Note: Descriptions are shown in the official language in which they were submitted.
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SCREENING AND THERAPY FOR
LYMPHATIC DISORDERS INVOLVING THE
FLT4 RECEPTOR TYROSINE KINASE (VEGFR-3)
This application is a Continuation-in-Part of International Patent
Application No. PCT/LTS99/06133, filed March 26, 1999, incorporated herein by
reference in its entirety.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with United States and Finnish government
support, including support under contract R03-HD35174, awarded by the U.S.
National Institutes ofHealth. The U.S. Government has certain rights in this
invention.
FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular
biology and medicine; more particularly to the areas of genetic screening and
the
identification and treatment of hereditary disorders; and more particularly to
identification and treatment of hereditary lymphedema.
DESCRIPTION OF RELATED ART
The lymphatic system is a complex structure organized in parallel
fashion to the circulatory system. In contrast to the circulatory system,
which utilizes
the heart to pump blood throughout the body, the lymphatic system pumps lymph
fluid
using the inherent contractility of the lymphatic vessels. The lymphatic
vessels are not
interconnected in the same manner as the blood vessels, but rather form a set
of
coordinated structures including the initial lymphatic sinuses [Jeltsch et
al." Science,
276:1423-1425 (1997); and Castenholz, A., in Olszewski, W.L. (ed.), Lymph
Stasis:
Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton, Florida
(1991),
pp.15-42] which drain into the lymphatic capillaries and subsequently to the
collecting
lymphatics which drain into the lymphatic trunks and the thoracic duct which
ultimately drains into the venous circulation. The composition of the channels
through
which lymph passes is varied [Olszewski, W.L., in Olszewski, W.L. (ed), Lymph
Stasis: Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton,
Florida
CA 02283470 1999-09-29
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(1991), pp. 235-258; and Kinmonth, J.B., in Kinmonth, J.B. (ed), The
Lymphatics:
Diseases, Lymphography and Surgery. Edward Arnold Publishers: London, England
(1972), pp. 82-86], including the single endothelial layers of the initial
lymphatics, the
multiple layers of the collecting lymphatics including endothelium, muscular
and
adventitial layers, and the complex organization of the lymph node. The
various
organs of the body such as skin, lung, and GI tract have components of the
lymphatics
with various unique features. [See Ohkuma, M., in Olszewski (1991), supra, at
pp.
157-190; Uhley, H. and Leeds, S., in Olszewski (1991), supra, at pp. 191-210;
and
Barrowman, J.A., in Olszewski (1991), at pp. 221-234).]
Molecular biology has identified at least a few genes and proteins
postulated to have roles mediating the growth and/or embryonic development of
the
lymphatic system. One such gene/protein is the receptor tyrosine kinase
designated
Flt4 (fms-1_ike Iyrosine kinase 4), cloned from human erythroleukaemia cell
and
placental cDNA libraries. [See U.S. Patent No. 5,776,755; Aprelikova et al.,
Cancer
Res., 52: 746-748 (1992); Galland et al., Genomics, 13: 475-478 (1992);
Galland et
al., Oncogene, 8: 1233-1240 (1993); and Pajusola et al., Cancer Res., 52: 5738-
5743
(1992), all incorporated herein by reference.] Studies showed that, in mouse
embryos,
a targeted disruption of the Flt4 gene leads to a failure of the remodeling of
the
primary vascular network, and death after embryonic day 9.5 [Dumont et al.,
Science,
282: 946-949 (1998)]. These studies suggested that Flt4 has an essential role
in the
development of the embryonic vasculature, before the emergence of the
lymphatic
vessels. However, additional studies indicated that, during further
development, the
expression of Flt4 becomes restricted mainly to lymphatic vessels [Kaipainen,
et al.,
Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)].
In humans, there are two isoforms of the Flt4 protein, designated as
Flt4s (short, Genbank Accession No. X68203) and F1t41 (long, Genbank Accession
Nos. X68203 and 566407, SEQ ID NO: 1). The sequence of these isoforms is
largely
identical, except for divergence that occurs at the carboxyl terminus of the
receptor as
a result of alternative mRNA splicing at the 3' end. The C-terminus of the
long form
contains three tyrosyl residues, and one of them (Y1337 (SEQ ID NO: 2)) serves
as an
autophosphorylation site in the receptor [Fournier et al., Oncogene, ll: 921-
931
(1995); and Pajusola, et al., Oncogene, 8: 2931-2937 (1993)]. Only the long
form is
detected in human erythroleukaemia (HEL) and in a megakaryoblastic cell line
(the
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DAMI cells), and the mouse Flt4 gene (Genbank Accession No. L07296) only
produces one mRNA transcript, corresponding to F1t41 [Galland et al.,
Oncogene, 8:
1233-1240 (1993); and Pajusola et al., Cancer Res., 52: 5738-5743 (1992)].
These
findings suggest that the long form of Flt4 may be responsible for most of the
biological properties of this receptor. The Flt4 protein is glycosylated and
proteolytically processed in transfected cells [Pajusola et al., Oncogene, 9:
3545-3555
(1994)]. During this process, the 175 kD form of the receptor matures to a 195
kD
form, which is subsequently cleaved into a 125 kD C-terminal fragment, and a
75 kD
extracellular domain-containing fragment, which are linked by disulphide
bonding in
the mature receptor.
Two growth factors, named vascular endothelial growth factors C and
D (VEGF-C and VEGF-D) due to amino acid sequence similarity to earlier-
discovered
vascular endothelial growth factor, have been shown to bind and activate the
tyrosine
phosphorylation of Flt4. [Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-
553
(1998); Joukov et al, EMBOJ., 16: 3898-3911; and Joukov et al., EMBO J., I5:
290-298 (1996)]. Because of Flt4's growth factor binding properties and the
fact that
Flt4 possesses amino acid sequence similarity to two previously identified
VEGF
receptors (Fltl/VEGFR-1 and KDR/VEGFR-2), Flt4 has also been designated
VEGFR-3, and these terms are used interchangeably herein.
When VEGF-C was intentionally over-expressed under a basal keratin
promoter in transgeruc mice, a hyperplastic lymphatic vessel network in the
skin was
observed. [Jeltsch et al., Science, 276:1423-1425 (1997).] The results of this
study,
when combined with the expression pattern of VEGFR-3 in the lymphatic
vasculature,
suggest that lymphatic growth may be induced by VEGF-C and mediated via
VEGFR-3. Notwithstanding the foregoing insights involving one cell surface
receptor
and the two apparent ligands therefor, little is known about the developmental
regulation of the lymphatic system.
Hereditary or primary lymphedema, first described by Milroy in 1892
[Milroy, N. Y. Med. J., 56:505-508 (1892)], is a developmental disorder ofthe
lymphatic system which leads to a disabling and disfiguring swelling of the
extremities.
Hereditary lymphedema generally shows an autosomal dominant pattern of
inheritance
with reduced penetrance, variable expression, and variable age-at-onset
[Greenlee
et al., Lymphology, 26:156-168 (1993)]. Swelling may appear in one or all
limbs,
CA 02283470 1999-09-29
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varying in degree and distribution. If untreated, such swelling worsens over
time. In
rare instances, angiosarcoma may develop in affected tissues [Offori et al.,
Clin. Exp.
