoting both vasculogenesis
and angiogenesis. VEGF plays an important regulatory function in
the formation of new blood vessels during embryonic vasculogenesis
and in angiogenesis during adult life.
[0004] The activities of VEGF are mediated primarily by its
interaction with two high-affinity receptor tyrosine kinases:
fms-like tyrosine kinase-1 (Flt-1/VEGFR-1) and kinase-insert domain
region (KDR/Flk-1/VEGFR-2) both of which are expressed on vascular
endothelial cell surfaces. Alternative splicing of Flt-1 results in
the production of an endogenously secreted protein referred to as
soluble Flt1 (sFlt1), which lacks the cytoplasmic and transmembrane
domains but retains the ligand-binding domain [He et al., Mol.
Endocrinol. (1999) 13: 537-545]. Thus, sFlt1 can antagonize
circulating VEGF by binding it and preventing the interaction of
VEGF with its endogenous receptors. sFlt1 also binds and
antagonizes placental growth factor (PlGF), another member of the
VEGF family, which is produced predominantly in the placenta, as
well as of another VEGF family member known as VEGF-B.
[0005] VEGF is an important mediator of angiogenesis in a number of
pathological conditions including tumor formation and metastasis of
solid tumors. Numerous inhibitors of the VEGF/VEGF receptor pathway
(e.g. monoclonal antibodies specific for VEGF) have been shown to
prevent tumor growth via an antiangiogenic mechanism [Kim et al.,
Nature (1993) 362(6423):841-4].
[0006] Preeclampsia, the most common, dangerous, unpredictable
complication of pregnancy is a major cause of maternal, fetal, and
neonatal mortality worldwide. While the cause of preeclampsia
remains unclear, the principle cause appears to be inadequate blood
supply to the placenta making it release hormones or chemical
agents that cause maternal endothelial dysfunction, alterations in
metabolism and inflammation [Drife J O, Magowan (eds) Clinical
Obstetrics and Gynecology, chapter 39, pp 367-370]. These
consequently lead to hypertension association with proteinuria in
the mother along with impaired placental blood flow, fetal growth
restriction and consequential fetal oxidative stress.
[0007] During pregnancy, the major source of circulating sFlt1 is
the placenta and only minor amounts are produced by other tissues
(e.g. by endothelial cells and monocytes). Recent investigations
have supported the finding that placental expression and serum
levels of sFlt-1 are upregulated in preeclamptic pregnancies, in
conjunction with decreased levels of circulating free VEGF and free
PlGF, compared to normal pregnancies [Maynard et al., J. Clin.
Invest. (2003) 111: 649-658]. The increase in sFlt1, followed by a
decrease in free PlGF and free VEGF, was found to precede the onset
of clinical disease by several weeks and appears to be more
pronounced in severe and early onset preeclampsia [Levine et al.,
N. Engl. J. Med. (2004) 350: 672-683]. Postpartum, sFlt-1 levels
decrease dramatically in both normal and preeclamptic pregnancies
[Maynard et al., supra]. Thus, excess sFlt1, by neutralizing VEGF
and PlGF, may play a crucial role in the pathogenesis of the
maternal syndrome in preeclampsia.
[0008] Lam et al. [Lam et al, Hypertension (2005) 46(5): 1077-85]
review the possibility of measuring circulating angiogenic proteins
(e.g. PlGF) or anti-angiogenic proteins (e.g. sFlt-1) in the blood
and urine of pregnant women as a diagnostic and screening tool for
predicting preeclampsia. They have examined odds ratios,
sensitivity and specificity for various sFlt-1 cutoff values in
different trimesters. Lam et al. describe a strong correlation
between high sFlt-1 levels and the risk and presence of
preeclampsia. Furthermore, they have yielded the conclusion that
the higher the sFlt-1 level, the more predictive it is of
preeclampsia.
[0009] PCT Publication No. WO 2006/069373 discloses methods,
compositions and kits for diagnosis of preeclampsia and
hypertensive disorders in pregnancy. More specifically, WO
2006/069373 teaches assessment of preeclampsia or predisposition to
preeclampsia by monitoring the levels of angiogenic factors,
specifically VEGF, PlGF and sFlt-1, in urinary samples of pregnant
women. WO 2006/069373 teaches that the higher the level of sFlt-1,
the more predictive it is of preeclampsia. Furthermore, according
to WO 2006/069373, preeclampsia is associated with a significant
decrease in PlGF and significant increase in VEGF urine
concentrations.
[0010] U.S. Publication No. 20050148040 discloses methods and
compositions for screening of gestational disorders (e.g.,
gestational diabetes, preeclampsia and gestational hypertension)
using specific biomarkers. The biomarkers taught are insulin
resistance biomarkers [e.g., sex hormone binding globulin (SHBG)]
and angiogenesis biomarkers including sFlt-1. More specifically,
alterations in two pathways, insulin resistance (e.g., as evidenced
by low serum levels of SHBG) and angiogenesis (e.g., as evidenced
by low PlGF or high sFlt1), when combined can be used to predict
gestational disorders.
[0011] U.S. Publication No. 20050025762 discloses methods for
diagnosing and treating preeclampsia and eclampsia. U.S.
Publication No. 20050025762 teaches treating or preventing
preeclampsia and eclampsia using compounds that increase VEGF or
PlGF levels (e.g., nicotine, adenosine), using compounds that
decrease sFlt-1 levels (e.g., purified sFlt-1 antibody, an sFlt-1
antigen-binding fragment, small interfering RNAs, or
double-stranded RNA) such as compounds that bind sFlt-1 and block
growth factor binding (e.g., chemical compound, polypeptide,
peptide, antibody).
SUMMARY OF THE INVENTION
[0012] According to an aspect of some embodiments of the present
invention there is provided an isolated polypeptide comprising an
amino acid sequence at least 70% homologous to SEQ ID NO: 4 as
determined by protein BLAST algorithm
(http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).
[0013] According to some embodiments of the invention, the isolated
polypeptide is capable of binding VEGF.
[0014] According to some embodiments of the invention, the isolated
polypeptide is soluble.
[0015] According to some embodiments of the invention, the isolated
polypeptide is as set forth in SEQ ID NO: 4.
[0016] According to some embodiments of the invention, the isolated
polypeptide is as set forth in SEQ ID NO: 2.
[0017] According to some embodiments of the invention, the isolated
polypeptide further comprises a heterologous amino acid sequence
attached to the amino acid sequence.
[0018] According to some embodiments of the invention, the
heterologous amino acid sequence is selected from the group
consisting of an immunoglobulin, a galactosidase, a glucuronidase,
a glutathione-S-transferase (GST), a carboxy terminal peptide (CTP)
from chorionic gonadotrophin (CG.beta.), and a chloramphenicol
acetyltransferase (CAT).
[0019] According to some embodiments of the invention, the isolated
polypeptide is attached to a non-proteinaceous moiety.
[0020] According to some embodiments of the invention, the
non-proteinaceous moiety is selected from the group consisting of
polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP),
poly(styrene comaleic anhydride) (SMA), and divinyl ether and
maleic anhydride copolymer (DIVEMA).
[0021] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding the isolated polypeptide, wherein
the isolated polynucleotide is not genomic Flt1.
[0022] According to some embodiments of the invention, the isolated
polynucleotide is an mRNA or a cDNA.
[0023] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide as set forth
in SEQ ID NO: 1 or 3.
[0024] According to an aspect of some embodiments of the present
invention there is provided a nucleic acid construct comprising the
nucleic acid sequence functionally attached to a cis-acting
regulatory element.
[0025] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence capable of specifically hybridizing to the
isolated polynucleotide and not to SEQ ID NO: 9.
[0026] According to an aspect of some embodiments of the present
invention there is provided an isolated antibody comprising an
antigen recognition domain which specifically binds the isolated
polypeptide and not to SEQ ID NO: 10.
[0027] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
the isolated polypeptide and a pharmaceutically acceptable
carrier.
[0028] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
the isolated polynucleotide and a pharmaceutically acceptable
carrier.
[0029] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
the antibody and a pharmaceutically acceptable carrier.
[0030] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
the isolated polynucleotide and a pharmaceutically acceptable
carrier.
[0031] According to an aspect of some embodiments of the present
invention there is provided a use of an agent capable of regulating
an activity and/or expression of sFlt-14 (SEQ ID NO: 1 or 2) and
not sFlt-1 (SEQ ID NO: 9 or 10), for the manufacture of a
medicament identified for treating a VEGF-associated medical
condition.
[0032] According to some embodiments of the invention, the
VEGF-associated medical condition is associated with reduced
activity and/or expression of VEGF and whereas the regulating
comprises downregulating the sFlt-14.
[0033] According to some embodiments of the invention, the
VEGF-associated medical condition is associated with excessive
activity and/or expression of VEGF and whereas the regulating
comprises upregulating the sFlt-14.
[0034] According to some embodiments of the invention, the
VEGF-associated medical condition is selected from the group
consisting of preeclampsia, gestational diabetes, gestational
hypertension, fetal growth restriction (FGR), fetal alcohol
syndrome (FAS), cancer, corneal neovascularization and
hypertension.
[0035] According to some embodiments of the invention, the agent
comprises the antibody.
[0036] According to some embodiments of the invention, the agent
comprises the isolated polynucleotide.
[0037] According to some embodiments of the invention, the agent
comprises the isolated polypeptide.
[0038] According to some embodiments of the invention, the agent
comprises the isolated polynucleotide.
[0039] According to an aspect of some embodiments of the present
invention there is provided a method of detecting sFlt-14 (SEQ ID
NO: 2) in a biological sample, the method comprising: (a)
contacting the biological sample with the antibody such that the
sFlt-14 and the antibody form a complex; and (b) measuring a
presence or a level of the complex to thereby detect sFlt-14 in the
biological sample.
[0040] According to an aspect of some embodiments of the present
invention there is provided a method of detecting sFlt-14 (SEQ ID
NO: 1) in a biological sample, the method comprising: (a)
contacting the biological sample with the isolated polynucleotide
so as to form a hybridization complex; and (b) measuring a presence
or a level of the complex to thereby detect sFlt-14 in the
biological sample.
[0041] According to some embodiments of the invention, the
measuring is effected by a method selected from the group
consisting of PCR, Real Time PCR, RT PCR, nucleic acid
sequence-based amplification (NASBA), Northern blot and in situ
hybridization.
[0042] According to an aspect of some embodiments of the present
invention there is provided a method of diagnosing a
pregnancy-associated medical condition associated with maternal or
fetal stress in a subject in need thereof, the method comprising
detecting expression level of sFlt-14 (SEQ ID NO: 1 or 2) in a
biological sample of the subject using an agent capable of
recognizing sFlt-14 (SEQ ID NO: 1 or 2) and not sFlt-1 (SEQ ID NO:
9 or 10), wherein an expression level of the sFlt-14 above a
predetermined threshold is indicative of the pregnancy-associated
medical condition associated with maternal or fetal stress.
[0043] According to an aspect of some embodiments of the present
invention there is provided a method of diagnosing a
pregnancy-associated medical condition associated with maternal or
fetal stress in a subject in need thereof, the method comprising
detecting expression level of sFlt-14 (SEQ ID NO: 1 or 2) in a
biological sample, wherein the biological sample is of a gestation
week 13 and on, and wherein an expression level of the sFlt-14
above a predetermined threshold is indicative of the
pregnancy-associated medical condition associated with maternal or
fetal stress.
[0044] According to some embodiments of the invention, the
condition is selected from the group consisting of preeclampsia,
gestational diabetes, gestational hypertension, fetal growth
restriction (FGR), and fetal alcohol syndrome (FAS).
[0045] According to some embodiments of the invention, the
biological sample is selected from the group consisting of a urine
sample, a blood sample, a serum sample, a placenta biopsy, a
chorionic villus sample, and an amniotic fluid sample.
[0046] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0048] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0049] In the drawings:
[0050] FIG. 1 is a schematic representation of the FLT1-full
transmembrane receptor (top), the well-known soluble sFlt1 variant
(middle), and the novel sFlt-14 variant of the present invention
(bottom). Arrows mark the beginning of translation (ATG) and the
stop (STOP) points. Exons are indicated by the dark numbered boxes
and introns 13 and 14 are indicated by the light numbered
boxes.
[0051] FIG. 2A depicts the unique cDNA sequence of the novel
sFlt-14 isoform. The sequence was cloned by 3' RACE from a human
preeclamptic placenta. The shown sequence starts near the beginning
of exon 14 and ends with a poly A tail. The italic letters
represent the coding region that is derived from exon 14 and intron
14. The stop codon is in bold. The underlined sequence is an Alu
repeat nested in the 3' UTR.
[0052] FIG. 2B depicts a window taken from the UCSC genome browser
after a blast search with the sequence that appears above (FIG.
2A). The search located three ESTs with a similar splicing pattern
to the one found in the above sequence: AI188382, N47911 and
AA035437.
[0053] FIG. 3A depicts the amino acid sequence of sFlt-14 as
deduced from its mRNA. The original reading frame was kept,
starting with the known translation start point of sFlt-1 and
transmembrane Flt. The amino acids shared with the full
transmembrane receptor (but not with sFlt-1) are underlined. The
unique 28 amino acids found only in the variant sFlt-14 of the
invention (from intron 14) are depicted in bold.
[0054] FIG. 3B depicts sequences of peptides synthesized in order
to create specific polyclonal antibodies which distinguish between
sFlt-1 and sFlt-14. Of note, whereas CESS is unique to sFlt-14,
CHFK can distinguish between the alternatively-spliced isoforms
only in conjunction with analysis of the protein size.
[0055] FIGS. 4A-B are pictures depicting the relative abundance of
the full membrane receptor Flt, sFlt-1 (also designated sFlt-13)
and the novel sFlt-14 in different cell types. FIG. 4A shows the
expression of the full receptor Flt and its two
alternatively-spliced variants sFlt-1 and sFlt-14 in endothelial
cells (left column), normal placentae (middle column) and
preeclampsia placentae (right column). RNA blots were hybridized
with a probe detecting an extracellular sequence common to all
three (each yields a band of equal intensity, irrespective of
size). Of note, endothelial cells preferentially produce the full
receptor and sFlt1, whereas placentae predominantly produce
sFlt-14. However, a dramatic upregulation of sFlt-14 is exhibited
in preeclampsia placentae; FIG. 4B shows the expression of sFlt-1
and sFlt-14 in Primary cultures of endothelial cells (EC) and in
vascular smooth muscle cells (VSMC) isolated from a human saphena
vein. Of note, sFlt1 and sFlt-14 are exclusively expressed by ECs
and VSMCs, respectively. The bottom panels of FIGS. 4A-B represent
28S and 18S RNA expression that was used as a loading control.
