U.S. patent application number 10/298794 was filed with the patent office on 2003-07-03 for novel nucleic acid and amino acid sequences.
This patent application is currently assigned to Compugen Ltd.. Invention is credited to Engel, Sharon, Mintz, Liat, Savitzky, Kinneret.
Application Number | 20030125295 10/298794 |
Document ID | / |
Family ID | 26323799 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030125295 |
Kind Code |
A1 |
Mintz, Liat ; et
al. |
July 3, 2003 |
Novel nucleic acid and amino acid sequences
Abstract
The invention concerns novel nucleic acid sequences and amino
acid sequences of a novel variant of vascular endothelial growth
factor (VEGF). The invention further concerns expression vectors
and host cells containing said sequences as well as pharmaceutical
compositions and detection methods using said sequences.
Inventors: |
Mintz, Liat; (Ramat
Hasharon, IL) ; Savitzky, Kinneret; (Tel Aviv,
IL) ; Engel, Sharon; (Ramat Hasharon, IL) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Compugen Ltd.
|
Family ID: |
26323799 |
Appl. No.: |
10/298794 |
Filed: |
November 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10298794 |
Nov 19, 2002 |
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09884050 |
Jun 20, 2001 |
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09884050 |
Jun 20, 2001 |
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09519476 |
Mar 6, 2000 |
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6506884 |
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Current U.S.
Class: |
514/44A ;
435/320.1; 435/6.16; 536/23.5 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 38/00 20130101; C07K 14/52 20130101 |
Class at
Publication: |
514/44 ;
435/320.1; 536/23.5; 435/6 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04; C12N 015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 1999 |
IL |
128852 |
Claims
1. An isolated nucleic acid molecule consisting essentially of a
sequence selected from: (i) a nucleic acid sequence coding the
amino acid sequence of SEQ ID NO:2; (ii) a nucleic acid sequence
that is complementary to the nucleic acid sequence of SEQ ID NO:1;
(iii) a nucleic acid sequence of SEQ ID NO:1.
2. An expression vector comprising a nucleic acid molecule of claim
1 and control elements for expression of the nucleic acid molecule
in a suitable host cell.
3. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and, as an active ingredient, an expression
vector comprising (i) a nucleic acid molecule of claim 1
operatively linked to (ii) a control element for the expression of
said nucleic acid molecule in a host cell within a treated
individual.
4. The pharmaceutical composition of claim 3, in which the nucleic
acid molecule (i) is oriented in the antisense direction.
5. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and, as an active ingredient, the nucleic acid
molecule (i) of claim 1.
6. A method for treatment of a disease in an individual, which
disease can be ameliorated or cured by raising the level of a
vascular endothelial growth factor variant (VEGFV) product,
comprising administering to the individual a pharmaceutical
composition of claim 3.
7. A method for treatment of a disease in an individual, which
disease can be ameliorated or cured by raising the level of a
vascular endothelial growth factor variant (VEGFV) product,
comprising administering to the individual a pharmaceutical
composition of claim 5.
8. A method for treatment of a disease in an individual, which
disease can be ameliorated or cured by decreasing the level of the
vascular endothelial growth factor variant (VEGFV) product,
comprising administering to the individual a pharmaceutical
composition of claim 4.
9. A method for treatment of a disease in an individual, which
disease can be ameliorated or cured by decreasing the level of the
vascular endothelial growth factor variant (VEGFV) product,
comprising administering to the individual a pharmaceutical
composition of claim 5.
10. A method for detecting a Vascular Endothelial Growth Factor
Variant nucleic acid sequence in a biological sample, comprising:
(a) contacting a probe nucleic acid comprising a nucleic acid
sequence of claim 1, with the biological sample and applying
conditions such that said probe nucleic acid will hybridize to
complementary nucleic acids if present in said sample; and (b)
detecting a hybridization complex.
11. The method of claim 10, wherein said biological sample
comprises mRNA transcripts.
12. The method of claim 10, wherein the probe nucleic acid sequence
is immobilized.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns novel nucleic acid sequences,
vectors and host cells containing them, amino acid sequences
encoded by said sequences, and antibodies reactive with said amino
acid sequences, as well as pharmaceutical compositions comprising
any of the above. The present invention further concerns methods
for screening for candidate activator or deactivators utilizing
said amino acid sequences.
BACKGROUND OF THE INVENTION
[0002] Vascular endothelial growth factor (VEGF) is a
heparin-binding growth factor specific for vascular endothelial
cells that is able to induce angiogenesis in vivo. DNA sequencing
suggests the existence of several molecular species of VEGF. VEGFs
are secreted proteins in contrast to other endothelial cell
mitogens such as acidic or basic fibroblast growth factors and
platelet-derived endothelial cell growth factors. VEGF was found to
augment human growth by inducing neovascularization. Thus it was
suggested that neutralization of VEGF activity may have clinical
application in inhibiting malignant cells-induced angiogenesis,
decreasing blood supply to the cancerous tissue, leading eventually
to its destruction. VEGF has various other functions on endothelial
cells, the most prominent of which is the induction of
proliferation and differentiation. It was found to be capable of
preventing serum starvation-induced apoptosis and this inhibition
may represent a major aspect of the regulatory activity of VEGF on
vascular endothelium.
[0003] VEGF was also found to be involved in the development and
the growth of ovarian corpus luteum (CL), since its development is
dependent on the growth of new capillary vessels. It has been
reported that Flt-1 receptors which inhibit vascular endothelial
growth factor bioactivity, resulted in complete separation of
corpus luteum angiogenesis in a rat model of hormonally induced
ovulation, indicated that VEGF is essential for CL angiogenesis and
may be involved in the control of fertility and treatment of
ovarian disorders characterized by hypervascularity and
hyperplasia.
[0004] The human VEGF gene has been recently assigned to chromosome
6p21.2. cDNA sequence analysis of a variety of human VEGF clones
had initially indicated that VEGF may exist as one of four
different molecular species, having respectively, 121, 165, 189 and
206 amino acids (VEGF.sub.121, VEGF.sub.165, BEGF.sub.206).
Alternative exon splicing of a single VEGF gene is the basis for
this molecular heterogeneity, VEGF.sub.165 lacks the residues
encoded by exon 6, while VEGF .sub.121 lacks the residues encoded
by exons 6 and 7. VEGF.sub.189 has an insertion of 24 amino acids
highly enriched in basic residues and VEGF.sub.206 has an
additional insertion of 17 amino acids. VEGF.sub.165 is the
predominant isoform secreted by a variety of normal and transformed
cells. Transcripts encoding VEGF.sub.121 and VEGF.sub.189 are
detected in the majority of cells and tissues expressing the VEGF
gene. In contrast, VEGF.sub.206 is a very rare form.
[0005] Native VEGF is a basic, heparin-binding, homodimeric
glycoprotein of 45 kDA. These properties correspond to those of
VEGF.sub.165. VEGF.sub.121 is an acidic polypeptide that fails to
bind to heparin. VEGF.sub.189 and VEGF.sub.200 are more basic and
bind to heparin with greater affinity than VEGF.sub.165.
VEGF.sub.121 is a freely soluble protein; VEGF.sub.165 is also
secreted, although a significant fraction remains bound to the cell
surface and the extracellular matrix (ECM). VEGF.sub.189 and
VEGF.sub.206 are almost completely sequestered in the ECM, but may
be released in a soluble form by heparin or heparinase. Also, these
long forms may be released by plasmin following cleavage at the
COOH terminus (Ferrara, N., European J of Cancer, 32A(14):2413-2422
(1996)).
[0006] Glossary
[0007] In the following description and claims use will be made, at
times, with a variety of terms, and the meaning of such terms as
they should be construed in accordance with the invention is as
follows:
[0008] "Vascular endothelial growth factor variant (VEGFV) nucleic
acid sequence"--the sequence shown in SEQ ID NO: 1, sequences
having at least 70% identity to said sequence and fragments of the
above sequences of least 20 b.p. long. This sequence is a sequence
coding for a novel alternative splice variant of the native VEGF.