Dermatol., 18:174-177 (1993)]. Despite having been described over a century
ago,
little progress has been made in understanding the mechanisms causing
lymphedema.
A long-felt need exists for the identification of the presumed genetic
variations that
underlie hereditary lymphedema, to permit better informed genetic counseling
in
affected families, earlier diagnosis and treatment, and the development of
more
targeted and effective lymphedema therapeutic regimens. In addition,
identification of
genetic markers and high risk members of lymphedema families facilitates the
identification and management of environmental factors that influence the
expression
and severity of a lymphedema phenotype.
SUMMARY OF THE INVENTION
The present invention provides materials and methods that address one
or more of the long-felt needs identified above by identifying a genetic
marker that
correlates and is posited to have a causative role in the development of
hereditary
lymphedema. The invention is based in part on the discovery that, in several
families
with members afflicted with hereditary lymphedema, the lymphedema phenotype
correlates with genetic markers localized to chromosome Sq34-q35; and that in
at least
some such families, a missense mutation in the VEGFR-3 gene (which maps to
chromosome Sq34-q35) exists that appears to behave in a loss-of function
dominant
negative manner to decrease tyrosine kinase signaling of the receptor. In view
of the
fact that VEGFR-3 acts as a high affinity receptor for vascular endothelial
growth
factor C (VEGF-C); a growth factor whose effects include modulation of the
growth
of the lymphatic vascular network, these linkage and biochemical studies
provide an
important marker for determining a genetic predisposition for lymphedema in
healthy
individuals; and for diagnosing hereditary lymphedema in symptomatic
individuals.
Materials and methods for performing such genetic analyses are considered
aspects of
the present invention.
Thus, the invention provides genetic screening procedures that entail
analyzing a person's genome -- in particular their VEGFR-3 alleles -- to
determine
whether the individual possesses a genetic characteristic found in other
individuals that
are considered to be afflicted with, or at risk for, developing hereditary
lymphedema.
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For example, in one embodiment, the invention provides a method for
determining a hereditary lymphedema development potential in a human subject
comprising the steps of analyzing the coding sequence of the VEGFR-3 genes
from the
human subject; and determining hereditary lymphedema development potential in
said
human subject from the analyzing step.
In another embodiment, the invention provides a method of screening a
human subject for an increased risk of developing a lymphatic disorder,
comprising the
steps of (a) assaying nucleic acid of a human subject to determine a presence
or an
absence of a mutation altering the encoded VEGFR-3 amino acid sequence or
expression of at least one VEGFR-3 allele; and (b) screening for an increased
risk of
developing a lymphatic disorder from the presence or absence of said mutation.
By "human subject" is meant any human being, human embryo, or
human fetus. It will be apparent that methods of the present invention will be
of
particular interest to individuals that have themselves been diagnosed with
lymphedema
or have relatives that have been diagnosed with lymphedema.
By "screening for an increased risk" is meant determination of whether
a genetic variation exists in the human subject that correlates with a greater
likelihood
of developing lymphedema than exists for the human population as a whole, or
for a
relevant racial or ethnic human sub-population to which the individual
belongs. Both
positive and negative determinations (i.e., determinations that a genetic
predisposition
marker is present or is absent) are intended to fall within the scope of
screening
methods of the invention. In preferred embodiments, the presence of a mutation
altering the sequence or expression of at least one Flt4 receptor tyrosine
kinase allele
in the nucleic acid is correlated with an increased risk of developing a
lymphatic
disorder, whereas the absence of such a mutation is reported as a negative
determination.
By "lymphatic disorder" is meant any clinical condition affecting the
lymphatic system, including but not limited to lymphedemas, lymphangiomas,
lymphangiosarcomas, lymphangiomatosis, lymphangiectasis, and cystic hygroma.
Preferred embodiments are methods of screening a human subject for an
increased risk
of developing a lymphedema disorder, i.e., any disorder that physicians would
diagnose as lymphedema and that is characterized by swelling associated with
lymph
accumulation, other than lymphedemas for which non-genetic causes (e.g.,
parasites,
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surgery) are known. By way of example, lymphedema disorders include Milroy-
Nonne
(OMIM 153100) syndrome-early onset lymphedema [Milroy, N. Y. Med. J., 56:505-
508 (1892); and Dale, J. Med. Genet., 22: 274-278 (1985)] and lymphedema
praecox
(Meige syndrome, OMIM 153200)-late onset lymphedema [Lewis et al., J. Ped.,
104:641-648 (1984); Holmes et al., Pediatrics 61: 575-579 (1978); and Wheeler
et al.,
Plastic Reconstructive Surg., 67:362-364 (1981)] which generally are described
as
separate entities, both characterized by dominant inheritance. However, there
is
confusion in the literature about the separation of these disorders. In
Milroy's
syndrome, the presence of edema, which is usually more severe in the lower
extremities, is seen from birth. Lymphedema praecox presents in a similar
fashion but
the onset of swelling is usually around puberty. Some cases have been reported
to
develop in the post-pubertal period. In the particular analyses described
herein, the
lymphedema families showing linkage to 5q34-q35 show an early onset for most
affected individuals, but individuals in these pedigrees have presented during
or after
puberty.
The "assaying" step of the invention may involve any techniques
available for analyzing nucleic acid to determine its characteristics,
including but not
limited to well-known techniques such as single-strand conformation
polymorphism
analysis (SSCP) [Orita et al., Proc Natl. Acad. Sci. USA, 86: 2766-2770
(1989)];
heteroduplex analysis [White et al., Genomics, 12: 301-306 (1992)]; denaturing
gradient gel electrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci.
USA, 80:
1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)];
DNA
sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)];
chemical
cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995);
and
Roberts et al., Nucl. Acids Res., 25: 3377-3378 (1997)]; restriction fragment
length
polymorphism analysis; single nucleotide primer extension analysis [Shumaker
et al.,
Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614
(1997)]; 5' nuclease assays [Pease et al., Proc. Natl. Acad. Sci. USA, 91:5022-
5026
(1994)]; DNA Microchip analysis [Ramsay, G., Nature Biotechnology, 16: 40-48
(1999); and Chee et al., U.S. Patent No. 5,837,832]; and ligase chain reaction
[Whiteley et al., U.S. Patent No. 5,521,065]. [See generally, Schafer and
Hawkins,
Nature Biotechnology, 16: 33-39 (1998).] All of the foregoing documents are
hereby
incorporated by reference in their entirety.