[0056] FIGS. 5A-D are pictures depicting sFlt-14 mRNA and protein
expression in the context of the preeclamptic placenta. FIGS. 5A-B
show immunohistochemical detection of the sFlt-14 protein using the
specific CESS antibody; FIGS. 5C-D show in-situ hybridization with
a sFlt-14-specific probe (derived from intron 14) identifying
sFlt-14-expressing cells. Of note, massive expression of sFlt-14
mRNA and protein in syncytial knots of the preeclamptic
placenta.
[0057] FIG. 6 is a western immunoblot image depicting
immunoprecipitation of the sFlt-14 protein in normal term placentae
with a CESS antibody or with a Flt1 antibody.
[0058] FIG. 7 shows a mass-spectrometry identification of sFlt-14
(SEQ ID NOs: 16-19).
[0059] FIG. 8 is a western immunoblot image depicting expression
levels of sFlt-14 proteins in serum and placentae samples of
preeclamptic subjects. Protein detection was carried out using a
specific sFlt-14 antibody (CESS, directed against SEQ ID NO:
5).
[0060] FIGS. 9A-B depict characterization of sFlt proteins during
the course of pregnancy. FIG. 9A illustrates RNA expression of
sFlt-1 and sFlt-14 during different time points of normal
gestation; and FIG. 9B shows quantification of the ratio of the two
sFlt1 isoforms (sFlt-1 and sFlt-14) during different time points of
normal gestation: weeks 9-11, week 13, and week 39.
[0061] FIGS. 10A-B depict sFlt-14 as a VEGF receptor. FIG. 10A
shows recombinant sFlt-14 and sFlt-1 proteins from the cellular
fraction (c) or from the media (m). sFlt-14 is located at 115 Kd
and 130 Kd, in the cellular fraction and media, respectively.
sFlt-1 is located at 100 Kd and 120 Kd, in the cellular fraction
and media, respectively; and FIG. 10B shows a VEGF inhibition assay
where VEGF was pre-incubated with sFlt-1 or sFlt-14 prior to
addition of VEGF-R2 (by addition of growth medium of Porcine Aortic
Endothelial cells). VEGF-R2 phosphorylation levels were measured as
a function of added sFlt-14/VEGF ratio or sFlt-1/VEGF ratio. Of
note, nearly complete inhibition of VEGF-R2 phosphorylation was
evident already at a 1:1 sFlt-14/VEGF ratio.
[0062] FIGS. 11A-B depict sFlt-14 expression in human cornea
sections. FIG. 11A shows immunohistochemistry of sFlt-14 using a
specific antibody (CESS directed against SEQ ID NO: 5); and FIG.
11A shows control immunohistochemistry using a pre immuned serum.
Of note, sFlt-14 was readily seen in the corneal epithelia.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0063] The present invention, in some embodiments thereof, relates
to isolated polypeptides and polynucleotides encoding same for the
diagnosis and treatment of VEGF-associated medical conditions.
[0064] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0065] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0066] The soluble VEGF receptor sFlt-1, which specifically binds
and antagonizes circulating VEGF and PlGF, has been previously
contemplated as the leading cause of preeclampsia and as such was
suggested as a marker of and target for treating this condition
[Maynard et al., supra; Levine et al., supra]. These findings were
based on the use of clinical tools such as antibodies and
oligonucleotides directed to sequences shared by the soluble and
non-soluble VEGF receptors.
[0067] While reducing some embodiments of the present invention to
practice, the present inventors have identified a novel VEGF
receptor variant. This variant is soluble, secreted, comprises a
unique amino acid sequence (SEQ ID NO: 4) and is expressed during
preeclampsia. Using antibodies or oligonucleotides specifically
directed to the unique sequence (SEQ ID NOs 4 or 3) of sFlt-14, the
present inventors were able to show that it is the sFlt-14 rather
than sFlt-1 (supra) that is highly expressed in preeclampsia. These
results prove beyond any doubt the clinical value of sFlt-14.
[0068] As is illustrated in the Examples section which follows, the
novel sFlt-14 (SEQ ID NOs: 1 and 2) of the present invention
differs from the full transmembrane receptor Flt1 and from the
known sFlt1 by comprising a unique 28 amino acid sequence (SEQ ID
NO: 4, see Example 1 and FIG. 1) derived by readthrough of intron
14. This novel sFlt-14 receptor is expressed in placentae and is
highly upregulated in preeclamptic placentae (see Example 3 and
FIG. 4A). Moreover, the results presented herein illustrate that
trophoblastic cells within the syncytial knots produce sFlt-14
(Example 4 and FIGS. 5A-D). Thus, sFlt-14 provides a valuable
indicator of preeclampsia or predisposition thereof. Furthermore,
since sFlt-14 functions in antagonizing VEGFR ligands (e.g., VEGF),
modulating sFlt-14 levels (e.g. downregulating or upregulating) may
serve as a powerful tool in treatment of VEGF associated conditions
(hyper angiogenesis e.g., cancer and neovascularized cornea).
[0069] Thus, according to one aspect of the present invention there
is provided an isolated polynucleotide comprising a nucleic acid
sequence encoding a polypeptide comprising an amino acid sequence
at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 87%, at least about
89%, at least about 90%, at least about 91%, at least about 93%, at
least about 95% or more say 100% identical or homologous to SEQ ID
NO: 4, wherein the isolated polynucleotide is not genomic Flt1.
[0070] As used herein the phrase "an isolated polynucleotide"
refers to a single or double stranded nucleic acid sequences which
is isolated and provided in the form of an RNA sequence, a
complementary polynucleotide sequence (Cdna) and/or a composite
polynucleotide sequences (e.g., a combination of the above).
[0071] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0072] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and partially genomic. A composite sequence can
include some exonal sequences required to encode the polypeptide of
the present invention, as well as some intronic sequences
interposing therebetween. The intronic sequences can be of any
source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements (further
explained in detail hereinbelow).
[0073] According to an exemplary embodiment of this aspect of the
present invention the isolated polypeptide encoded by the
polynucleotide described herein is capable of binding a VEGFR
ligand. Examples of such ligands include, without limitation, VEGF
(VEGF-A, GeneBank Accession No. NP.sub.--001020537), VEGF-B
(GeneBank Accession No. NP.sub.--003368) and Placenta growth factor
(PlGF, GeneBank Accession No. NP.sub.--002623).
[0074] According to an exemplary embodiment, binding of the
polypeptide is expected to be in a range of about 10.sup.-9
M-10.sup.-12 M.
[0075] According to an exemplary embodiment of this aspect of the
present invention, the isolated polynucleotide is as set forth in
SEQ ID NO: 1. Of note, it is suggested that naturally occurring
forms of the polynucleotide sequences of some embodiments of the
present invention are splice variants of the genomic Flt1. Examples
of genomic Flt1 are depicted in GeneBank Accession No.
NC.sub.--000013.9 region: complement (27773790 to 27967232)
GI:51511729 for human genomic Flt1 and GeneBank Accession No.
NC.sub.--006480.2 region: complement (27975879 to 28168596)
GI:114795054 for chimpanzee genomic Flt1 (see FIG. 1 showing
exon/intron organization).
[0076] The phrase "splice variant", as used herein, refers to
alternative forms of RNA transcribed from a VEGF receptor gene.
Splice variation arises naturally through use of alternative
splicing sites within a transcribed RNA molecule, or less commonly
between separately transcribed RNA molecules, and may result in
several different mRNAs transcribed from the same gene. Splice
variants may encode polypeptides having altered amino acid sequence
due to intron inclusion, exon exclusion or a combination of both.
The term splice variant is also used herein to denote a polypeptide
encoded by a splice variant of an mRNA transcribed from a gene.
[0077] According to an alternative embodiment the isolated
polynucleotide of the present invention is as set forth in SEQ ID
NO: 3.
[0078] It will be appreciated that homologues of the sequences
described hereinabove are also envisaged by the present invention.
Accordingly, the polynucleotide of this aspect of the present
invention may have a nucleic acid sequence at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 87%, at least about 89%, at least about 90%, at
least about 91%, at least about 93%, at least about 95% or more say
100% identical or homologous to SEQ ID NO: 1 or 3, as determined
using BlastN software of the National Center of Biotechnology
Information (NCBI) using default parameters.
[0079] Thus, the present invention encompasses nucleic acid
sequences described hereinabove; fragments thereof, sequences
hybridizable therewith, sequences homologous thereto, sequences
encoding similar polypeptides with different codon usage, altered
sequences characterized by mutations, such as deletion, insertion
or substitution of one or more nucleotides, either naturally
occurring or man induced, either randomly or in a targeted
fashion.
[0080] Since the polynucleotide sequences of the present invention
encode previously unidentified polypeptides, the present invention
also encompasses novel polypeptides or portions thereof, which are
encoded by the isolated polynucleotides and respective nucleic acid
fragments thereof described hereinabove.
[0081] Thus, according to another aspect of the present invention
there is provided an isolated polypeptide comprising an amino acid
sequence at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 87%, at least
about 89%, at least about 90% at least about 91%, at least about
93%, at least about 95% or more say 100% homologous to SEQ ID NO: 4
as determined by protein BLAST algorithm
(http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).
[0082] In an exemplary embodiment the isolated polypeptide is as
set forth in SEQ ID NO: 2 or 4.
[0083] The present invention also encompasses fragments (e.g., as
short as a specific antigenic determinant e.g., at least about 6,
at least about 8, at least about 10 at least about 20 amino acids
such as derived from SEQ ID NO: 4) of the above described
polypeptides and polypeptides having mutations, such as deletions,
insertions or substitutions of one or more amino acids, either
naturally occurring or man induced, either randomly or in a
targeted fashion. These fragments may be used to elicit antibody
production against the isolated polypeptides of the invention.
[0084] As used herein the phrase "an isolated polypeptide" refers
to isolated, native peptides (either degradation products,
synthetically synthesized peptides, or recombinant peptides),
peptidomimetics (typically, synthetically synthesized peptides),
and the peptide analogues peptoids and semipeptoids, and may have,
for example, modifications rendering the peptides more stable while
in a body or more capable of penetrating into cells. Such
modifications include, but are not limited to: N-terminus
modifications; C-terminus modifications; peptide bond
modifications, including but not limited to CH.sub.2--NH,
CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH, CH.sub.2--O,
CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH, and CF.dbd.CH; backbone
modifications; and residue modifications. Methods for preparing
peptidomimetic compounds are well known in the art and are
specified, for example, in Ramsden, C. A., ed. (1992), Quantitative
Drug Design, Chapter 17.2, F. Choplin Pergamon Press, which is
incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinbelow.
[0085] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)-CO--);
ester bonds (--C(R)H--C--O--O--C(R)--N--); ketomethylene bonds
(--CO--CH2-); .alpha.-aza bonds (--NH--N(R)--CO--), wherein R is
any alkyl group, e.g., methyl; carba bonds (--CH2-NH--);
hydroxyethylene bonds (--CH(OH)--CH2-); thioamide bonds
(--CS--NH--); olefinic double bonds (--CH.dbd.CH--); retro amide
bonds (--NH--CO--); and peptide derivatives (--N(R)--CH2-CO--),
wherein R is the "normal" side chain, naturally presented on the
carbon atom. These modifications can occur at any of the bonds
along the peptide chain and even at several (2-3) at the same
time.
[0086] Natural aromatic amino acids, Trp, Tyr, and Phe, may be
substituted for synthetic non-natural acids such as, for instance,
tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine
(Nol), ring-methylated derivatives of Phe, halogenated derivatives
of Phe, and o-methyl-Tyr.
[0087] In addition to the above, the polypeptides of the present
invention may also include one or more modified amino acids or one
or more non-amino acid monomers (e.g., fatty acids, complex
carbohydrates, etc.).
[0088] The term "amino acid" or "amino acids" is understood to
include the 20 naturally occurring amino acids; those amino acids
often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine, and phosphothreonine; and
other less common amino acids, including but not limited to
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine, and ornithine. Furthermore, the term "amino acid"
includes both D- and L-amino acids.
[0089] Thus, polypeptides of the present invention can be of a
short length typically 5-10, 10-20, 20-50, 50-100 amino acids in
length and longer e.g., 100-200, 200-300, 300-400, 400-500,
500-600, 600-733 amino acids in length.
[0090] Mimetic technology may be used to generate peptides which
are engineered to have at least one modified feature as compared to
the naturally occurring polypeptide (e.g., SEQ ID NO: 2) while
maintaining a biological activity of interest e.g., VEGF binding,
antibody binding and the like.
[0091] Generation of peptide mimetics can be effected using various
approaches which are well known in the art, including, for example,
display techniques.
[0092] Thus, the present invention contemplates a display library
comprising a plurality of display vehicles (such as phages, viruses
or bacteria) each displaying at least 5, at least 7, at least 11,
at least 15, at least 20, at least 25 consecutive amino acids
derived from the isolated polypeptide sequence of sFlt-14 (e.g.,
SEQ ID NO: 2 and SEQ ID NO: 4).
[0093] Peptide mimetics can also be uncovered using computational
biology.
[0094] According to an embodiment of the present invention, the
isolated polypeptides are soluble.
[0095] As used herein the term "soluble" refers to the ability of
the molecules of the present invention to dissolve in a
physiological aqueous solution (pH about 7, e.g., solubility level
in aqueous media of >100 .mu.g/ml) without substantial
aggregation. Thus, it is readily understood that soluble sFlt-14
are preferably devoid of hydrophobic transmembrane domains.
[0096] Being soluble, the polypeptides of the present invention may
be secreted. As depicted in the Example section which follows, the
present inventors have revealed, for the first time, that sFlt-14
(and not sFlt-1) is the soluble receptor found in the serum of
preeclamptic subjects (Example 7 and FIG. 8). Thus, sFlt-14 is the
major VEGF receptor in the circulation of preeclamptic
subjects.