While the known VEGF peptides include 206, 189, 105 or 121 amino
acids, the novel VEGF variant peptide of the invention includes
only 141 amino acids,--27 of which being in the signal peptide and
114 being present in the mature protein. According to the
terminology used in the publication of Ferrara (supra) this new
variant should be termed VEGF.sub.114.
[0009] "Vascular endothelial growth factor variant (VEGFV
product)--also referred at times as the "VEGFV protein" or "VEGFV
polypeptide"--is an amino acid sequence having the first 141 amino
acids of the native VEGF. This naturally occurring sequence is the
result of alternative splicing. The amino acid sequence may be a
peptide, a protein, as well as peptides or proteins having
chemically modified amino acids (see below) such as a glycopeptide
or glycoprotein. An example of an VEGFV product is shown in SEQ ID
NO: 2. The term also includes analogues of said sequences in which
one or more amino acids has been added, deleted, substituted (see
below) or chemically modified (see below) as well as fragments of
this sequence having at least 10 amino acids.
[0010] "Nucleic acid sequence"--a sequence composed of DNA
nucleotides, RNA nucleotides or a combination of both types and may
includes natural nucleotides, chemically modified nucleotides and
synthetic nucleotides.
[0011] "Amino acid sequence"--a sequence composed of any one of the
20 naturally appearing amino acids, amino acids which have been
chemically modified (see below), or composed of synthetic amino
acids.
[0012] "Fragment of VEGFV product"--a polypeptide which has an
amino acid sequence which is the same as part of but not all of the
amino acid sequence of the VEGFV product.
[0013] "Fragments of VEGFV nucleic acid sequence" a continuous
portion, preferably of about 20 nucleic acid sequences of the VEGFV
nucleic acid sequence.
[0014] "Conservative substitution"--refers to the substitution of
an amino acid in one class by an amino acid of the same class,
where a class is defined by common physicochemical amino acid side
chain properties and high substitution frequencies in homologous
proteins found in nature, as determined, for example, by a standard
Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general
classes of amino acid side chains have been categorized and
include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class
III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile,
Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,
substitution of an Asp for another class III residue such as Asn,
Gln, or Glu, is a conservative substitution.
[0015] "Non-conservative substitution"--refers to the substitution
of an amino acid in one class with an amino acid from another
class; for example, substitution of an Ala, a class II residue,
with a class III residue such as Asp, Asn, Glu, or Gln.
[0016] "Cliemically modified"--when referring to the product of the
invention, means a product (protein) where at least one of its
amino acid resides is modified either by natural processes, such as
processing or other post-translational modifications, or by
chemical modification techniques which are well known in the art.
Among the numerous known modifications typical, but not exclusive
examples include: acetylation, acylation, amidation,
ADP-ribosylation, glycosylation, GPI anchor formation, covalent
attachment of a lipid or lipid derivative, methylation,
myristlyation, pegylation, prenylation, phosphorylation,
ubiqutination, or any similar process.
[0017] "Biologically active"--refers to the VEGFV product having
structural, regulatory or biochemical functions of the naturally
occurring VEGFV product, for example the same effect on vascular
endothelial cells.
[0018] "Immunologically active" defines the capability of a
natural, recombinant or synthetic VEGFV product, or any fragment
thereof, to induce a specific immune response in appropriate
animals or cells and to bind with specific antibodies. Thus, for
example, a biologically active fragment of VEGFV product denotes a
fragment which retains some or all of the immunological properties
of the VEGFV product, e.g can bind specific anti-VEGFV product
antibodies or which can elicit an immune response which will
generate such antibodies or cause proliferation of specific immune
cells which produce VEGFV.
[0019] "Optimal alignment"--is defined as an alignment giving the
highest percent identity score. Such alignment can be performed
using a variety of commercially available sequence analysis
programs, such as the local alignment program LALIGN using a ktup
of 1, default parameters and the default PAM. A preferred alignment
is the one performed using the CLUSTAL-W program from MacVector
(TM), operated with an open gap penalty of 10.0, an extended gap
penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to
be inserted into a first sequence to optimally align it with a
second sequence, the percent identity is calculated using only the
residues that are paired with a corresponding amino acid residue
(i.e., the calculation does not consider residues in the second
sequences that are in the "gap" of the first sequence).
[0020] "Having at least X % identity"--with respect to two amino
acid or nucleic acid sequence sequences, refers to the percentage
of residues that are identical in the two sequences when the
sequences are optimally aligned. Thus, 70% amino acid sequence
identity means that 70% of the amino acids in two or more optimally
aligned polypeptide sequences are identical.
[0021] "Isolated nucleic acid molecule having an VEGFV nucleic acid
sequence"--is a nucleic acid molecule that includes the coding
VEGFV nucleic acid sequence. Said isolated nucleic acid molecule
may include the VEGFV nucleic acid sequence as an independent
insert; may include the VEGFV nucleic acid sequence fused to an
additional coding sequences, encoding together a fusion protein in
which the VEGFV coding sequence is the dominant coding sequence
(for example, the additional coding sequence may code for a signal
peptide); the VEGFV nucleic acid sequence may be in combination
with non-coding sequences, e.g., introns or control elements, such
as promoter and terminator elements or 5' and/or 3' untranslated
regions, effective for expression of the coding sequence in a
suitable host; or may be a vector in which the VEGFV protein coding
sequence is a heterologous.
[0022] "Expression vector"--refers to vectors that have the ability
to incorporate and express heterologous DNA fragments in a foreign
cell. Many prokaryotic and eukaryotic expression vectors are known
and/or commercially available. Selection of appropriate expression
vectors is within the knowledge of those having skill in the
art.
[0023] "Deletion"--is a change in either nucleotide or amino acid
sequence in which one or more nucleotides or amino acid residues,
respectively, are absent.
[0024] "Insertion" or "addition"--is that change in a nucleotide or
amino acid sequence which has resulted in the addition of one or
more nucleotides or amino acid residues, respectively, as compared
to the naturally occurring sequence.
[0025] "Substitution"--replacement of one or more nucleotides or
amino acids by different nucleotides or amino acids, respectively.
As regards amino acid sequences the substitution may be
conservative or non-conservative.
[0026] "Antibody"--refers to IgG, IgM, IgD, IgA, and IgG antibody.
The definition includes polyclonal antibodies or monoclonal
antibodies. This term refers to whole antibodies or fragments of
the antibodies comprising the antigen-binding domain of the
anti-VEGFV product antibodies, e.g. antibodies without the Fe
portion, single chain antibodies, fragments consisting of
essentially only the variable, antigen-binding domain of the
antibody, etc.
[0027] "Activator"--as used herein, refers to a molecule which
mimics the effect of the natural VEGFV product or at times even
increases or prolongs the duration of the biological activity of
said product, as compared to that induced by the natural product.
The mechanism may be by binding to the VEGFV receptor, by
prolonging the lifetime of the VEGFV, by increasing the activity of
the VEGFV on its target (vascular endothelial cells), by increasing
the affinity of VEGFV to its receptor, etc. Activators may be
polypeptides, nucleic acids, carbohydrates, lipids, or derivatives
thereof, or any other molecules which can bind to and activate the
VEGFV product.
[0028] "Deactivator" or ("Inhibitor")--refers to a molecule which
modulates the activity of the VEGFV product in an opposite manner
to that of the activator, by decreasing or shortening the duration
of the biological activity of the VEGFV product. This may be done
by blocking the binding of the VEGFV to its receptor (competitive
or non-competitive inhibition), by causing rapid degradation of the
VEGFV, etc. Deactivators may be polypeptides, nucleic acids,
carbohydrates, lipids, or derivatives thereof, or any other
molecules which bind to and modulate the activity of said
product.
[0029] "Treating a disease"--refers to administering a therapeutic
substance effective to ameliorate symptoms associated with a
disease, to lessen the severity or cure the disease, or to prevent
the disease from occurring.
[0030] "Detection"--refers to a method of detection of a disease.
This term may refer to detection of a predisposition to a
disease.
[0031] "Probe"--the VEGFV nucleic acid sequence, or a sequence
complementary therewith, when used to detect presence of other
similar sequences in a sample. The detection is carried out by
identification of hybridization complexes between the probe and the
assayed sequence. The probe may be attached to a solid support or
to a detectable label.