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In one preferred embodiment, the assaying involves sequencing of
nucleic acid to determine nucleotide sequence thereof, using any available
sequencing
technique. [See, e.g., Sanger et al., Proc. Natl. Acad Sci. (USA), 74: 5463-
5467
(1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32
(1994)
(sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58
(1998); U.S. Patent No. 5,202,231; and Science, 260: 1649-1652 (1993)
(sequencing
by hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992)
(sequencing by
primer walking); (Douglas et al., Biotechniques, 14: 824-828 (1993) (Direct
sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241
(1994);
Maxam and Gilbert, Meth. Enrymol., 65: 499-560 (1977) (chemical termination
sequencing), all incorporated herein by reference.] The analysis may entail
sequencing
of the entire VEGFR-3 gene genomic DNA sequence, or portions thereof; or
sequencing of the entire VEGFR-3 coding sequence or portions thereof. In some
circumstances, the analysis may involve a determination of whether an
individual
possesses a particular VEGFR-3 allelic variant, in which case sequencing of
only a
small portion of nucleic acid -- enough to determine the sequence of a
particular codon
characterizing the allelic variant -- is su~cient. This approach is
appropriate, for
example, when assaying to determine whether one family member inherited the
same
allelic variant that has been previously characterized for another family
member, or,
more generally, whether a person's genome contains an allelic variant that has
been
previously characterized and correlated with heritable lymphedema. More
generally,
the sequencing may be focused on those portions of the VEGFR-3 sequence that
encode a VEGFR-3 kinase domain, since several different and apparently
causative
mutations in affected individuals that have been identified correspond to
residues
within an intracellular VEGFR-3 kinase domain. Referring to SEQ ID NOs: 1 and
2,
the two kinase domains of human wild type VEGFR-3 correspond to nucleotides
2546
to 2848 and 3044 to 3514 of SEQ ID NO: 1, which encode residues 843 to 943 and
1009 to 1165 of SEQ ID NO: 2. Such kinase domains are localized to exons 17-20
and 22-26 in the VEGFR-3 gene, so the sequencing/analysis may be focused on
those
exons in particular. Molecular modeling suggests that, within these domains,
residues
6852, 6854, 6857, K879, E896, H1035, D1037, N1042, D1055, F1056, 61057,
E 1084, D 1096, and Rl 159 are of particular importance in comprising or
shaping the
catalytic pocket of the VEGFR-3 kinase domains, so the sequencing may focus on
CA 02283470 1999-09-29
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these residues (in addition to residues described herein for which mutations
have
already been identified).
In a related embodiment, the invention provides PCR primers useful for
amplifying particular exon sequences of human VEGFR-3 genomic DNA. The
Examples below identify preferred primers for amplifying Exon 17, Exon 22, and
Exon
24 sequences, where specific missense mutations described herein map. In
addition,
the Examples below describe the Exon-Intron junctions of human VEGFR-3, which,
in
combination with the VEGFR-3 cDNA sequence provided herein, permit the
manufacture of appropriate oligonucleotide primers for other exons. Any such
primers
of, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, or
more nucleotides that are identical or exactly complementary to a human VEGFR-
3
genomic sequence and that includes or is within 50 nucleotides of a VEGFR-3
exon-
intron splice site is intended to be within the scope of the invention.
In another embodiment, the assaying step comprises performing a
hybridization assay to determine whether nucleic acid from the human subject
has a
nucleotide sequence identical to or different from one or more reference
sequences. In
a preferred embodiment, the hybridization involves a determination of whether
nucleic
acid derived from the human subject will hybridize with one or more
oligonucleotides,
wherein the oligonucleotides have nucleotide sequences that correspond
identically to
a portion of the VEGFR-3 gene sequence, preferably the VEGFR-3 coding sequence
set forth in SEQ ID NO: 1, or that correspond identically except for one
mismatch.
The hybridization conditions are selected to differentiate between perfect
sequence
complementarity and imperfect matches differing by one or more bases. Such
hybridization experiments thereby can provide single nucleotide polymorphism
sequence information about the nucleic acid from the human subject, by virtue
of
knowing the sequences of the oligonucleotides used in the experiments.
Several of the techniques outlined above involve an analysis wherein
one performs a polynucleotide migration assay, e.g., on a polyacrylamide
electrophoresis gel, under denaturing or non-denaturing conditions. Nucleic
acid
derived from the human subject is subjected to gel electrophoresis, usually
adjacent to
one or more reference nucleic acids, such as reference VEGFR-3 sequences
having a
coding sequence identical to all or a portion of SEQ ID NO: 1, or identical
except for
CA 02283470 1999-09-29
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one known polymorphism. The nucleic acid from the human subject and the
reference
sequences) are subjected to similar chemical or enzymatic treatments and then
electrophoresed under conditions whereby the polynucleotides will show a
differential
migration pattern, unless they contain identical sequences. [See generally
Ausubel et
al. (eds.), Current Protocols in Molecular Biology, New York: John Wiley &
Sons,
Inc. (1987-1999); and Sambrook et al., (eds.), Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press
(1989), both incorporated herein by reference in their entirety.]
In the context of assaying, the term "nucleic acid of a human subject" is
intended to include nucleic acid obtained directly from the human subject
(e.g., DNA
or RNA obtained from a biological sample such as a blood, tissue, or other
cell or fluid
sample); and also nucleic acid derived from nucleic acid obtained directly
from the
human subject. By way of non-limiting examples, well known procedures exist
for
creating cDNA that is complementary to RNA derived from a biological sample
from a
human subject, and for amplifying (e.g., via polymerase chain reaction (PCR))
DNA or
RNA derived from a biological sample obtained from a human subject. Any such
derived polynucleotide which retains relevant nucleotide sequence information
of the
human subject's own DNA/RNA is intended to fall within the definition of
"nucleic
acid of a human subject" for the purposes of the present invention.
In the context of assaying, the term "mutation" includes addition,
deletion, and/or substitution of one or more nucleotides in the VEGFR-3 gene
sequence. The invention is demonstrated by way of non-limiting examples set
forth
below that identify several mutations in VEGFR-3, including single nucleotide
polymorphisms that introduce missense mutations into the YEGFR-3 coding
sequence
(as compared to the VEGFR-3 cDNA sequence set forth in SEQ ID NO: 1) and other
polymorphisms that occur in introns and that are identifiable via sequencing,
restriction
fragment length polymorphism, or other techniques. Example 2 provides an assay
to
determine whether a VEGFR-3 mutation inhibits VEGFR-3 signaling. Additional
assays to study both ligand binding and signaling activities of VEGFR-3 are
disclosed,
e.g., in U.S. Patent No. 5,776,755 and International Patent Publication No. WO
98/33917, published 06 August 1998, both ofwhich are incorporated herein by
reference in their entirety. Evidence that a VEGFR-3 mutation inhibits VEGFR-3
signaling is evidence that the mutation may have a causative role in
lymphedema
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phenotype. However, even mutations that have no apparent causative role may
serve
as useful markers for heritable lymphedema, provided that the appearance of
the
mutation correlates reliably with the appearance of lymphedema.
In a related embodiment, the invention provides a method of screening
for a VEGFR-3 hereditary lymphedema genotype in a human subject, comprising
the
steps of (a) providing a biological sample comprising nucleic acid from a
human
subject; (b) analyzing the nucleic acid for the presence of a mutation or
mutations in a
VEGFR-3 allele in the nucleic acid of the human subject; (c) determining a
VEGFR-3
genotype from said analyzing step; and (d) correlating the presence of a
mutation in a
VEGFR-3 allele with a hereditary lymphedema genotype. In a preferred
embodiment,
the biological sample is a cell sample containing human cells that contain
genomic
DNA of the human subject.