[0097] The peptides of the present invention are preferably
utilized in a linear form, although it will be appreciated that in
cases where cyclization does not severely interfere with peptide
characteristics, cyclic forms of the peptide can also be
utilized.
[0098] The peptides of the present invention may be synthesized by
any techniques that are known to those skilled in the art of
peptide synthesis. For solid phase peptide synthesis, a summary of
the many techniques may be found in: Stewart, J. M. and Young, J.
D. (1963), "Solid Phase Peptide Synthesis," W. H. Freeman Co. (San
Francisco); and Meienhofer, J (1973). "Hormonal Proteins and
Peptides," vol. 2, p. 46, Academic Press (New York). For a review
of classical solution synthesis, see Schroder, G. and Lupke, K.
(1965). The Peptides, vol. 1, Academic Press (New York).
[0099] In general, peptide synthesis methods comprise the
sequential addition of one or more amino acids or suitably
protected amino acids to a growing peptide chain. Normally, either
the amino or the carboxyl group of the first amino acid is
protected by a suitable protecting group. The protected or
derivatized amino acid can then either be attached to an inert
solid support or utilized in solution by adding the next amino acid
in the sequence having the complimentary (amino or carboxyl) group
suitably protected, under conditions suitable for forming the amide
linkage. The protecting group is then removed from this newly added
amino acid residue and the next amino acid (suitably protected) is
then added, and so forth; traditionally this process is accompanied
by wash steps as well. After all of the desired amino acids have
been linked in the proper sequence, any remaining protecting groups
(and any solid support) are removed sequentially or concurrently,
to afford the final peptide compound. By simple modification of
this general procedure, it is possible to add more than one amino
acid at a time to a growing chain, for example, by coupling (under
conditions which do not racemize chiral centers) a protected
tripeptide with a properly protected dipeptide to form, after
deprotection, a pentapeptide, and so forth.
[0100] Further description of peptide synthesis is disclosed in
U.S. Pat. No. 6,472,505. A preferred method of preparing the
peptide compounds of the present invention involves solid-phase
peptide synthesis, utilizing a solid support. Large-scale peptide
synthesis is described by Andersson Biopolymers 2000, 55(3),
227-50.
[0101] In cases where large amounts of the peptides of the present
invention are desired, the polypeptides of the present invention
can be generated using recombinant techniques such as described by
Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et
al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984)
Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311;
Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984)
Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol.
6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant
Molecular Biology, Academic Press, NY, Section VIII, pp
421-463.
[0102] Briefly, an expression construct (i.e., expression vector),
which includes the isolated polynucleotide of the present invention
(e.g., SEQ ID NO: 1, 3), optionally in frame fused to a nucleic
acid sequence encoding a heterologous amino acid sequence (e.g.
immunoglobulin sequence, as further described hereinbelow),
positioned under the transcriptional control of a regulatory
element, such as a promoter (as explained in detail hereinbelow),
is introduced into host cells.
[0103] For expression in mammalian cells, pRK5-based vectors
[Schall et al., Cell, 61:361-370 (1990)]; and CDM8-based vectors
[Seed, Nature, 329:840 (1989)] can be used.
[0104] Methods of introducing the expression construct into a host
cell are well known in the art and include electroporation,
lipofection and chemical transformation (e.g., calcium
phosphate).
[0105] The "transformed" cells are cultured under suitable
conditions, which allow the expression of the polypeptide encoded
by the nucleic acid sequence.
[0106] Following a predetermined time period, the expressed
chimeric molecule is recovered from the cell or cell culture, and
purification is effected according to the end use of the
recombinant polypeptide.
[0107] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements including
constitutive and inducible promoters, transcription enhancer
elements, transcription terminators, and the like, can be used in
the expression vector [see, e.g., Bitter et al., (1987) Methods in
Enzymol. 153:516-544].
[0108] Other than containing the necessary elements for the
transcription and translation of the inserted coding sequence
(encoding the chimera), the expression construct of the present
invention can also include sequences engineered to optimize
stability, production, purification, yield or toxicity of the
expressed fusion protein.
[0109] A variety of prokaryotic or eukaryotic cells can be used as
host-expression systems to express the fusion protein coding
sequence. These include, but are not limited to, microorganisms,
such as bacteria transformed with a recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vector containing the chimera
coding sequence; yeast transformed with recombinant yeast
expression vectors containing the chimera coding sequence; plant
cell systems infected with recombinant virus expression vectors
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or transformed with recombinant plasmid expression vectors, such as
Ti plasmid, containing the chimera coding sequence. Mammalian
expression systems are preferably used to express the chimera of
the present invention.
[0110] The choice of host cell line for the expression of the
molecules depends mainly on the expression vector. Eukaryotic
expression systems are preferred (e.g., mammalian and insects)
since they allow post translational modifications (e.g.,
glycosylation). Another consideration is the amount of protein that
is required. Milligram quantities often can be produced by
transient transfections. For example, the adenovirus
EIA-transformed 293 human embryonic kidney cell line can be
transfected transiently with pRK5-based vectors by a modification
of the calcium phosphate method to allow efficient expression.
CDM8-based vectors can be used to transfect COS cells by the
DEAE-dextran method (Aruffo et al., Cell, 61:1303-1313 (1990);
Zettmeissl et al., DNA Cell Biol. US, 9:347-353 (1990)]. If larger
amounts of protein are desired, the molecules can be expressed
after stable transfection of a host cell line. It will be
appreciated that the presence of a hydrophobic leader sequence at
the N-terminus of the molecule will ensure processing and secretion
of the molecule by the transfected cells.
[0111] It will be appreciated that the use of bacterial or yeast
host systems may be preferable to reduce cost of production.
However since bacterial host systems are devoid of protein
glycosylation mechanisms, a post production glycosylation may be
needed.
[0112] In any case, transformed cells are cultured under effective
conditions, which allow for the expression of high amounts of
recombinant polypeptide. Effective culture conditions include, but
are not limited to, effective media, bioreactor, temperature, pH
and oxygen conditions that permit protein production. An effective
medium refers to any medium in which a cell is cultured to produce
the recombinant chimera molecule of the present invention. Such a
medium typically includes an aqueous solution having assimilable
carbon, nitrogen and phosphate sources, and appropriate salts,
minerals, metals and other nutrients, such as vitamins. Cells of
the present invention can be cultured in conventional fermentation
bioreactors, shake flasks, test tubes, microtiter dishes, and petri
plates. Culturing can be carried out at a temperature, pH and
oxygen content appropriate for a recombinant cell. Such culturing
conditions are within the expertise of one of ordinary skill in the
art.
[0113] Depending on the vector and host system used for production,
resultant proteins of the present invention may either remain
within the recombinant cell, secreted into the fermentation medium,
secreted into a space between two cellular membranes, such as the
periplasmic space in E. coli; or retained on the outer surface of a
cell or viral membrane.
[0114] Following a predetermined time in culture, recovery of the
recombinant protein is effected.
[0115] Molecules of the present invention are preferably retrieved
in "substantially pure" form. As used herein, "substantially pure"
refers to a purity that allows for the effective use of the protein
in the applications, described hereinbelow.
[0116] As mentioned, the isolated polypeptide of this aspect of the
present invention may further comprise a heterologous amino acid
sequence.
[0117] As used herein the phrase "heterologous amino acid sequence"
refers to an amino acid sequence which does not form a part of a
naturally occurring sFlt-14 (e.g., SEQ ID NO: 2) amino acid
sequence. This sequence preferably confers solubility to the
molecule of this embodiment of the present invention, and
preferably increases the half-life of the chimeric molecule in the
serum.
[0118] The heterologous amino acid sequence is generally localized
at the amino- or carboxyl-terminus of the isolated polypeptide of
the present invention.
[0119] One or more heterologous amino acid sequences can be
conjugated to the sFlt-14 amino acid sequence of the present
invention. Examples of heterologous amino acid sequences commonly
used in fusion protein construction include, but are not limited
to, immunoglobulin, galactosidase, glucuronidase,
glutathione-S-transferase (GST), carboxy terminal peptide (CTP)
from chorionic gonadotrophin (CG.beta.) and chloramphenicol
acetyltransferase (CAT).
[0120] The exact site at which fusion (conjugation) between the
heterologous sequence and the sFlt-14 amino acid sequence is not
critical and the optimal site can be determined by routine
experimentation as long as functionality of the polypeptide is
maintained (e.g., VEGF binding). Methods of ligand binding
assessment are well known in the art (e.g., using a radiolabeled
ligand in a binding assay, or an ELISA).
[0121] Additionally or alternatively as mentioned hereinabove the
isolated polypeptide of the present invention may be attached to a
non-proteinaceous moiety.
[0122] Thus, embodiments of the present invention provide an
isolated polypeptide or polynucleotide being attached to a
non-proteinaceous moiety.
[0123] Such a conjugate molecule is highly stable (resistant to
in-vivo proteolytic activity probably due to steric hindrance
conferred by the non-proteinaceous moiety) and may be produced
using common solid phase synthesis methods which are inexpensive
and highly efficient, as further described hereinbelow. However, it
will be appreciated that recombinant techniques may still be used,
whereby the recombinant peptide product is subjected to in-vitro
modification (e.g., PEGylation).
[0124] The phrase "non-proteinaceous moiety" as used herein refers
to a molecule not including peptide bonded amino acids that is
attached to the above-described sFlt-14 amino acid sequence.
According to an embodiment the non-proteinaceous moiety of this
aspect of the present invention is a polymer or a co-polymer
(synthetic or natural). Non-limiting examples of the
non-proteinaceous moiety of the present invention include
polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), divinyl
ether and maleic anhydride copolymer (DIVEMA; see for example,
Kaneda Y, et al., 1997, Biochem. Biophys. Res. Commun. 239: 160-5)
and poly(styrene comaleic anhydride) (SMA; see for example, Mu Y,
et al., 1999, Biochem Biophys Res Commun. 255: 75-9).
[0125] Conjugation of such a non-proteinaceous moiety confers the
polypeptide of this aspect of the present invention with stability
(e.g., against protease activities) and/or solubility (e.g., within
a biological fluid such as blood, digestive fluid) while preserving
its biological activity and prolonging its half-life. Such a
conjugation is advantageous particularly in cases of therapeutic
proteins which exhibit short half-life and rapid clearance from the
blood. The increased half-lives of conjugated proteins, in the
plasma, results from increased size of protein conjugates (which
limits their glomerular filtration) and decreased proteolysis due
to polymer steric hindrance. Generally, the more polymer chains
attached per peptide, the greater the extension of half-life.
However, measures are taken not to reduce the specific activity of
the sFlt-14 amino acid sequence of the present invention (e.g.,
sFlt-14 binding to VEGF). Methods of conjugating non-protein
moieties to amino acid sequences are well known in the art (as
described in, for example, Veronese F M, Biomaterials, Volume
22(5), 2001, pp. 405-417(13), Elsevier Publishing; and Haruhiko
Kamada, et al., 2000, Cancer Research 60: 6416-6420, which are
fully incorporated herein by reference).
[0126] Thus, the present inventors have uncovered a novel soluble
and secreted variant of VEGFR. This variant is expressed in serum
and placentae of preeclamptic subjects and as such detection of
same may be clinically valuable such as in the diagnosis of
preeclampsia.
[0127] Thus, according to some embodiments of yet another aspect of
the present invention there is provided a method of detecting
sFlt-14 (e.g., SEQ ID NO: 2) in a biological sample (including in
vivo detection).
[0128] Typically, the method is effected by determining sFlt-14
level, presence or ratio (such as with respect to other Flt-1
isoforms, e.g., sFlt-1 as shown in FIG. 9B).
[0129] In accordance with some embodiments of this aspect of the
present invention the methods comprising, contacting the biological
sample with an antibody comprising an antigen recognition domain
which specifically binds the isolated polypeptide of sFlt-14 (e.g.,
SEQ ID NO: 2) and not to SEQ ID NO: 10 such that the sFlt-14 and
the antibody form a complex; and measuring a presence or a level of
the complex to thereby detect sFlt-14 in the biological sample.
[0130] As used herein a "biological sample" refers to a biological
material, such as cells, tissues (e.g., placenta, chorionic villus
sample, solid tumor) and fluids such as amniotic fluid, blood,
serum, plasma, lymph, bile fluid, urine, saliva, sputum, synovial
fluid, semen, tears, cerebrospinal fluid, bronchioalveolar large
fluid, ascites fluid, pus, conditioned medium and the like in which
sFlt-14 may be present. The biological sample is a maternal or
fetal sample. The biological sample, may be ex vivo or in vitro
analyzed, but can also be analyzed without retrieval from the
subject's body.
[0131] As shown in Example 8 and in FIGS. 9A-B (in the Examples
section which follows), sFlt-14 is exclusively expressed from week
13 of gestation. Thus, when analysis of sFlt-14 level or presence
is effected prior to this week, specific antibodies or
oligonucleotides are preferably employed. From week 13 and on, the
use of antibodies or oligonucleotides directed at common sequence
regions of Flt-1 variants may also be contemplated.
[0132] Thus, antibodies of some embodiments of this aspect of the
present invention may be directed to the amino acid sequence
CELYTSTSPSSSSSS (SEQ. ID. NO: 5). This peptide comprises amino
acids derived from the unique 28 amino acid sequence of sFlt-14
(SEQ. ID. NO: 4) which are not present in other Flt polypeptides
(i.e. in the transmembrane and soluble sFlt-1), as depicted in SEQ
ID NO: 10. Alternatively, antibodies may be directed to the amino
acid sequence CHANGVPEPQITWFK (SEQ. ID. NO: 6). This peptide
comprises amino acids derived from an amino acid sequence shared by
sFlt-14 and the transmembrane Flt-1, but not by the sFlt-1 (SEQ ID
NO: 10). Conversely, antibodies may be directed to the bridging
region which comprises both the common amino acid sequence and the
unique amino acid sequence. An exemplary bridging region which
antibodies can be directed to is HKIQQEPELYTSTS (SEQ. ID. NO: 15).
Measures are taken to select antibodies which are specific to
sFlt-14 and not Flt-1 or its soluble form.