SUMMARY OF THE INVENTION
[0032] The present invention is based on the surprising finding
that there exist in humans a novel variant of the VEGF protein,
having 141 amino acid (114 amino acids of the mature protein
without the signal peptide) than the known VEGF. The nucleic
sequence coding for this variant was identified as being from the
same locus as the known VEGF and thus it was concluded that the
variant is not encoded from a different gene than the known VEGF,
but is the result of alternative splicing of the known VEGF.
[0033] Thus the present invention provides by its first aspect, a
novel isolated nucleic acid molecule comprising or consisting of
the coding sequence SEQ ID NO: 1, fragments of said coding sequence
having at least 20 nucleic acids, or a molecule comprising a
sequence having at least 70%, preferably 80%, and most preferably
90% identity to SEQ ID NO: 1.
[0034] The present invention further provides a protein or
polypeptide comprising or consisting of an amino acid sequence
encoded by any of the above nucleic acid sequences, termed herein
"VEGFV product", for example, an amino acid sequence having the
sequence as depicted in SEQ ID NO: 2, fragments of the above amino
acid sequence having a length of at least 10 amino acids, as well
as homologs of the amino acid sequences SEQ ID NO.:2 in which one
or more of the amino acid residues has been substituted (by
conservative or non-conservative substitution) added, deleted, or
chemically modified.
[0035] The present invention further provides nucleic acid molecule
comprising or consisting of a sequence which encodes the above
amino acid sequences, (including the fragments and analogs of the
amino acid sequences). Due to the degenerative nature of the
genetic code, a plurality of alternative nucleic acid sequences,
beyond SEQ ID NO:1, can code for the amino acid sequence of the
invention. Those alternative nucleic acid sequences which code for
the same amino acid sequences codes by the sequence SEQ ID NO: 1
are also an aspect of the of the present invention.
[0036] The present invention further provides expression vectors
and cloning vectors comprising any of the above nucleic acid
sequences, as well as host cells transfected by said vectors.
[0037] The present invention still further provides pharmaceutical
compositions comprising, as an active ingredient, said nucleic acid
molecules, said expression vectors, or said protein or
polypeptide.
[0038] These pharmaceutical compositions are suitable for the
treatment of diseases and pathological conditions, which can be
ameliorated or cured by raising the level of the VEGFV product.
[0039] By a second aspect, the present invention provides a nucleic
acid molecule comprising or consisting of a non-coding sequence
which is complementary to that of SEQ ID NO:1, or complementary to
a sequence having at least 70% identity to said sequence or a
fragment of said two sequences. The complementary sequence may be a
DNA sequence which hybridizes with the SEQ of ID NO:1 or hybridizes
to a portion of that sequence having a length sufficient to inhibit
the transcription of the complementary sequence. The complementary
sequence may be a DNA sequence which can be transcribed into an
mRNA being an antisense to the mRNA transcribed from SEQ ID NO:1 or
into an mRNA which is an antisense to a fragment of the mRNA
transcribed from SEQ ID NO.:1 which has a length sufficient to
hybridize with the mRNA transcribed from SEQ ID NO:. 1, so as to
inhibit its translation. The complementary sequence may also be the
mRNA or the fragment of the mRNA itself.
[0040] The nucleic acids of the second aspect of the invention may
be used for therapeutic or diagnostic applications for example for
detection of the expression of VEGFV. The proportion of expression
of the VEGF variant of the present invention as compared to the
known VEGF variants may be indicative to a variety of physiological
or pathological conditions.
[0041] The present invention also provides expression vectors
comprising any one of the above defined complementary nucleic acid
sequences and host cells transfected with said nucleic acid
sequences or vectors, being complementary to those specified in the
first aspect of the invention.
[0042] The invention also provides anti-VEGFV product antibodies,
namely antibodies directed against the VEGFV product which
specifically bind to said VEGFV product. Said antibodies are useful
both for diagnostic and therapeutic purposes. For example said
antibody may be as an active ingredient in a pharmaceutical
composition as will be explained below.
[0043] The present invention also provides pharmaceutical
compositions comprising, as an active ingredient, the nucleic acid
molecules which comprise or consist of said complementary
sequences, or of a vector comprising said complementary sequences.
The pharmaceutical composition thus provides pharmaceutical
compositions comprising, as an active ingredient, said anti-VEGFV
product antibodies.
[0044] The pharmaceutical compositions comprising said anti-VEGFV
product antibodies or the nucleic acid molecule comprising said
complementary sequence, are suitable for the treatment of diseases
and pathological conditions where a therapeutically beneficial
effect may be achieved by neutralizing the VEGFV or decreasing the
amount of the VEGFV product or blocking its binding to the
receptor, for example, by the neutralizing effect of the
antibodies, or by the decrease of the effect of the antisense mRNA
in decreasing expression level of the VEGFV product.
[0045] According to the third aspect of the invention the present
invention provides methods for detecting the level of the
transcript (mRNA) of said VEGFV product in a body fluid sample, or
in a specific tissue sample, for example by use of probes
comprising or consisting of said coding sequences; as well as
methods for detecting levels of expression of said product in
tissue, e.g. by the use of antibodies capable of specifically
reacting with the above amino acid sequences. Detection of the
level of the expression of the VEGF variant of the invention in
particular as compared to that of the known VEGF variants may be
indicative of a plurality of physiological or pathological
conditions.
[0046] The method, according to this latter aspect, for detection
of a nucleic acid sequence which encodes the VEGFV product in a
biological sample, comprises the steps of:
[0047] (a) providing a probe comprising at least one of the nucleic
acid sequence defined above;
[0048] (b) contacting the biological sample with said probe under
conditions allowing hybridization of nucleic acid sequences thereby
enabling formation of hybridization complexes;
[0049] (c) detecting hybridization complexes, wherein the presence
of the complex indicates the presence of nucleic acid sequence
encoding the VEGFV product in the biological sample.
[0050] By a preferred embodiment the probe is part of a nucleic
acid chip used for detection purposes, i.e. the probe is a part of
an array of probes each present in a known location on a solid
support.
[0051] The nucleic acid sequence used in the above method may be a
DNA sequence an RNA sequence, etc; it may be a coding or a sequence
or a sequence complementary thereto (for respective detection of
RNA transcripts or coding-DNA sequences). By quantization of the
level of hybridization complexes and calibrating the quantified
results it is possible also to detect the level of the transcript
in the sample.
[0052] Methods for detecting mutations in the region coding for the
VEGFV product are also provided, which may be methods carried-out
in a binary fashion, namely merely detecting whether there is any
mismatches between the normal VEGFV nucleic acid sequence and the
one present in the sample, or carried-out by specifically detecting
the nature and location of the mutation.
[0053] The present invention also concerns a method for detecting
VEGFV product in a biological sample, comprising the steps of:
[0054] (a) contacting with said biological sample the antibody of
the invention, thereby forming an antibody-antigen complex; and
[0055] (b) detecting said antibody-antigen complex
[0056] wherein the presence of said antibody-antigen complex
correlates with the presence of VEGFV product in said biological
sample.
[0057] By yet another aspect the invention also provides a method
for identifying candidate compounds capable of binding to the VEGFV
product and modulating its activity (being either activators or
deactivators). The method includes:
[0058] (i) providing a protein or polypeptide comprising an amino
acid sequence substantially as depicted in SEQ ID NO: 2, or a
fragment of such a sequence;
[0059] (ii) contacting a candidate compound with said amino acid
sequence;
[0060] (iii) measuring the physiological effect of said candidate
compound on the activity of the amino acid sequences and selecting
those compounds which show a significant effect on said
physiological activity.
[0061] The activity of the amino acid which should be changed by
the modulator (being either the activator or deactivator) may be
for example the binding of the VEGFV product to its native
receptor, effect on the modulation in the effect of VEGFV on
vascular endothelial cells, etc. Any modulator which changes such
an activity has an intersecting potential.