Although more time consuming and expensive than methods involving
nucleic acid analysis, the invention also may be practiced by assaying protein
of a
human subject to determine the presence or absence of an amino acid sequence
variation in VEGFR-3 protein from the human subject. Such protein analyses may
be
performed, e.g., by fragmenting VEGFR-3 protein via chemical or enzymatic
methods
and sequencing the resultant peptides; or by Western analyses using an
antibody having
specificity for a particular allelic variant of VEGFR-3.
The invention also provides materials that are useful for performing
methods of the invention. For example, the present invention provides
oligonucleotides useful as probes in the many analyzing techniques described
above.
In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10 , 11, 12, 13,
14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a
sequence that
is identical, or exactly complementary, to a portion of a human VEGFR-3 gene
sequence, or that is identical or exactly complementary except for one
nucleotide
substitution. In a preferred embodiment, the oligonucleotides have a sequence
that
corresponds in the foregoing manner to a human VEGFR-3 coding sequence, and in
particular, the VEGFR-3 coding sequence set forth in SEQ B7 NO: 1. In one
variation, an oligonucleotide probe of the invention is purified and isolated.
In another
variation, the oligonucleotide probe is labeled, e.g., with a radioisotope,
chromophore,
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or fluorophore. In yet another variation, the probe is covalently attached to
a solid
support. [See generally Ausubel et al. And Sambrook et al., supra.]
In preferred embodiments, the invention comprises an oligonucleotide
probe useful for detecting one or more of several mutations that have been
characterized herein in affected individuals, including:
(1) a missense mutation at nucleotide 3360 of SEQ 117 NO: 1, causing a
proline to leucine change at residue 1114 in SEQ ID NO: 2;
(2) a missense mutation at nucleotide 2588 of SEQ ID NO: 1, causing a
glycine to arginine change at residue 857 in SEQ ID NO: 2;
(3) a missense mutation at nucleotide 3141 of SEQ ID NO: 1, causing an
arginine to proline change at residue 1041 in SEQ ID NO: 2;
(4) a missense mutation at nucleotide 3150 in SEQ ID NO: 1, causing a
leucine to proline change at residue 1044 in SEQ ID NO: 2; and
(5) a missense mutation at nucleotide 3164 of SEQ 117 NO: 1, causing an
aspartic acid to asparagine change at residue 1049 in SEQ ID NO: 2.
For example, the invention provides oligonucleotides comprising anywhere from
6 to
50 nucleotides that have a sequence that is identical to, or exactly
complementary to, a
portion of the human VEGFR-3 coding sequence set forth in SEQ ID NO: 1, except
for a nucleotide substitution corresponding to nucleotide 3360 of SEQ 117 NO:
1.
Such oligonucleotides may be generically described by the formula X"YZm or its
complement; where n and m are integers from 0 to 49; where 5 <_ (n + m) <_ 49;
where
X" is a stretch of n nucleotides identical to a first portion of SEQ ID NO: 1
and Zm is a
stretch of m nucleotides identical to a second portion of SEQ ID NO: 1,
wherein the
first and second portions are separated in SEQ ID NO: 1 by one nucleotide; and
wherein Y represents a nucleotide other than the nucleotide that separates the
first and
second portions of SEQ 117 NO: 1. For example, where XI, represents 0 to 49
nucleotides immediately upstream (S') of nucleotide 3360 of SEQ B7 NO: 1 and
Zm
represents 0 to 49 nucleotides immediately downstream (3') of nucleotide 3360
of
SEQ ID NO: 1, Y represents a nucleotide other than cytosine, since a cytosine
nucleotide is found at position 3360 of SEQ ID NO: 1. In a preferred
embodiment, Y
is a thymine nucleotide. Similar examples are contemplated for the other
specific
mutations identified immediately above.
CA 02283470 1999-09-29
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In a related embodiment, the invention provides a kit comprising at
least two such oligonucleotide probes. Preferably, the two or more probes are
provided in separate containers, or attached to separate solid supports, or
attached
separately to the same solid support, e.g., on a DNA microchip.
In still another related embodiment, the invention provides an array of
oligonucleotide probes immobilized on a solid support, the array having at
least 4
probes, preferably at least 100 probes, and preferably up to 100,000, 10,000,
or 1000
probes, wherein each probe occupies a separate known site in the array. In a
preferred
embodiment, the array includes probe sets comprising two to four probes,
wherein one
probe is exactly identical or exactly complementary to a human VEGFR-3 coding
sequence, and the other one to three members of the set are exactly identical
to the
first member , but for at least one different nucleotide, which different
nucleotide is
located in the same position in each of the one to three additional set
members. In one
preferred embodiment, the array comprises several such sets of probes, wherein
the
sets correspond to different segments of the human VEGFR-3 gene sequence. In a
highly preferred embodiment, the array comprises enough sets of
oligonucleotides of
length N to correspond to every particular N-mer sequence of the VEGFR-3 gene,
where N is preferably 6 to 25 and more preferably 9 to 20. Materials and
methods for
making such probes are known in the art and are described, for example, in
U.S. Patent
Nos. 5,837,832, 5,202,231, 5,002,867, and 5,143,854.
Moreover, the discoveries which underlie the present invention identify
a target for therapeutic intervention in cases of hereditary lymphedema. The
causative
mutations in the families that have been studied in greatest detail are
mutations that
appear to result in VEGFR-3 signaling that is reduced in heterozygous affected
individuals, but not completely eliminated. This data supports a therapeutic
indication
for administration of agents, such as VEGFR-3 ligand polypeptides, that will
induce
VEGFR-3 signaling in the lymphatic endothelia of affected individuals to
effect
improvement in the structure and function of the lymphatic vasculature of such
individuals. In addition, therapeutic gene therapy, to replace defective VEGFR-
3
alleles or increase production of VEGFR-3 ligand polypeptides in vivo, is
envisioned as
an aspect of the invention.
Thus, in yet another aspect, the invention provides a therapeutic or
prophylactic method of treatment for lymphedema, comprising the step of
CA 02283470 1999-09-29
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administering to a mammalian subject in need of therapeutic or prophylactic
treatment
for lymphedema a composition comprising a compound effective to induce
intracellular
signaling of VEGFR-3 in lymphatic endothelial cells that express said
receptor. In a
preferred embodiment, the compound comprises a polypeptide ligand for VEGFR-3,
or a polynucleotide encoding such a ligand, wherein the polynucleotide is
administered
in a form that results in transcription and translation of the polynucleotide
in the
mammalian subject to produce the ligand in vivo. In another preferred
embodiment,
the compound comprises any small molecule that is capable of binding to the
VEGFR-
3 receptor extracellular or intracellular domain and inducing intracellular
signaling.
For example, the invention provides a therapeutic or prophylactic
method of treatment for lymphedema, comprising the step of administering to a
mammalian subject in need of therapeutic or prophylactic treatment for
lymphedema a
composition comprising a polynucleotide, the polynucleotide comprising a
nucleotide
sequence that encodes a vascular endothelial growth factor C (VEGF-C)
polypeptide.
In a preferred embodiment, the subject is a human subject.