[0133] Specific peptides chosen for antibody generation are
preferably selected immunogenic (i.e., capable of stimulating an
antibody response). Parameters for testing peptide immunogenicity
are well known in the art including, but not limited to,
foreginess, molecular size, chemical composition and heterogeneity
and susceptibility to antigen processing and presentation. Various
sequence analysis software applications are known in the art, which
provide an immunogenicity index according to, for example, the
Jameson-Wolf algorithm. Examples include, but are not limited to,
Sciprot (available from
wwwdotasiaonlinedotnetdothk/.about.twcbio/DOCS/1/scPrteindothtm)
and Macvector (available from
wwwdotaccelrysdotcom/products/macvector/) as well as the widely
utilized GCG package (Genetics Computer Group, Wisconsin).
[0134] The term "antibody" as used herein includes whole antibody
molecules as well as functional fragments thereof, such as Fab,
F(ab').sub.2, and Fv that are capable of binding with antigenic
portions of the target polypeptide. These functional antibody
fragments constitute preferred embodiments of the present
invention, and are defined as follows:
[0135] (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0136] (2) Fab', the fragment of an antibody molecule that can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0137] (3) (Fab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds;
[0138] (4) Fv, defined as a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and
[0139] (5) Single chain antibody ("SCA"), a genetically engineered
molecule containing the variable region of the light chain and the
variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule as
described in, for example, U.S. Pat. No. 4,946,778.
[0140] Methods of generating such antibody fragments are well known
in the art (See for example, Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988,
incorporated herein by reference).
[0141] Purification of serum immunoglobulin antibodies (polyclonal
antisera) or reactive portions thereof can be accomplished by a
variety of methods known to those of skill in the art including,
precipitation by ammonium sulfate or sodium sulfate followed by
dialysis against saline, ion exchange chromatography, affinity or
immunoaffinity chromatography as well as gel filtration, zone
electrophoresis, etc. (see Goding in, Monoclonal Antibodies:
Principles and Practice, 2nd ed., pp. 104-126, 1986, Orlando, Fla.,
Academic Press). Under normal physiological conditions antibodies
are found in plasma and other body fluids and in the membrane of
certain cells and are produced by lymphocytes of the type denoted B
cells or their functional equivalent. Antibodies of the IgG class
are made up of four polypeptide chains linked together by disulfide
bonds. The four chains of intact IgG molecules are two identical
heavy chains referred to as H-chains and two identical light chains
referred to as L-chains. Additional classes include IgD, IgE, IgA,
IgM and related proteins.
[0142] Methods for the generation and selection of monoclonal
antibodies are well known in the art, as summarized for example in
reviews such as Tramontano and Schloeder, Methods in Enzymology
178, 551-568, 1989. A sFlt-14 polypeptide (or fragment thereof) of
the present invention may be used to generate antibodies in vitro.
More preferably, the sFlt-14 polypeptide of the present invention
is used to elicit antibodies in vivo. In general, a suitable host
animal is immunized with the sFlt-14 polypeptide of the present
invention. Advantageously, the animal host used is a mouse of an
inbred strain. Animals are typically immunized with a mixture
comprising a solution of the sFlt-14 polypeptide of the present
invention in a physiologically acceptable vehicle, and any suitable
adjuvant, which achieves an enhanced immune response to the
immunogen. By way of example, the primary immunization conveniently
may be accomplished with a mixture of a solution of the sFlt-14
polypeptide of the present invention and Freund's complete
adjuvant, the mixture being prepared in the form of a water in oil
emulsion. Typically the immunization will be administered to the
animals intramuscularly, intradermally, subcutaneously,
intraperitoneally, into the footpads, or by any appropriate route
of administration. The immunization schedule of the immunogen may
be adapted as required, but customarily involves several subsequent
or secondary immunizations using a milder adjuvant such as Freund's
incomplete adjuvant. Antibody titers and specificity of binding to
the sFlt-14 polypeptide can be determined during the immunization
schedule by any convenient method including by way of example
radioimmunoassay, or enzyme linked immunosorbant assay, which is
known as the ELISA assay. When suitable antibody titers are
achieved, antibody-producing lymphocytes from the immunized animals
are obtained, and these are cultured, selected and cloned, as is
known in the art. Typically, lymphocytes may be obtained in large
numbers from the spleens of immunized animals, but they may also be
retrieved from the circulation, the lymph nodes or other lymphoid
organs. Lymphocytes are then fused with any suitable myeloma cell
line, to yield hybridomas, as is well known in the art.
Alternatively, lymphocytes may also be stimulated to grow in
culture, and may be immortalized by methods known in the art
including the exposure of these lymphocytes to a virus, a chemical
or a nucleic acid such as an oncogene, according to established
protocols. After fusion, the hybridomas are cultured under suitable
culture conditions, for example in multi-well plates, and the
culture supernatants are screened to identify cultures containing
antibodies that recognize the hapten of choice. Hybridomas that
secrete antibodies that recognize the sFlt-14 polypeptides of the
present invention are cloned by limiting dilution and expanded,
under appropriate culture conditions. Monoclonal antibodies are
purified and characterized in terms of immunoglobulin type and
binding affinity.
[0143] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment.
[0144] Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647, and
references contained therein, which patents are hereby incorporated
by reference in their entirety (see also Porter, R. R., Biochem.
J., 73: 119-126, 1959). Other methods of cleaving antibodies, such
as separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0145] Fv fragments comprise an association of V.sub.H and V.sub.L
chains. This association may be noncovalent, as described in Inbar
et al. (Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972).
Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. Preferably, the Fv fragments comprise V.sub.H and
V.sub.L chains connected by a peptide linker. These single-chain
antigen binding proteins (sFv) are prepared by constructing a
structural gene comprising DNA sequences encoding the V.sub.H and
V.sub.L domains connected by an oligonucleotide. The structural
gene is inserted into an expression vector, which is subsequently
introduced into a host cell such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFvs are
described, for example, by Whitlow and Filpula, Methods, 2: 97-105,
1991; Bird et al., Science 242:423-426, 1988; Pack et al.,
Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No.
4,946,778, all of which are hereby incorporated, by reference, in
entirety.
[0146] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick and Fry Methods, 2: 106-10, 1991).
[0147] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues, which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)].
[0148] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source, which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann 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.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0149] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human monoclonal
antibodies can be made by introducing human immunoglobulin loci
into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, 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).
[0150] Thus the antibodies may be contacted with the biological
sample so at to form an immunocomplex. Detection of the level and
presence of the complex may be effected using methods which are
well known in the art. Examples of such methods include, but are
not limited to, Western blot, Radio-immunoassay (RIA), Fluorescence
activated cell sorting (FACS), and Immunohistochemical
analysis.
[0151] Alternatively, detection of sFlt-14 may be at the
polynucleotide level. To this end, the sample is contacted with an
isolated polynucleotide (e.g., oligonucleotide) which comprises a
nucleic acid sequence which specifically binds to sFlt-14 (SEQ ID
NO: 1 or 3 or to a bridging sequence as, for example, as set forth
in SEQ ID NO: 8) and not to sFlt-1 (SEQ ID NO: 9) so as to form a
hybridization complex.
[0152] Following a sufficient time of incubation the presence or
level of the complex is measured to thereby detect sFlt-14 in the
biological sample.
[0153] For example, oligonucleotides can be used which are capable
of binding to sequences which are specific to sFlt-14
polynucleotides and not to other Flt-1 polynucleotides (e.g.
membrane-anchored Flt-1 and soluble Flt-1) such as sFlt-1 (SEQ ID
NO: 9). Such sequences may be present in the untranslated region or
the open reading frame of the isolated polynucleotides.
[0154] As used herein, the term "oligonucleotide" refers to a
single-stranded or double-stranded oligomer or polymer of
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics
thereof. This term includes oligonucleotides composed of naturally
occurring bases, sugars, and covalent internucleoside linkages
(e.g., backbone), as well as oligonucleotides having non-naturally
occurring portions, which function similarly to respective
naturally occurring portions.
[0155] As used herein, the phrase "capable of specifically
hybridizing" refers to forming a double strand molecule such as
RNA:RNA, RNA:DNA and/or DNA:DNA molecules.
[0156] Oligonucleotides designed according to the teachings of the
present invention can be generated according to any oligonucleotide
synthesis method known in the art, such as enzymatic synthesis or
solid-phase synthesis. Equipment and reagents for executing
solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be
employed; the actual synthesis of the oligonucleotides is well
within the capabilities of one skilled in the art and can be
accomplished via established methodologies as detailed in, for
example: Sambrook, J. and Russell, D. W. (2001), "Molecular
Cloning: A Laboratory Manual"; Ausubel, R. M. et al., eds. (1994,
1989), "Current Protocols in Molecular Biology," Volumes I-III,
John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), "A
Practical Guide to Molecular Cloning," John Wiley & Sons, New
York; and Gait, M. J., ed. (1984), "Oligonucleotide Synthesis";
utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite
followed by deprotection, desalting, and purification by, for
example, an automated trityl-on method or HPLC.
[0157] The oligonucleotide of the present invention is of at least
17, at least 18, at least 19, at least 20, at least 22, at least
25, at least 30 or at least 40, bases specifically hybridizable
with polynucleotide sequences of the present invention.
[0158] Hybridization based assays which allow the detection of a
DNA or RNA of interest in a biological sample rely on the use of
oligonucleotide which can be 10, 15, 20, or 30 to 100 nucleotides
long preferably from 10 to 50, more preferably from 40 to 50
nucleotides.
[0159] Hybridization of short nucleic acids (below 200 bp in
length, e.g. 17-40 bp in length) can be effected using the
following exemplary hybridization protocols which can be modified
according to the desired stringency; (i) hybridization solution of
6.times.SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH
6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 .mu.g/ml denatured salmon
sperm DNA and 0.1% nonfat dried milk, hybridization temperature of
1-1.5.degree. C. below the T.sub.m, final wash solution of 3 M
TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5%
SDS at 1-1.5.degree. C. below the T.sub.m; (ii) hybridization
solution of 6.times.SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium
phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 .mu.g/ml
denatured salmon sperm DNA and 0.1% nonfat dried milk,
hybridization temperature of 2-2.5.degree. C. below the T.sub.m,
final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8),
1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5.degree. C. below the T.sub.m,
final wash solution of 6.times.SSC, and final wash at 22.degree.
C.; (iii) hybridization solution of 6.times.SSC and 1% SDS or 3 M
TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5%
SDS, 100 .mu.g/ml denatured salmon sperm DNA and 0.1% nonfat dried
milk, hybridization temperature.
[0160] The detection of hybrid duplexes can be carried out by a
number of methods. Typically, hybridization duplexes are separated
from unhybridized nucleic acids and the labels bound to the
duplexes are then detected. Such labels refer to radioactive,
fluorescent, biological or enzymatic tags or labels of standard use
in the art. A label can be conjugated to either the oligonucleotide
probes or the nucleic acids derived from the biological sample
(target).
[0161] For example, oligonucleotides of the present invention can
be labeled subsequent to synthesis, by incorporating biotinylated
dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a
psoralen derivative of biotin to RNAs), followed by addition of
labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin)
or the equivalent. Alternatively, when fluorescently-labeled
oligonucleotide probes are used, fluorescein, lissamine,
phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5,
Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others [e.g., Kricka et al.
(1992), Academic Press San Diego, Calif] can be attached to the
oligonucleotides.
[0162] Traditional hybridization assays include PCR, RT-PCR, RNase
protection, in-situ hybridization, primer extension, Northern Blot
and dot blot analysis (see Examples section hereinbelow).
[0163] It will be appreciated that detection of sFlt-14 may also be
effected by other methods which do not include the use of
antibodies or oligonucleotides. These methods, even if more
laborious at time, include but are not limited to molecular
weight-based identification and mass spectrometry.
[0164] It will be appreciated that the above described methods of
detecting sFlt-14 is mostly desired for the diagnosis of a
pregnancy associated medical condition associated with maternal or
fetal stress.
[0165] As used herein the term "diagnosis" refers to classifying a
disease or a symptom, determining a severity of such a disease,
monitoring disease progression, monitoring the effectiveness of a
therapeutic regime, forecasting (prognosing) an outcome of a
disease and/or prospects of recovery.
[0166] As used herein the phrase "a pregnancy associated medical
condition associated with maternal or fetal stress" refers to a
disease or a syndrome in which there are clinical symptoms in the
mother of fetus which are associated with upregulation of sFlt-14.
The pregnancy may be at any stage or phase. The medical condition
may include any hypertensive disorders: preeclampsia, eclampsia,
mild preeclampsia, chronic hypertension, EPH gestosis, gestational
hypertension, superimposed preeclampsia (including preeclampsia
superimposed on chronic hypertension, chronic nephropathy or
lupus), HELLP syndrome (hemolysis, elevated liver enzymes, low
platelet count) or nephropathy. The medical condition may also
include gestational diabetes, fetal growth restriction (FGR) and
fetal alcohol syndrome (FAS).
[0167] As used herein, the phrase "maternal or fetal stress" refers
to any condition in which the mother or the fetus is at risk of
developing a pregnancy related complication. Fetal stress includes,
without being limited to, inadequate nutrient supply and cessation
of fetal growth. Maternal stress includes, without being limited
to, hypertension and diabetes. Fetal and maternal stress may affect
fetal development and brain functions and plays a significant role
in pregnancy outcomes related to prematurity and urgent deliveries
(e.g. c-section).
[0168] As used herein the phrase "subject in need thereof" refers
to a mammal preferably a human subject (e.g., pregnant female or a
fetus).
[0169] Although this invention is described with respect to
pregnant women, methods described herein may also be utilized to
assess the risk to non-pregnant women of developing hypertensive
disorders during pregnancy.
[0170] As mentioned herein above, the present inventors have shown
that sFlt-14 is significantly upregulated in preeclampsia (see
Example 3 and FIGS. 4A-B). Furthermore, the present inventors have
shown that the novel VEGFR variant (sFlt-14) is expressed from
early pregnancy (weeks 9) and is the dominant VEGFR starting from
the second trimester (week 13, see Example 8 and FIGS. 9A-B). Thus,
the present inventors envision the use of agents capable of
downregulating sFlt-14 for the treatment of pregnancy associated
medical conditions. In addition the present inventors have
successfully shown that the novel variant competes with VEGFR (see
Example 9 and FIG. 10B) to binding of VEGF and as such regulation
of the novel variant is critical for the treatment of
VEGF-associated medical conditions.