[0062] The present invention also concerns compounds identified by
the above methods described above, which compound may either be an
activator of the serotonin-receptor like product or a deactivator
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] In order to understand the invention and to see how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0064] FIGS. 1 and 2 show a Northern blot analysis of RNA obtained
from various tissues and tested with a probe specific for known
VEGFs but lacking the VEGF variant of the invention (termed "VEGF")
(left), as well as a unique probe from the 3'UTR region of VEGF-114
(right).
[0065] FIG. 3 shows a Western blot analysis of Ac-VEGF-114 infected
insect cells using the VEGF(147) polyclonal antibody (Santa Cruz
Biotechnology).
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
EXAMPLE I
VEGFV--Nucleic Acid Sequence
[0066] The nucleic acid sequences of the invention include nucleic
acid sequences which encode VEGFV product and fragments and analogs
thereof. The nucleic acid sequences may alternatively be sequences
complementary to the above coding sequence, or to a region of said
coding sequence. The length of the complementary sequence is
sufficient to avoid the expression of the coding sequence. The
nucleic acid sequences may be in the form of RNA or in the form of
DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and
genomic DNA. The DNA may be double-stranded or single-stranded, and
if single-stranded may be the coding strand or the non-coding
(anti-sense, complementary) strand. The nucleic acid sequences may
also both include dNTPs, rNTPs as well as non naturally occurring
sequences. The sequence may also be a part of a hybrid between an
amino acid sequence and a nucleic acid sequence.
[0067] In a general embodiment, the nucleic acid sequence has at
least 70%, preferably 80% or 90% sequence identity with the
sequence identified as SEQ ID NO:1.
[0068] The nucleic acid sequences may include the coding sequence
by itself By another alternative the coding region may be in
combination with additional coding sequences, such as those coding
for fusion protein or signal peptides, in combination with
non-coding sequences, such as introns and control elements,
promoter and terminator elements or 5' and/or 3' untranslated
regions, effective for expression of the coding sequence in a
suitable host, and/or in a vector or host environment in which the
VEGFV nucleic acid sequence is introduced as a heterologous
sequence.
[0069] The nucleic acid sequences of the present invention may also
have the product coding sequence fused in-frame to a marker
sequence which allows for purification of the VEGFV product. The
marker sequence may be, for example, a hexahistidine tag to provide
for purification of the mature polypeptide fused to the marker in
the case of a bacterial host, or, the marker sequence may be a
hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is
used. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767
(1984)).
[0070] Also included in the scope of the invention are fragments
also referred to herein as oligonucleotides, typically having at
least 20 bases, preferably 20-30 bases corresponding to a region of
the coding-sequence nucleic acid sequence. The fragments may be
used as probes, primers, and when complementary also as antisense
agents, and the like, according to known methods.
[0071] As indicated above, the nucleic acid sequence may be
substantially a depicted in SEQ ID NO:1 or fragments thereof or
sequences having at least 70%, preferably 70-80%, most preferably
90% identity to the above sequence. Alternatively, due to the
degenerative nature of the genetic code, the sequence may be a
sequence coding the amino acid sequence of SEQ ID NO:2, or
fragments or analogs of said amino acid sequence.
[0072] A. Preparation of Nucleic Acid Sequences
[0073] The nucleic acid sequences may be obtained by screening cDNA
libraries using oligonucleotide probes which can hybridize to or
PCR-amplify nucleic acid sequences which encode the VEGFV products
disclosed above. cDNA libraries prepared from a variety of tissues
are commercially available and procedures for screening and
isolating cDNA clones are well-known to those of skill in the art.
Such techniques are described in, for example, Sambrook et al
(1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold
Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y.
[0074] The nucleic acid sequences may be extended to obtain
upstream and downstream sequences such as promoters, regulatory
elements, and 5' and 3' untranslated regions (UTRs). Extension of
the available transcript sequence may be performed by numerous
methods known to those of skill in the art, such as PCR or primer
extension (Sambrook et al., supra), or by the RACE method using,
for example, the Marathon RACE kit (Clontech, Cat. # K1802-1).
[0075] Alternatively, the technique of "restriction-site" PCR
(Gobinda et al. PCR Methods Applic. 2:318-22, (1993)), which uses
universal primers to retrieve flanking sequence adjacent a known
locus, may be employed. First, genomic DNA is amplified in the
presence of primer to a linker sequence and a primer specific to
the known region. The amplified sequences are subjected to a second
round of PCR with the same linker primer and another specific
primer internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase.
[0076] Inverse PCR can be used to amplify or extend sequences using
divergent primers based on a known region (Triglia, T. et al.,
Nucleic Acids Res. 16:8186, (1988)). The primers may be designed
using OLIGO(R) 4.06 Primer Analysis Software (1992; National
Biosciences Inc, Plymouth, Minn.), or another appropriate program,
to be 22-30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68-72.degree. C. The method uses several restriction enzymes to
generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used
as a PCR template.
[0077] Capture PCR (Lagerstrom, M. et al., PCR Methods Applic.
1:111-19, (1991)) is a method for PCR amplification of DNA
fragments adjacent to a i5 known sequence in human and yeast
artificial chromosome DNA. Capture PCR also requires multiple
restriction enzyme digestions and ligations to place an engineered
double-stranded sequence into a flanking part of the DNA molecule
before PCR.
[0078] Another method which may be used to retrieve flanking
sequences is that of Parker, J. D., et al., Nucleic Acids Res.,
19:3055-60, (1991)). Additionally, one can use PCR, nested primers
and PromoterFinder.TM. libraries to "walk in" genomic DNA
(PromoterFinder.TM.; Clontech, Palo Alto, Calif.). This process
avoids the need to screen libraries and is useful in finding
intron/exon junctions. Preferred libraries for screening for full
length cDNAs are ones that have been size-selected to include
larger cDNAs. Also, random primed libraries are preferred in that
they will contain more sequences which contain the 5' and upstream
regions of genes.
[0079] A randomly primed library may be particularly useful if an
oligo d(T) library does not yield a full-length cDNA. Genomic
libraries are useful for extension into the 5' nontranslated
regulatory region.
[0080] The nucleic acid sequences and oligonucleotides of the
invention can also be prepared by solid-phase methods, according to
known synthetic methods. Typically, fragments of up to about 100
bases are individually synthesized, then joined to form continuous
sequences up to several hundred bases.
[0081] B. Use of VEGFV Nucleic Acid Sequence for the Production of
VEGFV Products
[0082] In accordance with the present invention, nucleic acid
sequences specified above may be used as recombinant DNA molecules
that direct the expression of VEGFV products.
[0083] As will be understood by those of skill in the art, it may
be advantageous to produce VEGFV product-encoding nucleotide
sequences possessing codons other than those which appear in SEQ ID
NO:1 which are those which naturally occur in the human genome.
Codons preferred by a particular prokaryotic or eukaryotic host
(Murray, E. et al. Nuc Acids Res., 17:477-508, (1989)) can be
selected, for example, to increase the rate of VEGFV product
expression or to produce recombinant RNA transcripts having
desirable properties, such as a longer half-life, than transcripts
produced from naturally occurring sequence.
[0084] The nucleic acid sequences of the present invention can be
engineered in order to alter a VEGFV product coding sequence for a
variety of reasons, including but not limited to, alterations which
modify the cloning, processing and/or expression of the product.
For example, alterations may be introduced using techniques which
are well known in the art, e.g., site-directed mutagenesis, to
insert new restriction sites, to alter glycosylation patterns, to
change codon preference, to produce splice variants, etc.
[0085] The present invention also includes recombinant constructs
comprising one or more of the sequences as broadly described above.
The constructs comprise a vector, such as a plasmid or viral
vector, into which a nucleic acid sequence of the invention has
been inserted, in a forward or reverse orientation. In a preferred
aspect of this embodiment, the construct further comprises
regulatory sequences, including, for example, a promoter, operably
linked to the sequence. Large numbers of suitable vectors and
promoters are known to those of skill in the art, and are
commercially available. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are also described in
Sambrook, et al., (supra).