While it is contemplated that the VEGF-C polynucleotide could be
administered purely as a prophylactic treatment to prevent lymphedema in
subjects at
risk for developing lymphedema, it is contemplated in a preferred embodiment
that the
polynucleotide be administered to subjects afflicted with lymphedema, for the
purpose
of ameliorating its symptoms (e.g., swelling due to the accumulation of
lymph). The
polynucleotide is included in the composition in an amount and in a form
effective to
promote expression of a VEGF-C polypeptide in or near the lymphatic endothelia
of
the mammalian subject, to stimulate VEGFR-3 signaling in the lymphatic
endothelia of
the subject.
In a preferred embodiment, the mammalian subject is a human subject.
Practice of methods of the invention in other mammalian subjects, especially
mammals
that are conventionally used as models for demonstrating therapeutic efFlcacy
in
humans (e.g., primate, porcine, canine, equine, murine, or rabbit animals),
also is
contemplated. Several potential animal models for hereditary lymphedema have
been
described in the literature. [See, e.g., Lyon et al., Mouse News Lett. 71: 26
(1984),
Mouse News Lett. 74: 96 (1986), and Genetic variants and strains of the
laboratory
mouse, 2nd ed., New York: Oxford University Press (1989), p. 70 (Chylous
ascites
mouse); Dumont et al., Science, 282: 946-949 (1998) (heterozygous VEGFR-3
CA 02283470 1999-09-29
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knockout mouse); Patterson et al., "Hereditary Lymphedema," Comparative
Pathology Bulletin, 3: 2 (1971) (canine hereditary lymphedema model); van der
Putte,
"Congenital Hereditary Lymphedema in the Pig," Lympho, ll: 1-9 (1978); and
Campbell-Beggs et al., "Chyloabdomen in a neonatal foal," Veterinary Record,
137:
96-98 (1995).] Those models which are determined to have analogous mutations
to
the VEGFR-3 gene, such as the Chylous ascetei (Chy) mouse, are preferred. The
present inventors have analyzed the VEGFR-3 genes of the Chy mouse and
determined
that affected mice contain a missense mutation that results in a phenylalanine
(rather
than an isoleucine) in the VEGFR-3 sequence at a position corresponding to the
isoleucine at position 1053 of SEQ 1D NO: 2. This mutation maps to the
catalytic
pocket region of the tyrosine kinase domain of the VEGFR-3 protein, and may
represent a viable model for identical mutations in human (if discovered) or
other
mutations in humans that similarly affect the tyrosine kinase catalytic
domain. The Chy
mouse has peripheral swelling (oedema) after birth and chyle ascites. In
another
embodiment, "knock in" homologous recombination genetic engineering strategies
are
used to create an animal model (e.g., a mouse model) having a VEGFR-3 allelic
variation analogous to the human variations described herein. [See, e.g.,
Partanen et
al., Genes & Development, 12: 2332-2344 (1998) (gene targeting to introduce
mutations into a receptor protein (FGFR-1) in mice).] Such mice can also be
bread to
the heterozygous VEGFR-3 knockout mice or Chy mice described above to further
modify the phenotypic severity of the lymphedema disease.
For the practice of methods of the invention, the term "VEGF-C
polypeptide" is intended to include any polypeptide that has a VEGF-C or VEGF-
C
analog amino acid sequence (as defined elsewhere herein in greater detail) and
that is
able to bind the VEGFR-3 extracellular domain and stimulate VEGFR-3 signaling
in
vivo. The term "VEGF-C polynucleotide" is intended to include any
polynucleotide
(e.g., DNA or RNA, single- or double-stranded) comprising a nucleotide
sequence that
encodes a VEGF-C polypeptide. Due to the well-known degeneracy of the genetic
code, multiple VEGF-C polynucleotide sequences exist that encode any selected
VEGF-C polypeptide. Preferred VEGF-C polynucleotides, polypeptides, and VEGF-
C variants and analogs for use in this invention are disclosed in
International Patent
Application No. PCTlUS98/01973, published as WO 98/33917, incorporated herein
by
reference in its entirety.
CA 02283470 1999-09-29
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For treatment of humans, VEGF-C polypeptides with an amino acid
sequence of a human VEGF-C are highly preferred, and polynucleotides
comprising a
nucleotide sequence of a human VEGF-C cDNA are highly preferred. By "human
VEGF-C" is meant a polypeptide corresponding to a naturally occurring protein
(prepro-protein, partially-processed protein, or fully-processed mature
protein)
encoded by any allele of the human VEGF-C gene, or a polypeptide comprising a
biologically active fragment of a naturally-occurring mature protein. By way
of
example, a human VEGF-C comprises a continuous portion of the amino acid
sequence set forth in SEQ ID NO: 4 sufficient to permit the polypeptide to
bind and
stimulate VEGFR-3 phosphorylation in cells that express such receptors. A
polypeptide comprising amino acids 131-211 of SEQ ID NO: 4 is specifically
contemplated. For example, polypeptides having an amino acid sequence
comprising a
continuous portion of SEQ ID NO: 4, the continuous portion having, as its
amino
terminus, an amino acid selected from the group consisting of positions 30-131
of SEQ
ID NO: 4, and having, as its carboxyl terminus, an amino acid selected from
the group
consisting of positions 211-419 of SEQ ID NO: 4 are contemplated. An amino
terminus selected from the group consisting of positions 102-131 of SEQ ID NO:
4 is
preferred, and an amino terminus selected from the group consisting of
positions 103-
113 of SEQ ID NO: 4 is highly preferred. Likewise, a carboxyl terminus
selected
from the group consisting of positions 211-227 of SEQ ID NO: 4 is preferred.
As
stated above, the term "human VEGF-C" also is intended to encompass
polypeptides
encoded by allelic variants of the human VEGF-C characterized by the sequences
set
forth in SEQ ID NOs: 3 & 4.
Moreover, since the therapeutic VEGF-C is to be administered as
recombinant VEGF-C or indirectly via somatic gene therapy, it is within the
skill in the
art to make and use analogs of human VEGF-C (and polynucleotides that encode
such
analogs) wherein one or more amino acids have been added, deleted, or replaced
with
other amino acids, especially with conservative replacements, and wherein the
VEGFR-3-stimulatory biological activity has been retained. Analogs that retain
VEGFR-3-stimulatory VEGF-C biological activity are contemplated as VEGF-C
polypeptides for use in the present invention. In a preferred embodiment,
analogs
having l, 2, 3, 4, S, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24,
or 25 such modifications and that retain VEGFR-3-stimulatory VEGF-C biological
CA 02283470 1999-09-29
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activity are contemplated as VEGF-C polypeptides for use in the present
invention.
Analogs having a deletion of or substitution for the cysteine residue at
position 156 of
SEQ ID NO: 4 and that retain VEGFR-3 stimulatory activity but have reduced
activity
toward the receptor VEGFR-2, which is expressed in blood vessels, are
specifically
contemplated. See WO 98/33917. Polynucleotides encoding such analogs are
generated using conventional PCR, site-directed mutagenesis, and chemical
synthesis
techniques.
Also contemplated as VEGF-C polypeptides are non-human mammalian
or avian VEGF-C polypeptides and polynucleotides. By "mammalian VEGF-C" is
meant a polypeptide corresponding to a naturally occurring protein (prepro-
protein,
partially-processed protein, or fully-processed mature protein) encoded by any
allele of
a VEGF-C gene of any mammal, or a polypeptide comprising a biologically active
fragment of a mature protein. The term "mammalian VEGF-C polypeptide" is
intended to include analogs of mammalian VEGF-C's that possess the in vivo
VEGFR-
3-stimulatory effects of the mammalian VEGF-C.