[0171] As used herein "a VEGF associated medical condition" refers
to a disease, disorder or condition which onset or progression of
depend on reduced or excessive activity or expression of VEGFR
ligands as described above.
[0172] Thus, according to some embodiments of the present invention
there is provided a method of treating a VEGF-associated medical
condition in which there is a reduced activity and/or expression of
VEGF. The method comprising administering to a subject in need
thereof a therapeutically effective amount of an agent capable of
downregulating sFlt-14 to thereby treat the VEGF-associated medical
condition in the subject.
[0173] Examples of medical conditions associated with reduced
activity and/or expression of VEGF include, but are not limited to,
preeclampsia, gestational diabetes, gestational hypertension, fetal
growth restriction (FGR), fetal alcohol syndrome (FAS) and
hypertension.
[0174] As used herein "treating" refers to preventing, curing,
reversing, attenuating, alleviating, minimizing, suppressing or
halting the deleterious effects of a medical condition.
[0175] Examples of such agents include the above-described
antibodies and polynucleotides (e.g., capable of specifically
binding and inhibiting sFlt-14 and not sFlt-1).
[0176] For example, the agent of this aspect of the present
invention may be capable of reducing activity and/or expression
(i.e. downregulating) sFlt-14 by affecting the cells which produce
the sFlt-14 polypeptides (e.g. trophoblasts).
[0177] Thus, an agent capable of downregulating sFlt-14 of the
present invention is an oligonucleotide capable of specifically
hybridizing (e.g., in cells under physiological conditions) to a
polynucleotide comprising a nucleic acid sequence encoding a
sFlt-14 polypeptide. Such oligonucleotides have been described
hereinabove.
[0178] Delivery strategies which can be used to efficiently deliver
oligonucleotides into a wide variety of cell types are well known
in the art [see, for example, Luft J Mol Med 76: 75-6 (1998);
Kronenwett et al., Blood 91: 852-62 (1998); Rajur et al., Bioconjug
Chem 8: 935-40 (1997); Lavigne et al., Biochem Biophys Res Commun
237: 566-71 (1997) and Aoki et al., (1997) Biochem Biophys Res
Commun 231: 540-5 (1997].
[0179] An example of an oligonucleotide agent capable of
downregulating the expression of sFlt-14 polypeptides is a small
interfering RNA (siRNA) molecule. RNA interference is a two-step
process. During the first step, which is termed the initiation
step, input dsRNA is digested into 21-23 nucleotide (nt) small
interfering RNAs (siRNA), probably by the action of Dicer, a member
of the RNase III family of dsRNA-specific ribonucleases, which
cleaves dsRNA (introduced directly or via an expressing vector,
cassette or virus) in an ATP-dependent manner. Successive cleavage
events degrade the RNA to 19-21 bp duplexes (siRNA), each strand
with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin.
Genetics and Development 12:225-232 (2002); and Bernstein Nature
409:363-366 (2001)].
[0180] In the effector step, the siRNA duplexes bind to a nuclease
complex to form the RNA-induced silencing complex (RISC). An
ATP-dependent unwinding of the siRNA duplex is required for
activation of the RISC. The active RISC then targets the homologous
transcript by base pairing interactions and cleaves the mRNA into
12 nucleotide fragments from the 3' terminus of the siRNA
[Hutvagner and Zamore Curr. Opin. Genetics and Development
12:225-232 (2002); Hammond et al., (2001) Nat. Rev. Gen. 2:110-119
(2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the
mechanism of cleavage is still to be elucidated, research indicates
that each RISC contains a single siRNA and an RNase [Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
[0181] Because of the remarkable potency of RNAi, an amplification
step within the RNAi pathway has been suggested. Amplification
could occur by copying of the input dsRNAs, which would generate
more siRNAs, or by replication of the siRNAs formed. Alternatively
or additionally, amplification could be effected by multiple
turnover events of the RISC [Hammond et al., Nat. Rev. Gen.
2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For
more information on RNAi see the following reviews Tuschl
ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599
(2002); and Brantl Biochem. Biophys. Act. 1575:15-25 (2002).
[0182] Synthesis of RNAi molecules suitable for use with the
present invention can be effected as follows. First, the sFlt-14
polynucleotide sequence target is scanned downstream for AA
dinucleotide sequences. Occurrence of each AA and the 3' adjacent
19 nucleotides is recorded as potential siRNA target sites.
[0183] Second, potential target sites are compared to an
appropriate genomic database (e.g., human, mouse, rat etc.) using
any sequence alignment software, such as the BLAST software
available from the NCBI server
(wwwdotncbidotnlmdotnihdotgov/BLAST/). Putative target sites that
exhibit significant homology to other coding sequences are filtered
out.
[0184] Qualifying target sequences are selected as template for
siRNA synthesis. Preferred sequences are those including low G/C
content as these have proven to be more effective in mediating gene
silencing as compared to those with G/C content higher than 55%.
Several target sites are preferably selected along the length of
the target gene for evaluation. For better evaluation of the
selected siRNAs, a negative control is preferably used in
conjunction. Negative control siRNA preferably include the same
nucleotide composition as the siRNAs but lack significant homology
to the genome. Thus, a scrambled nucleotide sequence of the siRNA
is preferably used, provided it does not display any significant
homology to any other gene.
[0185] Other nucleic acid agents which can be used to downregulate
expression of sFlt-14 include but are not limited to a DNAzyme
molecule capable of specifically cleaving its encoding
polynucleotide. DNAzymes are single-stranded polynucleotides which
are capable of cleaving both single and double stranded target
sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995;
2: 655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci.
USA 1997; 94:4262); a ribozyme molecule capable of specifically
cleaving its encoding polynucleotide. Ribozymes are being
increasingly used for the sequence-specific inhibition of gene
expression by the cleavage of mRNAs encoding proteins of interest
[Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The
possibility of designing ribozymes to cleave any specific target
RNA has rendered them valuable tools in both basic research and
therapeutic applications; a triplex forming oligonucleotides
(TFOs). In the last decade, studies have shown that TFOs can be
designed which can recognize and bind to polypurine/polypirimidine
regions in double-stranded helical DNA in a sequence-specific
manner. Thus the DNA sequence encoding the polypeptide of the
present invention can be targeted thereby down-regulating the
polypeptide.
[0186] Downregulating sFlt-14 can also be effected at the protein
level.
[0187] Thus, another example of an agent capable of downregulating
a polypeptide of the present invention is an antibody or antibody
fragment capable of specifically binding sFlt-14 or a homologue
thereof, preferably to its active site, thereby preventing its
function. Methods of producing such antibodies are described
hereinabove.
[0188] Regardless of the agents employed, the effect of same on
sFlt-14 activity and/or expression (and indirectly VEGF activity or
expression) may be determined using well known molecular biology,
biochemical or cell biology techniques. The specific assay will be
selected according to the particular researcher's needs and
expertise.
[0189] As mentioned hereinabove, soluble VEGF receptors bind and
antagonize VEGF activity. Indeed, the present inventors have
further shown that sFlt-14 antagonizes VEGF (Example 9 and FIG.
10B).
[0190] Thus, according to some embodiments of the present invention
there is provided a method of treating a VEGF-associated medical
condition in which there is an excessive activity and/or expression
of VEGF (such an excessive activity or expression often results in
hyperangiogenesis). The method comprising administering to a
subject in need thereof a therapeutically effective amount of an
agent capable of upregulating sFlt-14 to thereby treat the
VEGF-associated medical condition in the subject.
[0191] Examples of medical conditions associated with excessive
activity and/or expression of VEGF include, but are not limited to,
cancer, eye disorders such as neovascularization of the cornea,
polycystic ovary disease and endometriosis.
[0192] As such, the present invention envisions use of the novel
sFlt-14 for antagonizing VEGF activity and thereby reducing
angiogenesis which may be harnessed for the treatment of VEGF
associated conditions (e.g., cancer).
[0193] As used herein the term "angiogenesis" refers to the
production or development of blood vessels.
[0194] As used herein the term "cancer" refers to any tumoral
disease including metastasis. Examples of cancer include but are
not limited to carcinoma, lymphoma, blastoma, sarcoma, and
leukemia. Particular examples of cancerous diseases but are not
limited to: Myeloid leukemia such as Chronic myelogenous leukemia.
Acute myelogenous leukemia with maturation. Acute promyelocytic
leukemia, Acute nonlymphocytic leukemia with increased basophils,
Acute monocytic leukemia. Acute myelomonocytic leukemia with
eosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's;
Lymphoctyic leukemia, such as Acute lymphoblastic leukemia. Chronic
lymphocytic leukemia; Myeloproliferative diseases, such as Solid
tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic
adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney,
Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma,
myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar),
Extraskeletel myxoid chonodrosarcoma, Ewing's tumor; other include
Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor,
Neuroblastoma, Endometrial cancer, Malignant melanoma,
Mesothelioma, breast, skin, prostate, and ovarian.
[0195] As used herein the phrase "neovascularized cornea" refers to
the abnormal, pathological condition in which the cornea becomes
vascular.
[0196] Other medical conditions, diseases and disease processes in
which angiogenesis plays a role can be treated according to the
teachings of the present invention. These include, but are not
limited to, diabetic retinopathy, neovascular glaucoma, rheumatoid
arthritis and hemangiomas.
[0197] Agents capable of upregulating the polypeptides of the
present invention, which may be used for the treatment of VEGF
associated conditions (e.g., cancer or corneal neovascularization),
comprise the isolated polypeptides per se or polynucleotides of the
present invention.
[0198] Thus, polynucleotides of the present invention can be
administered to the subject employing any suitable mode of
administration, described hereinbelow (i.e., in vivo gene therapy).
Alternatively, the nucleic acid construct is introduced into a
suitable cell via an appropriate gene delivery vehicle/method
(transfection, transduction, homologous recombination, etc.) and an
expression system as needed and then the modified cells are
expanded in culture and returned to the individual (i.e., ex vivo
gene therapy).
[0199] Such polynucleotide sequences are typically inserted into
expression vectors to enable expression of the recombinant
polypeptide. The expression vector of the present invention
includes additional sequences which render this vector suitable for
replication and integration in prokaryotes, eukaryotes, or
preferably both (e.g., shuttle vectors). Typical cloning vectors
contain transcription and translation initiation sequences (e.g.,
promoters, enhances) and transcription and translation terminators
(e.g., polyadenylation signals).
[0200] To enable cellular expression of the polynucleotides of the
present invention, the nucleic acid construct of the present
invention further includes at least one cis acting regulatory
element. As used herein, the phrase "cis acting regulatory element"
refers to a polynucleotide sequence, preferably a promoter, which
binds a trans acting regulator and regulates the transcription of a
coding sequence located downstream thereto.
[0201] Any suitable promoter sequence can be used by the nucleic
acid construct of the present invention.
[0202] Preferably, the promoter utilized by the nucleic acid
construct of the present invention is active in the specific cell
population transformed. Examples of cell type-specific and/or
tissue-specific promoters include promoters such as albumin that is
liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277],
lymphoid specific promoters [Calame et al., (1988) Adv. Immunol.
43:235-275]; in particular promoters of T-cell receptors [Winoto et
al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
(1983) Cell 33729-740], neuron-specific promoters such as the
neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci.
USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.
(1985) Science 230:912-916] or mammary gland-specific promoters
such as the milk whey promoter (U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166). The nucleic acid
construct of the present invention can further include an enhancer,
which can be adjacent or distant to the promoter sequence and can
function in up regulating the transcription therefrom.
[0203] The nucleic acid construct of the present invention may
further include an appropriate selectable marker and/or an origin
of replication. For example, the nucleic acid construct utilized
may be a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells, or integration in a gene and a tissue of choice. The
construct according to the present invention can be, for example, a
plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an
artificial chromosome.
[0204] In addition to the elements already described, the
expression vector of the present invention may typically contain
other specialized elements intended to increase the level of
expression of cloned nucleic acids or to facilitate the
identification of cells that carry the recombinant DNA. For
example, a number of animal viruses contain DNA sequences that
promote the extra chromosomal replication of the viral genome in
permissive cell types. Plasmids bearing these viral replicons are
replicated episomally as long as the appropriate factors are
provided by genes either carried on the plasmid or with the genome
of the host cell.
[0205] The vector may or may not include a eukaryotic replicon. If
a eukaryotic replicon is present, then the vector is amplifiable in
eukaryotic cells using the appropriate selectable marker. If the
vector does not comprise a eukaryotic replicon, no episomal
amplification is possible. Instead, the recombinant DNA integrates
into the genome of the engineered cell, where the promoter directs
expression of the desired nucleic acid.
[0206] Examples of mammalian expression vectors include, but are
not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-),
pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5,
DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from
Invitrogen, pCI which is available from Promega, pMbac, pPbac,
pBK-RSV and pBK-CMV which are available from Strategene, pTRES
which is available from Clontech, and their derivatives.
[0207] Expression vectors containing regulatory elements from
eukaryotic viruses such as retroviruses can be also used. SV40
vectors include pSVT7 and pMT2. Vectors derived from bovine
papilloma virus include pBV-1MTHA, and vectors derived from Epstein
Bar virus include pHEBO, and p2O5. Other exemplary vectors include
pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any
other vector allowing expression of proteins under the direction of
the SV-40 early promoter, SV-40 later promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells.
[0208] Recombinant viral vectors may also be used to synthesize the
polynucleotides of the present invention. Viruses are very
specialized infectious agents that have evolved, in many cases, to
elude host defense mechanisms. Typically, viruses infect and
propagate in specific cell types. The targeting specificity of
viral vectors utilizes its natural specificity to specifically
target predetermined cell types and thereby introduce a recombinant
gene into the infected cell. Bone marrow cells can be targeted
using the human T cell leukemia virus type I (HTLV-I).