[0086] The present invention also relates to host cells which are
genetically engineered with vectors of the invention, and the
production of the product of the invention by recombinant
techniques. Host cells are genetically engineered (i.e.,
transduced, transformed or transfected) with the vectors of this
invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the form of a
plasmid, a viral particle, a phage, etc. The engineered host cells
can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying the expression of the VEGFV nucleic acid sequence. The
culture conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to those skilled in the art.
[0087] The nucleic acid sequences of the present invention may be
included in any one of a variety of expression vectors for
expressing a product. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids;
vectors derived from combinations of plasmids and phage DNA, viral
DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable
and viable in the host. The appropriate DNA sequence may be
inserted into the vector by a variety of procedures. In general,
the DNA sequence is inserted into an appropriate restriction
endonuclease site(s) by procedures known in the art. Such
procedures and related sub-cloning procedures are deemed to be
within the scope of those skilled in the art.
[0088] The DNA sequence in the expression vector is operatively
linked to an appropriate transcription control sequence (promoter)
to direct mRNA synthesis. Examples of such promoters include: LTR
or SV40 promoter, the E. coli lac or trp promoter, the phage lambda
PL promoter, and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their viruses. The
expression vector also contains a ribosome binding site for
translation initiation, and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more
selectable marker genes to provide a phenotypic trait for selection
of transformed host cells such as dihydrofolate reductase or
neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0089] The vector containing the appropriate DNA sequence as
described above, as well as an appropriate promoter or control
sequence, may be employed to transform an appropriate host to
permit the host to express the protein. Examples of appropriate
expression hosts include: bacterial cells, such as E. coli,
Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;
insect cells such as Drosophila and Spodoptera Sf9; animal cells
such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses; plant
cells, etc. The selection of an appropriate host is deemed to be
within the scope of those skilled in the art from the teachings
herein. The invention is not limited by the host cells
employed.
[0090] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the VEGFV product. For
example, when large quantities of VEGFV product are needed for the
induction of antibodies, vectors which direct high level expression
of fusion proteins that are readily purified may be desirable. Such
vectors include, but are not limited to, multifunctional E. coli
cloning and expression vectors such as Bluescript(R) (Stratagene),
in which the VEGFV polypeptide coding sequence may be ligated into
the vector in-frame with sequences for the amino-terminal Met and
the subsequent 7 residues of beta-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke & Schuster J. Biol.
Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison Wis.);
and the like.
[0091] In the yeast Saccharomyces cerevisiae a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al., (Methods in Enzymology
153:516-544, (1987)).
[0092] In cases where plant expression vectors are used, the
expression of a sequence encoding VEGFV product may be driven by
any of a number of promoters. For example, viral promoters such as
the 35S and 19S promoters of CaMV (Brisson et al., Nature
310:511-514. (1984)) may be used alone or in combination with the
omega leader sequence from TMV (Takamatsu et al., EMBO J.,
6:307-311, (1987)). Alternatively, plant promoters such as the
small subunit of RUBISCO (Coruzzi et al., EMBO J. 3:1671-1680,
(1984); Broglie et al., Science 224:838-843, (1984)); or heat shock
promoters (Winter J and Sinibaldi R. M., Results Probl. Cell
Differ., 17:85-105, (1991)) may be used. These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. For reviews of such techniques, see
Hobbs S. or Murry L. E. (1992) in McGraw Hill Yearbook of Science
and Technology, McGraw Hill, New York, N.Y., pp 191-196; or
Weissbach and Weissbach (1988) Methods for Plant Molecular Biology,
Academic Press, New York, N.Y., pp 421-463.
[0093] VEGFV product may also be expressed in an insect system. In
one such system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes in Spodoptera
frugiperda cells or in Trichoplusia larvae. The VEGFV product
coding sequence may be cloned into a nonessential region of the
virus, such as the polyhedrin gene, and placed under control of the
polyhedrin promoter. Successful insertion of VEGFV coding sequence
will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein coat. The recombinant viruses are then
used to infect S. frugiperda cells or Trichoplusia larvae in which
VEGFV protein is expressed (Smith et al., J. Virol. 46:584, (1983);
Engelhard, E. K. et al., Proc. Nat. Acad. Sci. 91:3224-7,
(1994)).
[0094] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a VEGFV product coding sequence may be ligated
into an adenovirus transcription/translation complex consisting of
the late promoter and tripartite leader sequence. Insertion in a
nonessential E1 or E3 region of the viral genome will result in a
viable virus capable of expressing VEGFV protein in infected host
cells (Logan and Shenk, Proc. Natl. Acad. Sci. 81:3655-59, (1984).
In addition, transcription enhancers, such as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in
mammalian host cells.
[0095] Specific initiation signals may also be required for
efficient translation of a VEGFV protein coding sequence. These
signals include the ATG initiation codon and adjacent sequences. In
cases where VEGFV product coding sequence, its initiation codon and
upstream sequences are inserted into the appropriate expression
vector, no additional translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous transcriptional control signals including
the ATG initiation codon must be provided. Furthermore, the
initiation codon must be in the correct reading frame to ensure
transcription of the entire insert. Exogenous transcriptional
elements and initiation codons can be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate to the cell system in use
(Scharf, D. et al., (1994) Results Probl. Cell Differ., 20:125-62,
(1994); Bittner et al., Methods in Enzymol 153:516-544,
(1987)).
[0096] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M:, and Battey, I. (1986) Basic
Methods in Molecular Biology). Cell-free translation systems can
also be employed to produce polypeptides using RNAs derived from
the DNA constructs of the present invention.
[0097] A host cell strain may be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
protein include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing which cleaves a "pre-pro"
form of the protein may also be important for correct insertion,
folding and/or function. Different host cells such as CHO, HeLa,
MDCK, 293, WI38, etc. have specific cellular machinery and
characteristic mechanisms for such post-translational activities
and may be chosen to ensure the correct modification and processing
of the introduced, foreign protein.
[0098] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express VEGFV product may be transformed using
expression vectors which contain viral origins of replication or
endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clumps of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell type.
[0099] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler M., et al., Cell
11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I.,
et al., Cell 22:817-23, (1980)) genes which can be employed in tk-
or aprt-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler M.,
et al., Proc. Natl. Acad. Sci. 77:3567-70, (1980)); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al., J. Mol. Biol., 150:1-14, (1981)) and
als or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman S. C. and R. C. Mulligan, Proc. Natl. Acad. Sci.
85:8047-51, (1988)). The use of visible markers has gained
popularity with such markers as anthocyanins, beta-glucuronidase
and its substrate, GUS, and luciferase and its substrates,
luciferin and ATP, being widely used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system
(Rhodes, C. A. et. al., Methods Mol. Biol., 55:121-131,
(1995)).
[0100] Host cells transformed with a nucleotide sequence encoding
VEGFV product may be cultured under conditions suitable for the
expression and recovery of the encoded protein from cell-culture.
The product produced by a recombinant cell may be secreted or
contained intracellularly depending on the sequence and/or the
vector used. As will be understood by those of skill in the art,
expression vectors containing nucleic acid sequences encoding VEGFV
product can be designed with signal sequences which direct
secretion of VEGFV product through a prokaryotic or eukaryotic cell
membrane.
[0101] VEGFV product may also be expressed as a recombinant protein
with one or more additional polypeptide domains added to facilitate
protein purification. Such purification facilitating domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle,
Wash.). The inclusion of a protease-cleavable polypeptide linker
sequence between the purification domain and VEGFV protein is
useful to facilitate purification. One such expression vector
provides for expression of a fusion protein compromising a VEGFV
polypeptide fused to a polyhistidine region separated by an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography, as described in Porath, et al., Protein Expression
and Purification, 3:263-281, (1992)) while the enterokinase
cleavage site provides a means for isolating VEGFV polypeptide from
the fusion protein. pGEX vectors (Promega, Madison, Wis.) may also
be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption to ligand-agarose beads (e.g., glutathione-agarose in
the case of GST-fusions) followed by elution in the presence of
free ligand.
[0102] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period. Cells are typically harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents, or
other methods, which are well know to those skilled in the art.