Irrespective of which encoded VEGF-C polypeptide is chosen, any
VEGF-C polynucleotide gene therapy pharmaceutical encoding it preferably
comprises
a nucleotide sequence encoding a secretory signal peptide fused in-frame with
the
VEGF-C polypeptide sequence. The secretory signal peptide directs secretion of
the
VEGF-C polypeptide by the cells that express the polynucleotide, and is
cleaved by the
cell from the secreted VEGF-C polypeptide. For example, the VEGF-C
polynucleotide could encode the complete prepro-VEGF-C sequence set forth in
SEQ
117 NO: 4; or could encode the VEGF-C signal peptide fused in-frame to a
sequence
encoding a fully-processed VEGF-C (e.g., amino acids 103-227 of SEQ ID NO: 4)
or
VEGF-C analog. Moreover, there is no requirement that the signal peptide be
derived
from VEGF-C. The signal peptide sequence can be that of another secreted
protein, or
can be a completely synthetic signal sequence effective to direct secretion in
cells of
the mammalian subject.
In one embodiment, the VEGF-C polynucleotide of the invention
comprises a nucleotide sequence that will hybridize to a polynucleotide that
is
complementary to the human VEGF-C cDNA sequence specified in SEQ ID NO: 3
under the following exemplary stringent hybridization conditions:
hybridization at
42°C in 50% formamide, SX SSC, 20 mM Na~P04, pH 6.8; and washing in 1X
SSC at
CA 02283470 1999-09-29
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SS°C for 30 minutes; and wherein the nucleotide sequence encodes a
polypeptide that
binds and stimulates human VEGFR-3. It is understood that variation in these
exemplary conditions occur based on the length and GC nucleotide content of
the
sequences to be hybridized. Formulas standard in the art are appropriate for
S determining appropriate hybridization conditions. [See Sambrook et al.,
Molecular
Cloning: A Laboratory Manual (Second ed., Cold Spring Harbor, New York: Cold
Spring Harbor Laboratory Press, 1989) ~~ 9.47-9.51.]
In preferred embodiments, the VEGF-C polynucleotide further
comprises additional sequences to facilitate the VEGF-C gene therapy. In one
embodiment, a "naked" VEGF-C transgene (i.e., a transgene without a viral,
liposomal,
or other vector to facilitate transfection) is employed for gene therapy. In
this
embodiment, the VEGF-C polynucleotide preferably comprises a suitable promoter
and/or enhancer sequence (e.g., cytomegalovirus promoter/enhancer [Lehner et
al., J.
Clirz Microbiol., 29:2494-2502 (1991); Boshart et al., Cell, 41: 521-530
(1985)];
Rous sarcoma virus promoter [Davis et al., Hum. Gene Ther., 4:151 (1993)]; Tie
promoter [Korhonen et al., Blood, 8G(S): 1828-1835 (1995)]; or simian virus 40
promoter) for expression in the target mammalian cells, the promoter being
operatively
linked upstream (i.e., S') of the VEGF-C coding sequence. The VEGF-C
polynucleotide also preferably further includes a suitable polyadenylation
sequence
(e.g., the SV40 or human growth hormone gene polyadenylation sequence)
operably
linked downstream (i.e., 3') of the VEGF-C coding sequence. The polynucleotide
may
further optionally comprise sequences whose only intended function is to
facilitate
large-scale production of the vector, e.g., in bacteria, such as a bacterial
origin of
replication and a sequence encoding a selectable marker. However, in a
preferred
embodiment, such extraneous sequences are at least partially cleaved off prior
to
administration to humans according to methods of the invention. One can
manufacture
and administer such polynucleotides to achieve successful gene therapy using
procedures that have been described in the literature for other transgenes.
See, e.g.,
Isner et al., Circulation, 91: 2687-2692 (1995); and Isner et al., Human Gene
Therapy, 7: 989-1011 (1996); incorporated herein by reference in the entirety.
Any suitable vector may be used to introduce the VEGF-C transgene
into the host. Exemplary vectors that have been described in the literature
include
replication-deficient retroviral vectors, including but not limited to
lentivirus vectors
CA 02283470 1999-09-29
-18-
[Kim et al., .I. Virol., 72(I): 811-816 (1998); Kingsman & Johnson, Scrip
Magazine,
October, 1998, pp. 43-46.]; adeno-associated viral vectors [Gnatenko et al.,
J.
Investig. Med., 45: 87-98 (1997)]; adenoviral vectors [See, e.g., U.S. Patent
No.
5,792,453; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992);
Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630 (1992); and
Rosenfeld et al.,
Cell, 68: 143-155 (1992)]; Lipofectin-mediated gene transfer (BRL); liposomal
vectors
[See, e.g., U.S. Patent No. 5,631,237 (Liposomes comprising Sendai virus
proteins)] ;
and combinations thereof. All of the foregoing documents are incorporated
herein by
reference in the entirety. Replication-deficient adenoviral vectors constitute
a
preferred embodiment.
In embodiments employing a viral vector, preferred polynucleotides still
include a suitable promoter and polyadenylation sequence as described above.
Moreover, it will be readily apparent that, in these embodiments, the
polynucleotide
further includes vector polynucleotide sequences (e.g., adenoviral
polynucleotide
sequences) operably connected to the sequence encoding a VEGF-C polypeptide.
Thus, in one embodiment the composition to be administered comprises
a vector, wherein the vector comprises the VEGF-C polynucleotide. In a
preferred
embodiment, the vector is an adenovirus vector. In a highly preferred
embodiment, the
adenovirus vector is replication-deficient, i.e., it cannot replicate in the
mammalian
subject due to deletion of essential viral-replication sequences from the
adenoviral
genome. For example, the inventors contemplate a method wherein the vector
comprises a replication-deficient adenovirus, the adenovirus comprising the
VEGF-C
polynucleotide operably connected to a promoter and flanked on either end by
adenoviral polynucleotide sequences.
The composition to be administered according to methods of the
invention preferably comprises (in addition to the polynucleotide or vector) a
pharmaceutically-acceptable carrier solution such as water, saline, phosphate-
bui~ered
saline, glucose, or other Garners conventionally used to deliver therapeutics
intravascularly. Multi-gene therapy is also contemplated, in which case the
composition optionally comprises both the VEGF-C polynucleotide/vector and
another
polynucleotide/vector. As described in greater detail below, a VEGF-D
transgene is a
preferred candidate for co-administration with the VEGF-C transgene.
CA 02283470 1999-09-29
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The "administering" that is performed according to the present method
may be performed using any medically-accepted means for introducing a
therapeutic
directly or indirectly into a mammalian subject to reach the lymph or the
lymphatic
system, including but not limited to injections; oral ingestion; intranasal or
topical
administration; and the like. In a preferred embodiment, administration of the
composition comprising the VEGF-C polynucleotide is performed intravascularly,
such
as by intravenous or intra-arterial injection, or by subcutaneous injection or
local depot
administration. In a highly preferred embodiment, the composition is
administered
locally, e.g., to the site of swelling.