[0209] Currently preferred in vivo nucleic acid transfer techniques
include transfection with viral or non-viral constructs, such as
adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated
virus (AAV) and lipid-based systems. Useful lipids for
lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are
viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. A viral construct such as a retroviral construct
includes at least one transcriptional promoter/enhancer or
locus-defining element(s), or other elements that control gene
expression by other means such as alternate splicing, nuclear RNA
export, or post-translational modification of messenger. Such
vector constructs also include a packaging signal, long terminal
repeats (LTRs) or portions thereof, and positive and negative
strand primer binding sites appropriate to the virus used, unless
it is already present in the viral construct. In addition, such a
construct typically includes a signal sequence for secretion of the
peptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a mammalian signal sequence or
the signal sequence of the polypeptide variants of the present
invention. Optionally, the construct may also include a signal that
directs polyadenylation, as well as one or more restriction sites
and a translation termination sequence. By way of example, such
constructs will typically include a 5' LTR, a tRNA binding site, a
packaging signal, an origin of second-strand DNA synthesis, and a
3' LTR or a portion thereof. Other vectors can be used that are
non-viral, such as cationic lipids, polylysine, and dendrimers.
[0210] Various methods can be used to introduce the expression
vector of the present invention into cells. Such methods are
generally described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989,
1992), in Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic
Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene
Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, Butterworths, Boston
Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986]
and include, for example, stable or transient transfection,
lipofection, electroporation and infection with recombinant viral
vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992
for positive-negative selection methods.
[0211] Introduction of nucleic acids by viral infection offers
several advantages over other methods such as lipofection and
electroporation, since higher transfection efficiency can be
obtained due to the infectious nature of viruses.
[0212] It will be appreciated that up-regulating the expression
and/or function of a sFlt-14 polypeptide will typically result in a
reduced VEGF activity.
[0213] The above described agents of the present invention can be
provided to the individual per se, or as part of a pharmaceutical
composition where it is mixed with a pharmaceutically acceptable
carrier.
[0214] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0215] Herein the term "active ingredient" refers to the isolated
polypeptides, the isolated polynucleotides, or the antibody
preparations, which are accountable for the biological effect.
[0216] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases. One of the
ingredients included in the pharmaceutically acceptable carrier can
be for example polyethylene glycol (PEG), a biocompatible polymer
with a wide range of solubility in both organic and aqueous media
(Mutter et al. (1979).
[0217] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0218] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0219] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, transnasal, intestinal or parenteral
delivery, including intramuscular, subcutaneous and intramedullary
injections as well as intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular
injections.
[0220] Alternately, one may administer the preparation in a local
rather than systemic manner, for example, via injection of the
preparation directly into a specific region of a patient's
body.
[0221] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0222] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0223] For injection, the active ingredients of the invention may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0224] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries if desired,
to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable
polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0225] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0226] Pharmaceutical compositions, which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0227] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0228] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0229] The preparations described herein may be formulated for
parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0230] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0231] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0232] The preparation of the present invention may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0233] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients effective to prevent,
alleviate or ameliorate symptoms of disease or prolong the survival
of the subject being treated.
[0234] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art.
[0235] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro assays. For example, a dose can be
formulated in animal models and such information can be used to
more accurately determine useful doses in humans.
[0236] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. [See
e.g., Fingl, et al., (1975) "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1].
[0237] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0238] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0239] Compositions including the preparation of the present
invention formulated in a compatible pharmaceutical carrier may
also be prepared, placed in an appropriate container, and labeled
for treatment of an indicated condition.
[0240] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0241] It is expected that during the life of a patent maturing
from this application many relevant polypeptides and
polynucleotides encoding sFlt-14 will be developed and the scope of
the term polypeptides and polynucleotides encoding sFlt-14 is
intended to include all such new technologies a priori.
[0242] As used herein the term "about" refers to .+-.10%
[0243] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to". This term encompasses the terms "consisting of" and
"consisting essentially of".
[0244] The phrase "consisting essentially of" means that the
composition or method may include additional ingredients and/or
steps, but only if the additional ingredients and/or steps do not
materially alter the basic and novel characteristics of the claimed
composition or method.
[0245] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0246] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0247] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0248] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0249] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0250] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0251] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0252] Reference is now made to the following examples, which
together with the above descriptions, illustrate some embodiments
of the invention in a non limiting fashion.
[0253] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Sequence of the Novel sFlt-14
[0254] Materials and Experimental Procedures
[0255] RNA
[0256] Placental tissue was homogenized with a Polytron homogenizer
and preceded the total RNA extraction in TRI Reagent (Sigma)
according to the manufacturer's protocol. Cells were harvested and
RNA was extracted in TRI Reagent.
[0257] RACE
[0258] Rapid Amplification of cDNA Ends was preformed using BD
SMART.TM. RACE cDNA amplification kit. 3' RACE based on
preeclamptic placental RNA was used with a primer taken from the
beginning of FLT1's exon 14 used as the 5' primer
CCTCCTGCGAAACCTCAGTG (SEQ ID NO: 12) and a 3' primer that was
supplied with the RACE kit: AAGCAGTGGTATCAACGCAGAGTAC(T).sub.30 VN
(SEQ ID NO: 11).
[0259] Results
[0260] As is illustrated in FIGS. 1-3, the novel sFlt1 of the
present invention differs from the full transmembrane receptor Flt1
and from the known sFlt1 in the following three aspects:
1) sFlt-14 does not comprise the 31 amino acids unique to sFlt-1
(derived from intron 13) as clear from FIG. 1 (marked by the light
coloring). 2) sFlt-14 contains RNA sequences, as well as an amino
acid stretch, not present in sFlt-1. The amino acid sequence
includes amino acids derived from exon 14 (which are also present
in the full transmembrane receptor, see FIG. 1) as well as a unique
amino acid sequence derived from intron 14 (marked by the light
coloring, see FIG. 1). As shown in FIG. 3A, this unique sequence
comprises a stretch of 28 amino acids derive from intron 14. 3) As
shown in FIG. 2A, sFlt-14 also contains a unique regulatory
sequence, namely a 3'-UTR fully derived from intron 14, including
an Alu element contained within its 3'-UTR.
[0261] Furthermore, as is illustrated in FIG. 2A, the cDNA sequence
that is unique to the novel sFlt-14 comprises sequences not present
in cDNAs from either the full transmembrane Flt1 or from the
soluble sFlt-1. A search throw database revealed several EST
sequences deposited in data bases where only fragments of this
unique transcript were identified (FIG. 2B). However, this
alternatively-spliced sequence variance of Flt (namely sFlt-14 of
the present invention) was not mentioned previously in the
published literature, let alone association with any tissue or
pathology.
Example 2
Generation of sFlt-14 Specific Antibodies
[0262] Materials and Experimental Procedures
[0263] Generation of sFlt-14 Specific Polyclonal Antibodies
[0264] Polyclonal antibodies were generated as described in the
Sigma-Aldrich's protocol. In short, two peptides derived from
sFlt-14 were synthesized (CHFK, SEQ ID NO: 6 and CESS, SEQ ID NO:
5) and injected into rabbits in order to produce anti sFlt-14 sera.
Three injections for each peptide were performed, with a month
period kept between injections. At the end of this procedure rabbit
serums were evaluated for sFlt-14 reactivity.
[0265] Results
[0266] Two short peptides were generated from the amino acid
sequence which distinguishes the novel sFlt-14 of the present
invention from the previously described sFlt-1. As illustrated in
FIG. 3B, the first peptide, termed CHFK (SEQ ID NO: 6), which was
derived from exon 14, is not comprised in sFlt-1 but is present in
the full transmembrane receptor. The second peptide, termed CESS
(SEQ ID NO: 5), was derived from a sequence unique only to the
novel sFlt-14 (and is not comprised in either the transmembrane
Flt-1 or sFlt-1). These two peptides were used to elicit polyclonal
antibodies as described in the experimental description
hereinabove.
[0267] The polyclonal antibodies generated can distinguish between
the novel sFlt-14 and the previously described sFlt-1.
Example 3
Relative Abundance of Transmembrane Flt-1, sFlt-1 and sFlt-14 in
Different Cells
[0268] Materials and Experimental Procedures
[0269] Cells
[0270] Cells were obtained and cultured as previously described in
Gluzman et al. [Gluzman et al., Biochem Biophys Res Commun. (2007)
359:263-8].
[0271] RNA
[0272] Normal placenta tissue and preeclampsia placenta tissue were
homogenized with a Polytron homogenizer and preceded the total RNA
extraction in TRI Reagent (Sigma) according to the manufacturer's
protocol. Cells were harvested and RNA was extracted in TRI
Reagent.
[0273] Northern Blotting
[0274] Total RNA (5-20 .mu.g) was resolved by formaldehyde--agarose
(1%) denaturing gels and blotted to positively charged nylon
membrane by capillary elution. The RNA was UV crosslinked (1200
j/m2) and the membrane was stained with 0.1% methylene blue to
ensure equal loading and transfer. Blots were hybridized overnight
with a .sup.32P-labeled probe by a rediprime kit (Amersham). The
blots were subjected to two washes (with 2.times.SSC, 1% SDS) for
30 minute at 60.degree. C., after which they were exposed to MS
sensitive film (Kodak). Three different probes from different
regions of the FLT1 transcripts were used in order to allow
detection of the various FLT1 isoforms: 1) an extra-cellular coding
region for the detection of all the isoforms; 2) an intron 14
region for the detection of sFLT-14; and 3) an intron 13 region for
the detection of sFLT1.
[0275] Results
[0276] As illustrated in FIG. 4A, RNA obtained from HUVEC, normal
placenta and preeclampsia placenta were separated on an RNA blot
and hybridized with a probe common to Flt-1, sFlt-1, and sFlt-14
(which were distinguished by the band position).
As evident from the results, in endothelial cells, known as the
`traditional` cells expressing VEGF receptors, the predominant
soluble Flt is sFlt-1 (also termed herein sFlt-13). In sharp
contrast, in placenta the predominant soluble receptor is the novel
sFlt-14 of the present invention. The identity of the respective
splice variant was further validated through re-probing with probes
specific for sFlt1 and for sFlt-14 (data not shown).
[0277] Likewise, a comparison between endothelial cells and
vascular smooth muscle cells shows that whereas endothelial cells
predominantly express sFlt-1, vascular smooth muscle cells
predominantly express the novel sFlt-14 (FIG. 4B). sFlt-14 has also
been demonstrated as the predominant, if not the exclusive, Flt
isoform present in trophoblasts and dendritic cells (data not
shown). Taken together these results indicate that sFlt-14 may be
viewed as non-endothelial cell-specific, whereas, sFlt1 as the
variant present in endothelial cells.
[0278] It is important to note that since alternative splicing
which generates the full receptor or, alternatively, the soluble
receptor (either variant) are mutually exclusive, the following
occurs: In case sFlt1 is generated (e.g. by endothelial cells), the
ratio of membrane-spanning Flt1 receptor to soluble Flt1 receptor
is high. In contrast, the alternative splicing mode which generates
sFlt-14, allows the dominancy of sFlt14 over the membrane spanning
Flt1 receptor. This results in a net transition from pro- to
anti-VEGF signaling and renders the expressing cells irresponsive
to VEGF.
Example 4
In Situ Protein and mRNA Detection of sFlt-14 in Preeclamtic
Placenta
[0279] Materials and Experimental Procedures
[0280] In Situ Hybridization
[0281] Placental paraffin embedded sections were hybridized with a
S35 riboprobe taken from the intron 14 region of sFlt-14 (SEQ ID
NO: 13) as was previously described by Motro et al. [Motro et al.,
PNAS, 1990, 87(8), 3092-6].
[0282] Immunohistochemistry
[0283] A sFlt-14 specific rabbit polyclonal antibody at a 1:100
dilution was used on paraffin embedded placental sections. Antigen
retrieval was carried out using 25 mM citrate buffer pH=6.0. The
antibody was directed against a peptide derived from the C-terminus
of the sFLT-14 protein--CELYTSTSPSSSSSS (CESS antibody, SEQ ID NO:
5).
[0284] Results
[0285] FIGS. 5A-B demonstrate the major value of the CESS antibody
directed specifically against the unique section of sFlt-14 (the
amino acids of intron 14) as illustrated in immunohistological
detection of sFlt-14 proteins in placental sections. Likewise,
FIGS. 5C-D demonstrate the importance of the unique mRNA probe
(complementary to the unique intron 14 sequence) for specific
detection of sFlt-14 mRNA as illustrated by in situ hybridization
of placental sections with the specific probe. It should be
emphasized that there is no cross-reaction of the CESS antibody or
of the probe with the full receptor Flt and the soluble sFlt-1 so
that the reagents are truly exclusive for the novel sFlt-14.
[0286] FIGS. 5A-D also provide some mechanistic insights to the
pathogenic process as these results identify, for the first time,
which cells in the diseased placenta produce the soluble receptor
(cells that were not identified using the known sFlt1). These
findings illustrate that trophoblastic cells within the syncytial
knots produce the soluble sFlt-14. These results are consistent
with the fact that syncytial knots are much more abundant in
pre-eclampsia compared to normal pregnancy, and are a hallmark of a
degenerative placenta.
Example 5
Expression of sFlt-14 in Normal Term Placentae
[0287] Materials and Experimental Procedures
[0288] Western Blotting
[0289] Three normal term placentae were homogenized and separated
into two groups: group 1) subjected to a pre immune cleaning
treatment; and group 2) not subjected to cleaning treatment. The
cleaning treatment (used to clean the sample from proteins that
might interact with the irrelevant antibodies of the CESS serum in
the detection step) included 3 hour incubation with 20 .mu.l rabbit
pre-immune serum followed by an addition of Protein A beads (P3391,
Sigma), overnight incubation and precipitation. The non-cleaned
treatment was the same, without the addition of rabbit pre-immune
serum.
[0290] Each sample, cleaned or non-cleaned, was separated into two
different immunoprecipitations, one with the CESS antibody and the
other with the FLT11 antibody (V4262, Sigma). The precipitants were
loaded on a 6% acrylamide gel, run electrophoretically, transferred
to a membrane and detected with the CESS antibody.
[0291] Results
[0292] As clearly illustrated in FIG. 6, the size of the sFLT14
protein is approximately 110 Kd. It is precipitated by the FLT11
antibody, which targets the extracellular domain of Flt-1, and
visualized by the CESS antibody, which specifically targets the C'
terminus of the novel sFlt-14, validating the existence of a novel
variant of the soluble Flt1. These results also proved that the
unique CESS epitope is an integral part of a splice variant that
includes the extracellular binding domain of Flt1.