[0103] The VEGFV products can be recovered and purified from
recombinant cell cultures by any of a number of methods well known
in the art, including ammonium sulfate or ethanol precipitation,
acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
mature protein. Finally, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0104] C. Diagnostic Applications Utilizing Nucleic Acid
Sequences
[0105] The nucleic acid sequences of the present invention may be
used for a variety of diagnostic purposes. The nucleic acid
sequences may be used to detect and quantitate expression of VEGFV
in patient's cells, e.g. biopsied tissues, by detecting the
presence of mRNA coding for VEGFV product. Alternatively, the assay
may be used to detect soluble VEGFV in the serum or blood. This
assay typically involves obtaining total mRNA from the tissue or
serum and contacting the mRNA with a nucleic acid probe. The probe
is a nucleic acid molecule of at least 20 nucleotides, preferably
20-30 nucleotides, capable of specifically hybridizing with a
sequence included within the sequence of a nucleic acid molecule
encoding VEGFV under hybridizing conditions, detecting the presence
of mRNA hybridized to the probe, and thereby detecting the
expression of VEGFV. This assay can be used to distinguish between
absence, presence, and excess expression of VEGFV product and to
monitor levels of VEGFV expression during therapeutic
intervention.
[0106] The invention also contemplates the use of the nucleic acid
sequences as a diagnostic for diseases resulting from inherited
defective VEGFV sequences, or diseases in which the purpose of the
amount of the known VEGF to the novel VEGF variant of the invention
is altered. These sequences can be detected by comparing the
sequences of the defective (i.e., mutant) VEGFV coding region with
that of a normal coding region. Association of the sequence coding
for mutant VEGFV product with abnormal VEGFV product activity may
be verified. In addition, sequences encoding mutant VEGFV products
can be inserted into a suitable vector for expression in a
functional assay system (e.g., calorimetric assay, complementation
experiments in a VEGFV protein deficient strain of HEK293 cells) as
yet another means to verify or identify mutations. Once mutant
genes have been identified, one can then screen populations of
interest for carriers of the mutant gene.
[0107] Individuals carrying mutations in the nucleic acid sequence
of the present invention may be detected at the DNA level by a
variety of techniques. Nucleic acids used for diagnosis may be
obtained from a patient's cells, including but not limited to such
as from blood, urine, saliva, placenta, tissue biopsy and autopsy
material. Genomic DNA may be used directly for detection or may be
amplified enzymatically by using PCR (Saiki, et al., Nature
324:163-166, (1986)) prior to analysis. RNA or cDNA may also be
used for the same purpose. As an example, PCR primers complementary
to the nucleic acid of the present invention can be used to
identify and analyze mutations in the gene of the present
invention. Deletions and insertions can be detected by a change in
size of the amplified product in comparison to the normal
genotype.
[0108] Point mutations can be identified by hybridizing amplified
DNA to radiolabeled RNA of the invention or alternatively,
radiolabeled antisense DNA sequences of the invention. Sequence
changes at specific locations may also be revealed by nuclease
protection assays, such RNase and S1 protection or the chemical
cleavage method (e.g. Cotton, et al Proc. Natl. Acad. Sci. USA,
85:4397-4401, (1985)), or by differences in melting temperatures.
"Molecular beacons" (Kostrikis L. G. et al., Science 279:1228-1229,
(1998)), hairpin-shaped, single-stranded synthetic
oligo-nucleotides containing probe sequences which are
complementary to the nucleic acid of the present invention, may
also be used to detect point mutations or other sequence changes as
well as monitor expression levels of VEGFV product. Such
diagnostics would be particularly useful for prenatal testing.
[0109] Another method for detecting mutations uses two DNA probes
which are designed to hybridize to adjacent regions of a target,
with abutting bases, where the region of known or suspected
mutation(s) is at or near the abutting bases. The two probes may be
joined at the abutting bases, e.g., in the presence of a ligase
enzyme, but only if both probes are correctly base paired in the
region of probe junction. The presence or absence of mutations is
then detectable by the presence or absence of ligated probe.
[0110] Also suitable for detecting mutations in the VEGFV product
coding sequence are oligonucleotide array methods based on
sequencing by hybridization (SBH), as described, for example, in
U.S. Pat. No. 5,547,839. In a typical method, the DNA target
analyte is hybridized with an array of oligonucleotides formed on a
microchip. The sequence of the target can then be "read" from the
pattern of target binding to the array.
[0111] RNA was obtained from the following tissues: putamen,
temporal lobe, frontal lobe, occipital lobe, spinal cord, medulla,
cerebral cortex, brain, heart, skeletal muscle, colon, thymus,
spleen, kidney, liver, small intestine, placenta lung and
leukocytes and was hybridized either with an VEGF probe or with a
probe from the unique 3' UTR region of the VEGF-V of the invention.
The results are shown in FIGS. 1 and 2. As can be seen the new
VEGF-V probe of the invention shows, in general, similar
hybridization patterns to those of native VEGF.
[0112] D. Gene Mapping Utilizing Nucleic Acid Sequences
[0113] The nucleic acid sequences of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0114] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 20-30 bp) from the VEGFV cDNA. Computer
analysis of the 3' untranslated region is used to rapidly select
primers that do not span more than one exon in the genomic DNA,
which would complicate the amplification process. These primers are
then used for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids containing the
human gene corresponding to the primer will yield an amplified
fragment.
[0115] PCR mapping of somatic cell hybrids or using instead
radiation hybrids are rapid procedures for assigning a particular
DNA to a particular chromosome. Using the present invention with
the same oligonucleotide primers, sublocalization can be achieved
with panels of fragments from specific chromosomes or pools of
large genomic clones in an analogous manner. Other mapping
strategies that can similarly be used to map to its chromosome
include in situ hybridization, prescreening with labeled
flow-sorted chromosomes and preselection by hybridization to
construct chromosome specific-cDNA libraries.
[0116] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bases. For a review of this technique,
see Verma et al., Human Chromosomes: a Manual of Basic Techniques,
(1988) Pergamon Press, New York.
[0117] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in the OMIM database (Center for Medical Genetics, Johns
Hopkins University, Baltimore, Md. and National Center for
Biotechnology Information, National Library of Medicine, Bethesda,
Md.). The OMIM gene map presents the cytogenetic map location of
disease genes and other expressed genes. The OMIM database provides
information on diseases associated with the chromosomal location.
Such associations include the results of linkage analysis mapped to
this interval, and the correlation of translocations and other
chromosomal aberrations in this area with the advent of polygenic
diseases, such as cancer, in general and prostate cancer in
particular.
[0118] E. Therapeutic Applications of Nucleic Acid Sequences
[0119] Nucleic acid sequences of the invention may also be used for
therapeutic purposes. Turning first to the second aspect of the
invention (i.e. inhibition of expression of VEGFV), expression of
VEGFV product may be modulated through antisense technology, which
controls gene expression through hybridization of complementary
nucleic acid sequences, i.e. antisense DNA or RNA, to the control,
5' or regulatory regions of the gene encoding VEGFV product. For
example, the 5' coding portion of the nucleic acid sequence
sequence which codes for the product of the present invention is
used to design an antisense oligonucleotide of from about 10 to 40
base pairs in length. Oligonucleotides derived from the
transcription start site, e.g. between positions -10 and +10 from
the start site, are preferred. An antisense DNA oligonucleotide is
designed to be complementary to a region of the nucleic acid
sequence involved in transcription (Lee et al., Nucl. Acids, Res.,
6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan
et al., Science 251:1360, (1991)), thereby preventing transcription
and the production of the VEGFV products. An antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the VEGFV products (Okano J.
Neurochem. 56:560, (1991)). The antisense constructs can be
delivered to cells by procedures known in the art such that the
antisense RNA or DNA may be expressed in vivo. The antisense may be
antisense mRNA or DNA sequence capable of coding such antisense
mRNA. The antisense mRNA or the DNA coding thereof can be
complementary to the full sequence of nucleic acid sequences coding
to the VEGFV protein or to a fragment of such a sequence which is
sufficient to inhibit production of a protein product.
[0120] Turning now to the first aspect of the invention, i.e.
expression of VEGFV, expression of VEGFV product may be increased
by providing coding sequences for coding for said product under the
control of suitable control elements ending its expression in the
desired host.