In still another variation, endothelial cells or endothelial progenitor cells
are transfected ex vivo with a wild type VEGFR-3 transgene, and the
transfected cells
are administered to the mammalian subject.
In another aspect, the invention provides a therapeutic or prophylactic
method of treating for lymphedema, comprising the step of administering to a
mammalian subject in need of treatment for lymphedema a composition comprising
a
VEGF-C polypeptide, in an amount effective to treat or prevent swelling
associated
with lymphedema. Administration via one or more intravenous or subcutaneous
injections is contemplated. Co-administration of VEGF-C polynucleotides and
VEGF-
C polypeptides is also contemplated.
In yet another embodiment, the invention provides the use of a VEGF-
C polynucleotide or VEGF-C polypeptide for the manufacture of a medicament for
the
treatment or prevention of lymphedema.
In still another embodiment, the invention provides a therapeutic or
prophylactic method of treatment for lymphedema, comprising the step of
administering to a mammalian subject in need of therapeutic or prophylactic
treatment
of lymphedema a composition comprising a polynucleotide, the polynucleotide
comprising a nucleotide sequence that encodes a vascular endothelial growth
factor D
(VEGF-D) polypeptide. Such methods are practiced essentially as described
herein
with respect to VEGF-C-encoding polynucleotides, except that polynucleotides
encoding VEGF-D are employed. A detailed description of the human VEGF-D gene
and protein are provided in Achen, et al., Proc. Nat'l Acad. Sci. U.S.A.,
95(2): 548-
553 (1998); International Patent Publication No. WO 98/07832, published 26
February
1998; and in Genbank Accession No. AJ000185, all incorporated herein by
reference.
CA 02283470 1999-09-29
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A cDNA and deduced amino acid sequence for prepro-VEGF-D is set forth herein
in
SEQ ID NOs: 5 and 6. Of course, due to the well-known degeneracy of the
genetic
code, multiple VEGF-D encoding polynucleotide sequence exist, any of which may
be
employed according to the methods taught herein.
As described herein in detail with respect to VEGF-C, the use of
polynucleotides that encode VEGF-D fragments, VEGF-D analogs, VEGF-D allelic
and interspecies variants, and the like which possess in vivo stimulatory
effects of
human VEGF-D are all contemplated as being encompassed by the present
invention.
In yet another embodiment, the invention provides a therapeutic or
prophylactic method of treatment for lymphedema, comprising the step of
administering to a mammalian subject in need of treatment for lymphedema a
composition comprising a VEGF-D polypeptide, in an amount effective to treat
or
prevent swelling associated with lymphedema. Administration via one or more
intravenous or subcutaneous injections is contemplated.
The VEGFR-3 allelic variant polynucleotides and polypeptides
described herein that were discovered and characterized by the present
inventors are
themselves considered aspects of the invention. Such polynucleotides and
polypeptides are useful, for example, in screening assays (e.g., cell-based
assays or
assays involving transgenic mice that express the polynucleotide in lieu of a
native
VEGF-3 allele) to study the biological activities of VEGFR-3 variant alleles
and
identify compounds that are capable of modulating that activity, e.g., to
identify
therapeutic candidates for treatment of lymphedema. Such screening assays are
also
considered aspects of the invention.
The polypeptides of the invention are intended to include complete
VEGFR-3 polypeptides with signal peptide (e.g., approximately residues 1 to 20
of
SEQ ID NO: 2), mature VEGFR-3 polypeptides lacking any signal peptide, and
recombinant variants wherein a foreign or synthetic signal peptide has been
fused to
the mature VEGFR-3 polypeptide. Polynucleotides of the invention include all
polynucleotides that encode all such polypeptides. It will be understood that
for
essentially any polypeptide, many polynucleotides can be constructed that
encode the
polypeptide by virtue of the well known degeneracy of the genetic code. All
such
polynucleotides are intended as aspects of the invention.
CA 02283470 1999-09-29
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Thus, in yet another aspect, the invention provides a purified
polynucleotide comprising a nucleotide sequence encoding a human VEGFR-3
protein
variant, wherein said polynucleotide is capable of hybridizing to the
complement of
SEQ m NO: 1 under stringent hybridization conditions, and wherein the encoded
VEGFR-3 protein variant has an amino acid sequence that differs at position
1114,
857, 1041, 1044 or 1049 from the amino acid sequence set forth in SEQ ID NO:
1.
Exemplary conditions are as follows: hybridization at 42°C in 50%
formamide, SX
SSC, 20 mM Na~P04, pH 6.8; and washing in 0.2X SSC at 55 °C. It is
understood by
those of skill in the art that variation in these conditions occurs based on
the length and
GC nucleotide content of the sequences to be hybridized. Formulas standard in
the art
are appropriate for determining appropriate hybridization conditions. [See
Sambrook
et al. ( 1989), supra, ~ ~ 9.47-9. S 1. ]
In a related embodiment, the invention provides a purified
polynucleotide comprising a nucleotide sequence encoding a VEGFR-3 protein of
a
human that is affected with heritable lymphedema or other lymphatic disorder;
wherein
the polynucleotide is capable of hybridizing to the complement of SEQ 117 NO:
1
under stringent hybridization conditions, and wherein the encoded
polynucleotide has
an amino acid sequence that differs from SEQ ID NO: 1 at at least one codon.
It will
be understood that conventional recombinant techniques can be used to isolate
such
polynucleotides from individuals affected with heritable lymphedema or their
relatives.
The wildtype VEGFR-3 cDNA sequence set forth in SEQ ID NO: 1 (or its
complement, or fragments thereof) is used as a probe to identify and isolate
VEGFR-3
sequences from nucleic acid derived from the individuals. Alternatively, PCR
amplification primers based on the wildtype VEGFR-3 sequence are generated and
used to amplify either VEGFR-3 genomic DNA or VEGFR-3 mRNA from the human
subject. The resultant amplified genomic DNA or cDNA is sequenced to determine
the variations that characterize the VEGFR-3 lymphedema allele of the
individual.
Preferred VEGFR-3 lymphedema alleles include, but are not limited to the
P1114L,
G857R, R1041P, L1044P and D1049N alleles described in detail herein.
In addition, the invention provides vectors that comprise the
polynucleotides of the invention. Such vectors are useful for amplifying and
expressing the VEGFR-3 proteins encoded by the polynucleotides, and for
creating
recombinant host cells and/or transgenic animals that express the
polynucleotides. The
CA 02283470 1999-09-29
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invention further provides a host cell transformed or transfected with
polynucleotides
(including vectors) of the invention. In a preferred embodiment, the host cell
expresses
the encoded VEGFR-3 protein on its surface. Such host cells are useful in cell-
based
screening assays for identifying modulators that stimulate or inhibit
signaling of the
encoded VEGFR-3. Modulators that stimulate VEGFR-3 signaling have utility as
therapeutics to treat lymphedemas, whereas modulators that are inhibitory have
utility
for treating hyperplastic lymphatic conditions mediated by the allelic variant
VEGFR-3.