Example 6
Mass-Spectrometry Identification of sFLT14
[0293] Materials and Experimental Procedures
[0294] Mass Spectrometry
[0295] A preeclamptic placenta was homogenized in a protein lysis
buffer, and incubated for 3 hours with 20 .mu.l rabbit pre-immune
serum (in order to clean the sample from proteins that might
interact with the irrelevant antibodies of the CESS serum). Protein
A beads (P3391, Sigma) were added for overnight incubation and
precipitated. 15 .mu.l CESS antibody was added to the cleaned
homogenate, incubated for 3 hours, followed by an addition of
protein A beads and another overnight incubation. The beads were
precipitated, washed, and boiled with sample buffer. The samples
were loaded on a 6% acrylamide gel and run electrophoretically. The
gel was stained with coomassie blue and destained till bands
appeared. A 110 Kd band was cut for mass spectrometry analysis. The
band was digested by trypsin, analyzed by LC-MS/MS on DECA/LCQ and
identified by Pep-Miner and Sequest software against nr database of
human, mouse, rat, bovine and rabbit.
[0296] Results
[0297] As depicted in FIG. 7, a positive identification of Flt1 was
achieved by three peptides taken from the extracellular domain of
Flt 1. These results proved that the extracellular domain of Flt1
and the C' terminus of the novel sFlt-14 are on the same
continuity. Eight amino-acids of the DQEAPYLLR peptide (QEAPYLLR,
SEQ ID NO. 19) are encoded by exon 14, further strengthening the
inclusion of exon 14 in the novel C' terminus.
Example 7
Presence of sFlt-14 Isoforms in Serum of Preeclamptic Subjects
[0298] Materials and Experimental Procedures
[0299] Analysis of Serum Proteins
[0300] 20 ml serum samples from PE subjects were concentrated via
capture on FLT11-coated beads and elution. Affinity-purified
proteins were analyzed by Western-blotting. Accordingly, proteins
were separated on 6% acrylamide gel, electrophoretically
transferred to a membrane, and immunoblotted with the sFlt-14
specific CESS antibody. Protein detection was carried out using
CESS antibody or the ab9540 antibody.
[0301] Results
[0302] Following the novel results illustrating that PE placentae
express upregulated levels of sFlt-14 (Examples 3 and 4
hereinabove), inventors of the present invention investigated which
sFlt isoform accumulates in the serum of PE subjects. To identify
the characteristics of circulating soluble receptors, inventors of
the present invention analyzed preeclamptic serum specimens. This
was done by affinity purifying sFlt isoforms from serum of PE
patients with the FLT11 extracellular antibody. The purified
isoforms were western blotted next to a CESS immunoprecipitate of
the placenta using the specific sFlt-14 antibody (CESS antibody).
As illustrated in FIG. 8, the same two protein bands were detected
in the placenta and serum of PE subjects. These two proteins were
previously identified as sFlt-14 proteins (Example 3, hereinabove).
Furthermore, the sFlt-14 protein detected in the serum of PE
subjects was visualized as two bands identical in size to those
produced by cells transfected with sFlt-14 expression plasmid (see
Example 10 hereinbelow) and detected with the sFlt-14-specific
antibody. A second immunoblotting using the ab9540 extracellular
targeting antibody failed to give different bands than the two
mentioned above (data not shown), thus eliminating sFlt-1 existence
in the PE serums that were tested, indicating that sFlt-14 is the
major VEGF receptor in the circulation of PE subjects.
Example 8
sFLT-14 is the Exclusive sFLT1 Isoform from the Second Trimester of
Pregnancy
[0303] Materials and Experimental Procedures
[0304] Northern Blotting
[0305] Total RNA was generated from human placental biopsies at
different time points of gestation (weeks 9-11, 13 and 39 of
gestation). 10-15 .mu.g RNA of each sample was resolved by
formaldehyde--agarose (1%) denaturing gels and blotted to
positively charged nylon membrane by capillary elution. The RNA was
UV crosslinked (1200 j/m2) and the membrane was stained with 0.1%
methylene blue to ensure equal loading and transfer. Blots were
hybridized overnight with a .sup.32P-labeled probe by a rediprime
kit (Amersham). The probe used (SEQ ID NO: 14) targeted the shared
sequences of both isoforms, thus able to show their relative
abundance. The blots were subjected to two washes (with
2.times.SSC, 1% SDS) for 30 minute at 60.degree. C., after which
they were exposed to MS sensitive film (Kodak).
[0306] Results
[0307] In order to characterize the relative contribution of each
of the sFLT1 isoforms during the normal course of gestation, RNA
was generated from human placental biopsies at different time
points during the course of normal gestation and an extracellular
probe targeting the shared sequences of both isoforms was used for
hybridization (see materials and experimental section above). As
illustrated in FIGS. 9A-B, weeks 9-11 of gestation (first
trimester) are characterized by a 1:1 ratio of the sFlt-1 and
sFlt-14 isoforms. However, at week 13 of gestation (beginning of
the second trimester) sFlt-14 becomes the dominant, if not the
exclusive isoform expressed in placentae. Furthermore, at week 39
(third trimester) sFlt-14 remains the exclusive isoform. The
exclusive expression of sFLT-14 from the second trimester of
pregnancy and onward corresponds to the fact that sFLT-14 is
significantly upregulated in preeclampsia (a condition that usually
occurs during the third trimester of pregnancy).
Example 9
sFLT-14 is a Potent VEGF Inhibitor
[0308] Materials and Experimental Procedures
[0309] Expression of Recombinant sFlt1 and sflt-14 Proteins in Hela
Cells
[0310] cDNAs encompassing the entire coding region of both soluble
receptor isoforms sFlt-14 and sFlt-1 (SEQ ID NOs: 1 and 9,
respectively) were sub-cloned into Bluescript expression vectors
and transfected into T7 polymerase-expressing human Hela cells.
20-24 hours later, growth media were collected and cells were
harvested. Secreted proteins and cell associated proteins were
immunoprecipitated with the FLT11 antibody (V4262, Sigma) and
analyzed by immunoblotting with antibodies (Ab9540, Abcam) directed
against the extracellular domain of both sFlt-1 and sFlt-14.
[0311] ELISA
[0312] Analysis of sFlt-1 and sFlt-14 secretion of into the growth
medium was carried out using ELISA directed against a shared
extracellular epitope using DVR100 (R&D systems).
[0313] Immunoprecipitation and Western Blotting
[0314] sFlt-1 and sFlt-14 secretion into the growth medium was
analyzed by immunoprecipitation with the FLT11 antibody and western
immunoblotting was carried out with the ab9540 antibody. Western
blotting was further carried out as described in Example 5.
[0315] VEGF Inhibition Assay
[0316] Porcine Aortic Endothelial (PAE) cells engineered to express
high levels of human VEGF-R2 were acquired from Prof. Gera Neufeld
(Technion, Haifa, Israel). Cells were grown in 10% FCS DMEM growth
medium.
[0317] Increasing amounts of sFlt-14 or sFlt-1 (20, 40, 80 ng/ml)
were pre-incubated with a constant amount of VEGF (20 ng/ml) prior
to adding the growth medium of PAE cells. VEGF-R2 phosphorylation
levels were measured as a function of added sFlt-14/VEGF ratio or
sFlt-1/VEGF ratio. A reduction in VEGF-R2 phosphorylation was
determined using antibodies detecting phospho-VEGF-R2
(Cell-signaling, Cat. #2478) and standardized to total VEGF-R2
protein visualized by immunoblotting with anti-VEGF-R2 antibody
(Santa Cruz Cat. SC-504).
[0318] Results
[0319] Since sFlt1 and sFlt-14 are qualitatively different proteins
(sFlt-14 contains 75 amino acids not present in sFlt1 and sFlt1
contains 31 highly-conserved amino acids not present in sFlt-14),
inventors of the present invention wished to demonstrate that
sFlt-14 is in fact a VEGF receptor capable of specifically binding
and antagonizing VEGF.
[0320] To this end, inventors generated sFlt-14 expressing human
Hela cells and, for comparison, generated sFlt1-expressing Hela
cells. ELISA analysis (directed against a shared extracellular
epitope) has indicated that the secretion of sFlt-1 and sFlt-14
into the respective growth medium was comparable (concentrations of
100-200 ng/ml were detected for both, data not presented).
Furthermore, inventors confirmed the mutually exclusive presence of
either sFlt-14 or sFlt-1 in the respective growth media by
immunoprecipitation and western blots, as evident by the apparent
molecular size of the immunoreactive protein (130 Kd and 120 Kd,
respectively, FIG. 10A).
[0321] To determine whether sFlt-14 inhibits VEGF signaling,
increasing amounts of sFlt-14 were pre-incubated with a constant
amount of VEGF (20 ng/ml) prior to adding the growth medium of
Porcine Aortic Endothelial (PAE) cells engineered to express high
levels of human VEGF-R2. VEGF-R2 phosphorylation levels were
measured as a function of added sFlt-14/VEGF ratio. As shown in
FIG. 10B, nearly complete inhibition of VEGF-R2 phosphorylation was
evident already at a 1:1 sFlt-14/VEGF ratio. Conversely, at this
ratio, sFlt1 did not significantly inhibit VEGF-R2 phosphorylation
and, in fact, inhibited VEGF-R2 phosphorylation only at higher
sFlt-1/VEGF ratios. Taken together, these results conclusively
showed that sFlt-14 is a potent inhibitor of VEGF signaling and
notably more potent than sFlt-1.
Example 10
sFlt-14 is Expressed in Human Cornea
[0322] Materials and Experimental Procedures
[0323] Immunohistochemistry
[0324] Corneal sections were isolated from human corneas that were
removed due to a diseased state. A sFlt-14 specific rabbit
polyclonal antibody at a 1:100 dilution was used on paraffin
embedded corneal sections. Antigen retrieval was carried out using
25 mM citrate buffer pH=6.0. The antibody was directed against a
peptide derived from the C-terminus of the sFlt-14
protein--CELYTSTSPSSSSSS (CESS antibody, SEQ ID NO: 5).
[0325] Results
[0326] It is well known that sFlt-1 plays a major physiological
role in the cornea were it a crucial anti-VEGF factor, keeping the
cornea avascular, a state which is imperative for clear vision. In
mammals, sFlt-1 is expressed by the epithelia of the cornea. In
order for the inventors of the present invention to examine whether
sFlt-14 is also expressed in the human cornea, specific sFlt-14
antibodies were used for immunohistochemistry of human corneal
sections. As clear from FIG. 11A-B, sFlt-14 is highly expressed in
the corneal epithelia. The presence of sFlt-14 in the human corneal
epithelia was further validated by sFlt-14 PCR analysis of several
epithelia samples isolated from human corneas (data not shown).
[0327] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0328] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
1912202DNAHomo sapiens 1atggtcagct actgggacac cggggtcctg ctgtgcgcgc
tgctcagctg tctgcttctc 60acaggatcta gttcaggttc aaaattaaaa gatcctgaac
tgagtttaaa aggcacccag 120cacatcatgc aagcaggcca gacactgcat
ctccaatgca ggggggaagc agcccataaa 180tggtctttgc ctgaaatggt
gagtaaggaa agcgaaaggc tgagcataac taaatctgcc 240tgtggaagaa
atggcaaaca attctgcagt actttaacct tgaacacagc tcaagcaaac
300cacactggct tctacagctg caaatatcta gctgtaccta cttcaaagaa
gaaggaaaca 360gaatctgcaa tctatatatt tattagtgat acaggtagac
ctttcgtaga gatgtacagt 420gaaatccccg aaattataca catgactgaa
ggaagggagc tcgtcattcc ctgccgggtt 480acgtcaccta acatcactgt
tactttaaaa aagtttccac ttgacacttt gatccctgat 540ggaaaacgca
taatctggga cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa
600gaaatagggc ttctgacctg tgaagcaaca gtcaatgggc atttgtataa
gacaaactat 660ctcacacatc gacaaaccaa tacaatcata gatgtccaaa
taagcacacc acgcccagtc 720aaattactta gaggccatac tcttgtcctc
aattgtactg ctaccactcc cttgaacacg 780agagttcaaa tgacctggag
ttaccctgat gaaaaaaata agagagcttc cgtaaggcga 840cgaattgacc
aaagcaattc ccatgccaac atattctaca gtgttcttac tattgacaaa
900atgcagaaca aagacaaagg actttatact tgtcgtgtaa ggagtggacc
atcattcaaa 960tctgttaaca cctcagtgca tatatatgat aaagcattca
tcactgtgaa acatcgaaaa 1020cagcaggtgc ttgaaaccgt agctggcaag
cggtcttacc ggctctctat gaaagtgaag 1080gcatttccct cgccggaagt
tgtatggtta aaagatgggt tacctgcgac tgagaaatct 1140gctcgctatt
tgactcgtgg ctactcgtta attatcaagg acgtaactga agaggatgca
1200gggaattata caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa
cctcactgcc 1260actctaattg tcaatgtgaa accccagatt tacgaaaagg
ccgtgtcatc gtttccagac 1320ccggctctct acccactggg cagcagacaa
atcctgactt gtaccgcata tggtatccct 1380caacctacaa tcaagtggtt
ctggcacccc tgtaaccata atcattccga agcaaggtgt 1440gacttttgtt
ccaataatga agagtccttt atcctggatg ctgacagcaa catgggaaac
1500agaattgaga gcatcactca gcgcatggca ataatagaag gaaagaataa
gatggctagc 1560accttggttg tggctgactc tagaatttct ggaatctaca
tttgcatagc ttccaataaa 1620gttgggactg tgggaagaaa cataagcttt
tatatcacag atgtgccaaa tgggtttcat 1680gttaacttgg aaaaaatgcc
gacggaagga gaggacctga aactgtcttg cacagttaac 1740aagttcttat
acagagacgt tacttggatt ttactgcgga cagttaataa cagaacaatg
1800cactacagta ttagcaagca aaaaatggcc atcactaagg agcactccat
cactcttaat 1860cttaccatca tgaatgtttc cctgcaagat tcaggcacct
atgcctgcag agccaggaat 1920gtatacacag gggaagaaat cctccagaag
aaagaaatta caatcagaga tcaggaagca 1980ccatacctcc tgcgaaacct
cagtgatcac acagtggcca tcagcagttc caccacttta 2040gactgtcatg
ctaatggtgt ccccgagcct cagatcactt ggtttaaaaa caaccacaaa
2100atacaacaag agcctgaact gtatacatca acgtcaccat cgtcatcgtc
atcatcacca 2160ttgtcatcat catcatcatc gtcatcatca tcatcatcat ag
22022733PRTHomo sapiens 2Met Val Ser Tyr Trp Asp Thr Gly Val Leu
Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser
Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly
Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln
Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met
Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80
Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85
90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala
Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr
Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu
Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210
215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro
Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys
Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp
Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg
Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser
Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu
Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser
Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330
335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser
340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu
Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser
Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp
Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu
Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr
Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val
Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg
Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455
460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys
465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp
Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln
Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr
Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys
Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser
Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn
Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575
Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580
585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln
Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu
Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala
Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu
Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu Ala Pro Tyr
Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala Ile Ser Ser
Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro
Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700
Pro Glu Leu Tyr Thr Ser Thr Ser Pro Ser Ser Ser Ser Ser Ser Pro 705
710 715 720 Leu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 725
730 3233DNAHomo sapiensmisc_featureNucleic acid sequence encoding a
unique 28 a.a. polypeptide portion of sFlt-14 3atcaggaagc
accatacctc ctgcgaaacc tcagtgatca cacagtggcc atcagcagtt 60ccaccacttt
agactgtcat gctaatggtg tccccgagcc tcagatcact tggtttaaaa
120acaaccacaa aatacaacaa gagcctgaac tgtatacatc aacgtcacca
tcgtcatcgt 180catcatcacc attgtcatca tcatcatcat cgtcatcatc
atcatcatca tag 233428PRTHomo sapiensmisc_featureA unique 28 a.a.