[0121] The nucleic acid sequences of the invention may be employed
in combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of the
compound, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0122] The products of the invention as well as any activators and
deactivators compounds (see below) which are polypeptides, may also
be employed in accordance with the present invention by expression
of such polypeptides in vivo, which is often referred to as "gene
therapy." Cells from a patient may be engineered with a nucleic
acid sequence (DNA or RNA) encoding a polypeptide ex vivo, with the
engineered cells then being provided to a patient to be treated
with the polypeptide. Such methods are well-known in the art. For
example, cells may be engineered by procedures known in the art by
use of a retroviral particle containing RNA encoding a polypeptide
of the present invention.
[0123] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by procedures known in the art. As known in
the art, a producer cell for producing a retroviral particle
containing RNA encoding the polypeptide of the present invention
may be administered to a patient for engineering cells in vivo and
expression of the polypeptide in vivo. These and other methods for
administering a product of the present invention by such method
should be apparent to those skilled in the art from the teachings
of the present invention. For example, the expression vehicle for
engineering cells may be other than a retrovirus, for example, an
adenovirus which may be used to engineer cells in vivo after
combination with a suitable delivery vehicle.
[0124] Retroviruses from which the retroviral plasmid vectors
mentioned above may be derived include, but are not limited to,
Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses
such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor
virus.
[0125] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X,
VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAm12, and DAN cell
lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5-14,
(1990)). The vector may transduce the packaging cells through any
means known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0126] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0127] The genes introduced into cells may be placed under the
control of inducible promoters, such as the radiation-inducible
Egr-1 promoter, (Maceri, H. J., et al., Cancer Res., 56(19):4311
(1996)), to stimulate VEGFV production or antisense inhibition in
response to radiation, eg., radiation therapy for treating
tumors.
EXAMPLE II
VEGFV Product
[0128] The substantially purified VEGFV product of the invention
has been defined above as the product coded from the nucleic acid
sequence of the invention. Preferably the amino acid sequence is an
amino acid sequence having at least 70%, preferably at least 80% or
90% identity to the sequence identified as SEQ ID NO:2. The protein
or polypeptide may be in mature and/or modified form, also as
defined above. Also contemplated are protein fragments having at
least 10 contiguous amino acid residues, preferably at least 10-20
residues, derived from the VEGFV product.
[0129] The sequence variations are preferably those that are
considered conserved substitutions, as defined above. Thus, for
example, a protein with a sequence having at least 80% sequence
identity with the protein identified as SEQ ID NO:2, preferably by
utilizing conserved substitutions as defined above is also part of
the invention. In a more specific embodiment, the protein has or
contains the sequence identified SEQ ID NO:2. The VEGFV product may
be (i) one in which one or more of the amino acid residues in a
sequence listed above are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue), or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
VEGFV product is fused with another compound, such as a compound to
increase the half-life of the protein (for example, polyethylene
glycol (PEG)), or a moiety which serves as targeting means to
direct the protein to its target tissue or target cell population
(such as an antibody), or (iv) one in which additional amino acids
are fused to the VEGFV product. Such fragments, variants and
derivatives are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0130] A. Preparation of VEGFV Product
[0131] Recombinant methods for producing and isolating the VEGFV
product, and fragments of the protein are described above.
[0132] In addition to recombinant production, fragments and
portions of VEGFV product may be produced by direct peptide
synthesis using solid-phase techniques (cf. Stewart et al., (1969)
Solid-Phase Peptide Synthesis, W H Freeman Co, San Francisco;
Merrifield J., J. Am. Chem. Soc., 85:2149-2154, (1963)). In vitro
peptide synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster
City, Calif.) in accordance with the instructions provided by the
manufacturer. Fragments of VEGFV product may be chemically
synthesized separately and combined using chemical methods to
produce the full length molecule.
[0133] The VEGF-V of the invention was cloned into the Back to Back
Baculovirus Expression System(Invitrogen). Briefly, the gene was
inserted into the pFastBackHTa1 transfer vector (including a 6xHis
tag on its 5'). The resulting transfer vector (pFBHTa1-VEGF) was
confirmed for the correct insertion by sequencing. pFBHTa1-VEGF was
then transformed into DH10Bac competent cells (containing the ACNPV
viral genome in the form of bacmid DNA). White colonies obtained
from the transformation were verified by PCR with gene specific and
viral specific primers for the VEGF insertion into the viral genome
under the polh promoter. The bacmid DNA was extracted from a
positive colony and transfected into insect cells (Sf-9). Viral
particles were collected and used for two consecutive infections in
order to amplify the viral stock. The viral particles collected
from the second infection was used for infection of Sf-9 cells in
suspension. Samples were collected at 48 and 72 hours post
infection (hpi) and centrifuged in order to verify protein
expression in cells pellet and its secretion into the medium. The
different samples were subjected for western blotting using the
VEGF(147) polyclonal antibody (Santa Cruz Biotechnology), and the
results are shown in FIG. 3.
[0134] As can be seen the medium contains two bands of size around
11 KD and 15 KD. The 15 KD band corresponds for the 6XHis-VEGF
translation starting at the His tag AUG and the 11 KD band
corresponds for the VEGF translation starting from the VEGF gene
AUG.
[0135] B. Therapeutic Uses and Compositions Utilizing the VEGFV
Product
[0136] The VEGFV product of the invention is generally useful in
treating diseases and disorders which are characterized by a lower
than normal level of VEGFV expression, and or diseases which can be
cured or ameliorated by raising the level of the VEGFV product,
even if the level is normal.
[0137] VEGFV products or fragments may be administered by any of a
number of routes and methods designed to provide a consistent and
predictable concentration of compound at the target organ or
tissue. The product-containing compositions may be administered
alone or in combination with other agents, such as stabilizing
compounds, and/or in combination with other pharmaceutical agents
such as drugs or hormones.
[0138] VEGFV product-containing compositions may be administered by
a number of routes including, but not limited to oral, intravenous,
intramuscular, transdermal, subcutaneous, topical, sublingual, or
rectal means as well as by nasal application. VEGFV
product-containing compositions may also be administered via
liposomes. Such administration routes and appropriate formulations
are generally known to those of skill in the art.
[0139] The product can be given via intravenous or intraperitoneal
injection. Similarly, the product may be injected to other
localized regions of the body. The product may also be administered
via nasal insufflation. Enteral administration is also possible.
For such administration, the product should be formulated into an
appropriate capsule or elixir for oral administration, or into a
suppository for rectal administration.
[0140] The foregoing exemplary administration modes will likely
require that the product be formulated into an appropriate carrier,
including ointments, gels, suppositories. Appropriate formulations
are well known to persons skilled in the art.
[0141] Dosage of the product will vary, depending upon the potency
and therapeutic index of the particular polypeptide selected.
[0142] A therapeutic composition for use in the treatment method
can include the product in a sterile injectable solution, the
polypeptide in an oral delivery vehicle, the product in an aerosol
suitable for nasal administration, or the product in a nebulized
form, all prepared according to well known methods. Such
compositions comprise a therapeutically effective amount of the
compound, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The product of the invention may also be used to modulate
endothelial differentiation and proliferation as well as to
modulate apoptosis either ex vivo or in vitro, for example, in cell
cultures.
EXAMPLE III
Screening Methods for Activators and Deactivators (Inhibitors)
[0143] The present invention also includes an assay for identifying
molecules, such as synthetic drugs, antibodies, peptides, or other
molecules, which have a modulating effect on the activity of the
VEGFV product, e.g. activators or deactivators of the VEGFV product
of the present invention. Such an assay comprises the steps of
providing an VEGFV product encoded by the nucleic acid sequences of
the present invention, contacting the VEGFV protein with one or
more candidate molecules to determine the candidate molecules
modulating effect on the activity of the VEGFV product, and
selecting from the molecules a candidate's molecule capable of
modulating VEGFV product physiological activity.
[0144] VEGFV product, its catalytic or immunogenic fragments or
oligopeptides thereof, can be used for screening therapeutic
compounds in any of a variety of drug screening techniques. The
fragment employed in such a test may be free in solution, affixed
to a solid support, borne on a cell membrane or located
intracellularly. The formation of binding complexes, between VEGFV
product and the agent being tested, may be measured. Alternatively,
the activator or deactivator may work by serving as agonist or
antagonist, respectively, of the VEGFV receptor and their effect
may be determined in connection with the receptor.
[0145] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the VEGFV product is described in detail by
Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984.
In summary, large numbers of different small peptide test compounds
are synthesized on a solid substrate, such as plastic pins or some
other surface. The peptide test compounds are reacted with the full
VEGFV product or with fragments of VEGFV product and washed. Bound
VEGFV product is then detected by methods well known in the art.
Substantially purified VEGFV product can also be coated directly
onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0146] Antibodies to the VEGFV product, as described in Example IV
below, may also be used in screening assays according to methods
well known in the art. For example, a "sandwich" assay may be
performed, in which an anti-VEGFV antibody is affixed to a solid
surface such as a microtiter plate and VEGFV product is added. Such
an assay can be used to capture compounds which bind to the VEGFV
product. Alternatively, such an assay may be used to measure the
ability of compounds to influence with the binding of VEGFV product
to the VEGFV receptor, and then select those compounds which effect
the binding.
EXAMPLE IV
Anti-VEGFV Antibodies
[0147] A. Synthesis
[0148] In still another aspect of the invention, the purified VEGFV
product is used to produce anti-VEGFV antibodies which have
diagnostic and therapeutic uses related to the activity,
distribution, and expression of the VEGFV product, in particular
therapeutic applications in inhibiting the effect of the VEGFV on
vascular endothelial cells.
[0149] Antibodies to VEGFV product may be generated by methods well
known in the art. Such antibodies may include, but are not limited
to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab
fragments and fragments produced by an Fab expression library.
Antibodies, i.e., those which inhibit dimer formation, are
especially preferred for therapeutic use.
[0150] A fragment VEGFV product for antibody induction does not
require biological activity but have to feature immunological
activity; however, the protein fragment or oligopeptide must be
antigenic. Peptides used to induce specific antibodies may have an
amino acid sequence consisting of at least five amino acids,
preferably at least 10 amino acids of the sequences specified in
SEQ ID NO: 2. Preferably they should mimic a portion of the amino
acid sequence of the natural protein and may contain the entire
amino acid sequence of a small, naturally occurring molecule. Short
stretches of VEGFV protein amino acids may be fused with those of
another protein such as keyhole limpet hemocyanin and antibody
produced against the chimeric molecule. Procedures well known in
the art can be used for the production of antibodies to VEGFV
product.
[0151] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, etc may be immunized by injection with
VEGFV product or any portion, fragment or oligopeptide which
retains immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include but are not limited to Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are
potentially useful human adjuvants.
[0152] Monoclonal antibodies to VEGFV protein may be prepared using
any technique which provides for the production of antibody
molecules by continuous cell lines in culture. These include but
are not limited to the hybridoma technique originally described by
Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell
hybridoma technique (Kosbor et al., Immunol. Today 4:72, (1983);
Cote et al., Proc. Natl. Acad. Sci. 80:2026-2030, (1983)) and the
EBV-hybridoma technique (Cole. et al., Mol. Cell Biol. 62:109-120,
(1984)).
[0153] Techniques developed for the production of "chimeric
antibodies", the splicing of mouse antibody genes to human antibody
genes to obtain a molecule with appropriate antigen specificity and
biological activity can also be used (Morrison et al., Proc. Natl.
Acad. Sci. 81:6851-6855, (1984); Neuberger et al., Nature
312:604-608, (1984); Takeda et al., Nature 314:452-454, (1985)).
Alternatively, techniques described for the production of single
chain antibodies U.S. Pat. No. 4,946,778) can be adapted to produce
single-chain antibodies specific for the VEGFV protein.
[0154] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in Orlandi et al. (Proc. Natl. Acad. Sci.
86:3833-3837, 1989)), and Winter G and Milstein C., (Nature
349:293-299, (1991)).
[0155] Antibody fragments which contain specific binding sites for
VEGFV protein may also be generated. For example, such fragments
include, but are not limited to, the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse W. D. et al., Science 256:1275-1281, (1989)).
[0156] B. Diagnostic Applications of Antibodies
[0157] A variety of protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal
antibodies with established specificities are well known in the
art. Such immunoassays typically involve the formation of complexes
between VEGFV product and its specific antibody and the measurement
of complex formation. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two noninterfering
epitopes on a specific VEGFV product is preferred, but a
competitive binding assay may also be employed. These assays are
described in Maddox D. E., et al., (J. Exp. Med. 158:1211,
(1983)).
[0158] Antibodies which specifically bind VEGFV product are useful
for the diagnosis of conditions or diseases characterized by over
expression of VEGFV as well as for detection of diseases in which
the proportion between the amount of the known VEGF and the novel
VEGF variant of the invention is altered. Alternatively, such
antibodies may be used in assays to monitor patients being treated
with VEGFV product, its activators, or its deactivators. Diagnostic
assays for VEGFV protein include methods utilizing the antibody and
a label to detect VEGFV product in human body fluids or extracts of
cells or tissues. The products and antibodies of the present
invention may be used with or without modification. Frequently, the
proteins and antibodies will be labeled by joining them, either
covalently or noncovalently, with a reporter molecule. A wide
variety of reporter molecules are known in the art.
[0159] A variety of protocols for measuring VEGFV product, using
either polyclonal or monoclonal antibodies specific for the
respective protein are known in the art. Examples include
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
and fluorescent activated cell sorting (FACS). As noted above, a
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering epitopes on VEGFV
product is preferred, but a competitive binding assay may be
employed. These assays are described, among other places, in
Maddox, et al. (supra). Such protocols provide a basis for
diagnosing altered or abnormal levels of VEGFV product expression.
Normal or standard values for VEGFV product expression are
established by combining body fluids or cell extracts taken from
normal subjects, preferably human, with antibody to VEGFV product
under conditions suitable for complex formation which are well
known in the art. The amount of standard complex formation may be
quantified by various methods, preferably by photometric methods.
Then, standard values obtained from normal samples may be compared
with values obtained from samples from subjects potentially
affected by disease. Deviation between standard and subject values
establishes the presence of disease state.
[0160] The antibody assays are useful to determine the level of
VEGFV present in a body fluid sample, in order to determine whether
it is being overexpressed or underexpressed in the tissue, or as an
indication of how VEGFV levels are responding to drug
treatment.
[0161] C. Therapeutic Uses of Antibodies
[0162] In addition to their diagnostic use the antibodies may have
a therapeutical utility in blocking or decreasing the activity of
the VEGFV product in pathological conditions where beneficial
effect can be achieved by such a decrease.
[0163] The antibody employed is preferably a humanized monoclonal
antibody, or a human MAb produced by known globulin-gene library
methods. The antibody is administered typically as a sterile
solution by IV injection, although other parenteral routes may be
suitable. Typically, the antibody is administered in an amount
between about 1-15 mg/kg body weight of the subject. Treatment is
continued, e.g., with dosing every 1-7 days, until a therapeutic
improvement is seen.
[0164] Although the invention has been described with reference to
specific methods and embodiments, it is appreciated that various
modifications and changes may be made without departing from the
invention.
Sequence CWU 1
1
2 1 426 DNA Homo sapiens 1 atgaactttc tgctgtcttg ggtgcattgg
agccttgcct tgctgctcta cctccaccat 60 gccaagtggt cccaggctgc
acccatggca gaaggaggag ggcagaatca tcacgaagtg 120 gtgaagttca
tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac 180
atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg
240 atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac
tgaggagtcc 300 aacatcacca tgcagattat gcggatcaaa cctcaccaag
gccagcacat aggagagatg 360 agcttcctac agcacaacaa atgtgaatgc
agaccaaaga aagatagagc aagacaagaa 420 aagtaa 426 2 141 PRT Homo
sapiens 2 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu
Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro
Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys
Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu
Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu
Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly
Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu
Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110
Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115
120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys 130 135
140
* * * * *