polypeptide portion of sFlt-14 4Glu Leu Tyr Thr Ser Thr Ser Pro Ser
Ser Ser Ser Ser Ser Pro Leu 1 5 10 15 Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser 20 25 515PRTArtificial sequenceA synthetic
peptide used to generate a specific sFlt-14 antibody (CESS) 5Cys
Glu Leu Tyr Thr Ser Thr Ser Pro Ser Ser Ser Ser Ser Ser 1 5 10 15
615PRTArtificial sequenceA synthetic peptide used to generate a
non-specific sFlt-14 antibody (CHFK) 6Cys His Ala Asn Gly Val Pro
Glu Pro Gln Ile Thr Trp Phe Lys 1 5 10 15 7712DNAHomo
sapiensmisc_featureA portion of sFlt-14 starting from exon 14 to
the poly A tail 7cctcctgcga aacctcagtg atcacacagt ggccatcagc
agttccacca ctttagactg 60tcatgctaat ggtgtccccg agcctcagat cacttggttt
aaaaacaacc acaaaataca 120acaagagcct gaactgtata catcaacgtc
accatcgtca tcgtcatcat caccattgtc 180atcatcatca tcatcgtcat
catcatcatc atcatagcta tcatcattat catcatcatc 240atcatcatca
tcatagctac catttattga aaactattat gtgtcaactt caaagaactt
300atcctttagt tggagagcca agacaatcat aacaataaca aatggccggg
catggtggct 360cacgcctgta atcccagcac tttgggaggc caaggcaggt
ggatcatttg aggtcaggag 420tccaagacca gcctgaccaa gatggtgaaa
tgctgtctct attaaaaata caaaattagc 480caggcatggt ggctcatgcc
tgtaatgcca gctactcggg aggctgagac aggagaatca 540cttgaaccca
ggaggcagag gttgcaggga gccgagatcg tgtactgcac tccagcctgg
600gcaacaagag cgaaactccg tctcaaaaaa caaataaata aataaataaa
taaacagaca 660aaattcactt tttattctat taaacttaac atacatgcta
aaaaaaaaaa aa 712830DNAHomo sapiensmisc_featuresFlt-14 specific
bridging region 8aaattacaat cagagatcag gaagcaccat 3092065DNAHomo
sapiens 9atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg
tctgcttctc 60acaggatcta gttcaggttc aaaattaaaa gatcctgaac tgagtttaaa
aggcacccag 120cacatcatgc aagcaggcca gacactgcat ctccaatgca
ggggggaagc agcccataaa 180tggtctttgc ctgaaatggt gagtaaggaa
agcgaaaggc tgagcataac taaatctgcc 240tgtggaagaa atggcaaaca
attctgcagt actttaacct tgaacacagc tcaagcaaac 300cacactggct
tctacagctg caaatatcta gctgtaccta cttcaaagaa gaaggaaaca
360gaatctgcaa tctatatatt tattagtgat acaggtagac ctttcgtaga
gatgtacagt 420gaaatccccg aaattataca catgactgaa ggaagggagc
tcgtcattcc ctgccgggtt 480acgtcaccta acatcactgt tactttaaaa
aagtttccac ttgacacttt gatccctgat 540ggaaaacgca taatctggga
cagtagaaag ggcttcatca tatcaaatgc aacgtacaaa 600gaaatagggc
ttctgacctg tgaagcaaca gtcaatgggc atttgtataa gacaaactat
660ctcacacatc gacaaaccaa tacaatcata gatgtccaaa taagcacacc
acgcccagtc 720aaattactta gaggccatac tcttgtcctc aattgtactg
ctaccactcc cttgaacacg 780agagttcaaa tgacctggag ttaccctgat
gaaaaaaata agagagcttc cgtaaggcga 840cgaattgacc aaagcaattc
ccatgccaac atattctaca gtgttcttac tattgacaaa 900atgcagaaca
aagacaaagg actttatact tgtcgtgtaa ggagtggacc atcattcaaa
960tctgttaaca cctcagtgca tatatatgat aaagcattca tcactgtgaa
acatcgaaaa 1020cagcaggtgc ttgaaaccgt agctggcaag cggtcttacc
ggctctctat gaaagtgaag 1080gcatttccct cgccggaagt tgtatggtta
aaagatgggt tacctgcgac tgagaaatct 1140gctcgctatt tgactcgtgg
ctactcgtta attatcaagg acgtaactga agaggatgca 1200gggaattata
caatcttgct gagcataaaa cagtcaaatg tgtttaaaaa cctcactgcc
1260actctaattg tcaatgtgaa accccagatt tacgaaaagg ccgtgtcatc
gtttccagac 1320ccggctctct acccactggg cagcagacaa atcctgactt
gtaccgcata tggtatccct 1380caacctacaa tcaagtggtt ctggcacccc
tgtaaccata atcattccga agcaaggtgt 1440gacttttgtt ccaataatga
agagtccttt atcctggatg ctgacagcaa catgggaaac 1500agaattgaga
gcatcactca gcgcatggca ataatagaag gaaagaataa gatggctagc
1560accttggttg tggctgactc tagaatttct ggaatctaca tttgcatagc
ttccaataaa 1620gttgggactg tgggaagaaa cataagcttt tatatcacag
atgtgccaaa tgggtttcat 1680gttaacttgg aaaaaatgcc gacggaagga
gaggacctga aactgtcttg cacagttaac 1740aagttcttat acagagacgt
tacttggatt ttactgcgga cagttaataa cagaacaatg 1800cactacagta
ttagcaagca aaaaatggcc atcactaagg agcactccat cactcttaat
1860cttaccatca tgaatgtttc cctgcaagat tcaggcacct atgcctgcag
agccaggaat 1920gtatacacag gggaagaaat cctccagaag aaagaaatta
caatcagagg tgagcactgc 1980aacaaaaagg ctgttttctc tcggatctcc
aaatttaaaa gcacaaggaa tgattgtacc 2040acacaaagta atgtaaaaca ttaaa
206510687PRTHomo sapiens 10Met Val Ser Tyr Trp Asp Thr Gly Val Leu
Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser
Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly
Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln
Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met
Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80
Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85
90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala
Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr
Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr
Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu
Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr
Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210
215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro
Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys
Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp
Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg
Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser
Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu
Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser
Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330
335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser
340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu
Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser
Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp
Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu
Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr
Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val
Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg
Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455
460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys
465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp
Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln
Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr
Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys
Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser
Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn
Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575
Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580
585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln
Lys 595
600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile
Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg
Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys
Lys Glu Ile Thr Ile Arg 645 650 655 Gly Glu His Cys Asn Lys Lys Ala
Val Phe Ser Arg Ile Ser Lys Phe 660 665 670 Lys Ser Thr Arg Asn Asp
Cys Thr Thr Gln Ser Asn Val Lys His 675 680 685 1157DNAArtificial
sequenceRACE kit 3' oligonucleotide 11aagcagtggt atcaacgcag
agtacttttt tttttttttt tttttttttt tttttvn 571220DNAHomo sapiens
12cctcctgcga aacctcagtg 2013211DNAArtificial sequenceIntron 14
specific probe 13aactgtatac atcaacgtca ccatcgtcat cgtcatcatc
accattgtca tcatcatcat 60catcgtcatc atcatcatca tcatagctat catcattatc
atcatcatca tcatcatcat 120catagctacc atttattgaa aactattatg
tgtcaacttc aaagaactta tcctttagtt 180ggagagccaa gacaatcata
acaataacaa a 21114680DNAArtificial sequencesFLT probe specific for
both isoforms 14tcaccatggt cagctactgg gacaccgggg tcctgctgtg
cgcgctgctc agctgtctgc 60ttctcacagg atctagttca ggttcaaaat taaaagatcc
tgaactgagt ttaaaaggca 120cccagcacat catgcaagca ggccagacac
tgcatctcca atgcaggggg gaagcagccc 180ataaatggtc tttgcctgaa
atggtgagta aggaaagcga aaggctgagc ataactaaat 240ctgcctgtgg
aagaaatggc aaacaattct gcagtacttt aaccttgaac acagctcaag
300caaaccacac tggcttctac agctgcaaat atctagctgt acctacttca
aagaagaagg 360aaacagaatc tgcaatctat atatttatta gtgatacagg
tagacctttc gtagagatgt 420acagtgaaat ccccgaaatt atacacatga
ctgaaggaag ggagctcgtc attccctgcc 480gggttacgtc acctaacatc
actgttactt taaaaaagtt tccacttgac actttgatcc 540ctgatggaaa
acgcataatc tgggacagta gaaagggctt catcatatca aatgcaacgt
600acaaagaaat agggcttctg acctgtgaag caacagtcaa tgggcatttg
tataagacaa 660actatctcac acatcgacaa 6801514PRTHomo
sapiensmisc_featureAntigenic bridging region 15His Lys Ile Gln Gln
Glu Pro Glu Leu Tyr Thr Ser Thr Ser 1 5 10 161338PRTHomo
sapiensmisc_featureMass spec identified protein 16Met Val Ser Tyr
Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu
Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30
Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35
40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu
Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr
Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr
Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr
Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu
Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg
Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His
Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165
170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly
Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu
Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn
Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln
Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His
Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr
Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys
Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285
Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290
295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe
Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala
Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr
Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys
Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr
Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly
Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410
415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu
420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu
Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro
Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn
His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu
Glu Ser Ser Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg
Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys
Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile
Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535
540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His
545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu
Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val
Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His
Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His
Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln
Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr
Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655
Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660
665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val
Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile
Gln Gln Glu 690 695 700 Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr
Leu Phe Ile Glu Arg 705 710 715 720 Val Thr Glu Glu Asp Glu Gly Val
Tyr His Cys Lys Ala Thr Asn Gln 725 730 735 Lys Gly Ser Val Glu Ser
Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser 740 745 750 Asp Lys Ser Asn
Leu Glu Leu Ile Thr Leu Thr Cys Thr Cys Val Ala 755 760 765 Ala Thr
Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys Met Lys 770 775 780
Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp 785
790 795 800 Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro
Tyr Asp 805 810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys
Leu Gly Lys Ser 820 825 830 Leu Gly Arg Gly Ala Phe Gly Lys Val Val
Gln Ala Ser Ala Phe Gly 835 840 845 Ile Lys Lys Ser Pro Thr Cys Arg
Thr Val Ala Val Lys Met Leu Lys 850 855 860 Glu Gly Ala Thr Ala Ser
Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys 865 870 875 880 Ile Leu Thr
His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly 885 890 895 Ala
Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905
910 Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe
915 920 925 Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys
Glu Lys 930 935 940 Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg
Leu Asp Ser Val 945 950 955 960 Thr Ser Ser Glu Ser Phe Ala Ser Ser
Gly Phe Gln Glu Asp Lys Ser 965 970 975 Leu Ser Asp Val Glu Glu Glu
Glu Asp Ser Asp Gly Phe Tyr Lys Glu 980 985 990 Pro Ile Thr Met Glu
Asp Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005 Gly Met
Glu Phe Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020
Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025
1030 1035 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp
Tyr 1040 1045 1050 Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp
Met Ala Pro 1055 1060 1065 Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr
Lys Ser Asp Val Trp 1070 1075 1080 Ser Tyr Gly Val Leu Leu Trp Glu
Ile Phe Ser Leu Gly Gly Ser 1085 1090 1095 Pro Tyr Pro Gly Val Gln
Met Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110 Arg Glu Gly Met
Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125 Ile Tyr
Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140
Arg Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145
1150 1155 Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile
Asn 1160 1165 1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser
Thr Pro Ala 1175 1180 1185 Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile
Ser Ala Pro Lys Phe 1190 1195 1200 Asn Ser Gly Ser Ser Asp Asp Val
Arg Tyr Val Asn Ala Phe Lys 1205 1210 1215 Phe Met Ser Leu Glu Arg
Ile Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230 Asn Ala Thr Ser
Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245 Leu Leu
Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260
Lys Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265
1270 1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe
Cys 1280 1285 1290 His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg
Arg Phe Thr 1295 1300 1305 Tyr Asp His Ala Glu Leu Glu Arg Lys Ile
Ala Cys Cys Ser Pro 1310 1315 1320 Pro Pro Asp Tyr Asn Ser Val Val
Leu Tyr Ser Thr Pro Pro Ile 1325 1330 1335 1716PRTArtificial
sequenceMass spec sequenced peptide 17Ala Val Ser Ser Phe Pro Asp
Pro Ala Leu Tyr Pro Leu Gly Ser Arg 1 5 10 15 1811PRTArtificial
sequenceMass spec sequenced peptide 18Asn Val Tyr Thr Gly Glu Glu
Ile Leu Gln Lys 1 5 10 199PRTArtificial sequenceMass spec sequenced
peptide 19Asp Gln Glu Ala Pro Tyr Leu Leu Arg 1 5
* * * * *
References