In a preferred embodiment, host cells of the invention are co-transfected with
both a
wildtype and an allelic variant VEGFR-3 polynucleotide, such that the cells
express
both receptor types on their surface. Such host cells are preferred for
simulating a
heterozygous VEGFR-3 genotype of many individuals afl;'ected with lymphedema.
In yet another aspect, the invention provides a transgenic mammal, e.g.,
mouse, characterized by a non-native VEGFR-3 allele that has been introduced
into
the mouse, and the transgenic progeny thereof. Preferred allelic variants
include allelic
variants that correlate with hereditary lymphedema in human subjects, such as
an allelic
variant wherein a P1114L, G857R, R1041P, L1044P or D1049N missense mutation
has been introduced into the murine VEGFR-3 gene, or wherein the human P
1114L,
G857R, R1041P, L1044P or D1049N allelic variant has been substituted for a
murine
VEGFR-3 allele. Such mice are produced using standard methods. [See, e.g.,
Hogan
et al. (eds.), Manipulating the Mouse Embryo, Cold Spring Harbor, New York:
Cold
Spring Harbor Laboratory (1986).] The introduction of the human-like mutations
into
non-human sequences is readily achieved with standard techniques, such as site-
directed mutagenesis. The determination of which residues in a non-human
sequence
to alter to mimic the foregoing human mutations is routine since the foregoing
mutations all occur in regions of the VEGFR-3 sequence that contain residues
that are
highly conserved between species. See Figs. 3A-3B.
In yet another aspect, the invention provides assays for identifying
modulators of VEGFR-3 signaling, particularly modulators of the signaling of
allelic
variants of VEGFR-3 that correlate with lymphatic disorders such as heritable
lymphedema. For example, the invention provides a method for identifying a
modulator of intracellular VEGFR-3 signaling, comprising the steps o~
contacting a
cell expressing at least one mutant mammalian VEGFR-3 polypeptide in the
presence
and in the absence of a putative modulator compound; b) detecting VEGFR-3
CA 02283470 1999-09-29
- 23 -
signaling in the cell; and c) identifying a putative modulator compound in
view of
decreased or increased signaling in the presence of the putative modulator, as
compared to signaling in the absence of the putative modulator.
By "mutant mammalian VEGFR-3 polypeptide" is meant a VEGFR-3
polypeptide that varies from a wildtype mammalian VEGFR-3 polypeptide (e.g.,
by
virtue of one or more amino acid additions, deletions, or substitutions),
wherein the
variation is reflective of a naturally occurring variation that has been
correlated with a
lymphatic disorder, such as lymphedema. By way of example, the previously
described
substitution variations of human VEGFR-3, such as P1114L, have been correlated
with
heritable lymphedema. Any of the human allelic variants described above, or
analogous human allelic variants having a different substitution at the
indicated amino
acid positions, or a non-human VEGFR-3 into which a mutation at the position
corresponding to any of the described positions has been introduced are all
examples
of mutant mammalian VEGFR-3 polypeptides.
The detecting step can entail the detection of any parameter indicative
of VEGFR-3 signaling. For example, the detecting step can entail a measurement
of
VEGFR-3 autophosphorylation, or a measurement of VEGFR-3-mediated cell growth,
or a measurement of any step in the VEGFR-3 signaling cascade between VEGFR-3
autophosphorylation and cell growth.
In a preferred embodiment, the method is practiced with a cell that
expresses the mutant mammalian VEGFR-3 polypeptide and a wildtype mammalian
VEGFR-3 polypeptide. Such cells are thought to better mimic the conditions in
heterozygous individuals suffering from a VEGFR-3-mediated lymphatic disorder.
In
a highly preferred embodiment, the mutant and wildtype VEGFR-3 polypeptides
are
human. In the preferred embodiments, the mutant VEGFR-3 polypeptide comprises
a
leucine amino acid at the position corresponding to position 1114 of SEQ ID
NO: 2;
an arginine at the position corresponding to position 857 of SEQ ID NO: 2; a
proline
amino acid at the position corresponding to position 1041 of SEQ 117 NO: 2; a
proline
amino acid at the position corresponding to position 1044 of SEQ m NO: 2; or
an
asparagine at the position corresponding to position 1049 of SEQ ID NO: 2.
Additional features and variations of the invention will be apparent to
those skilled in the art from the entirety of this application, including the
drawing and
detailed description, and all such features are intended as aspects of the
invention.
CA 02283470 1999-09-29
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Likewise, features of the invention described herein can be re-combined into
additional
embodiments that are also intended as aspects of the invention, irrespective
of whether
the combination of features is specifically mentioned above as an aspect or
embodiment of the invention. Also, only such limitations which are described
herein as
critical to the invention should be viewed as such; variations of the
invention lacking
limitations which have not been described herein as critical are intended as
aspects of
the invention.
In addition to the foregoing, the invention includes, as an additional
aspect, all embodiments of the invention narrower in scope in any way than the
variations specifically mentioned above. Although the applicants) invented the
full
scope of the claims appended hereto, the claims appended hereto are not
intended to
encompass within their scope the prior art work of others. Therefore, in the
event that
statutory prior art within the scope of a claim is brought to the attention of
the
applicants by a Patent Office or other entity or individual, the applicants)
reserve the
right to exercise amendment rights under applicable patent laws to redefine
the subject
matter of such a claim to specifically exclude such statutory prior art or
obvious
variations of statutory prior art from the scope of such a claim. Variations
of the
invention defined by such amended claims also are intended as aspects of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-1F depict pedigrees of six hereditary lymphedema families
(Families 101, 106, 111, 135, 105, and 127, respectively) informative for
linkage.
Filled symbols represent individuals with clinically documented lymphedema.
Crossed
symbols represent individuals with an ambiguous phenotype. An ambiguous
phenotype is defined as self reported swelling of the limbs with no known
cause,
without a clinical diagnosis of lymphedema. Individuals of ambiguous phenotype
were
coded as disease status unknown for the linkage analysis. The proband in each
family
is indicated by an arrow.
Figure 2 is a graph summarizing VITESSE analysis of lymphedema
families with markers localized to chromosome Sq34-q35. In the graph, filled
circles
represent analyses for Families 101, 105, 106, and 111; open boxes represent
analyses
for Families 101, 106, and 111; open circles represent the VEGFR-3 gene; and
open
triangles represent Family 135. The one LOD confidence interval lies
completely
CA 02283470 1999-09-29
- 25 -
within the interval flanked by markers DSS1353 and DSS408 and overlaps the
most
likely location of Flt4 (VEGFR-3). Linkage is excluded for the entire region
for family
135.
Figure 3A-3B depict an alignment of portions of the human (top line,
SEQ ID NO: 2) and murine (bottom line, GenBank Acc. No. P35917, SEQ ID NO:
19) VEGFR-3 amino acid sequences to demonstrate similarity. Identical residues
are
marked with a line, and highly conserved and less conserved differences are
marked
with two dots or a single dot, respectively. The location of various mutations
that
have been observed to correlate with a heritable lymphedema phenotype are
indicated
immediately beneath the aligned sequences.
DETAILED DESCRIPTION OF THE INVENTION
Certain therapeutic aspects of the present invention involve the
administration of Vascular Endothelial Growth Factor C or D polynucleotides
and
polypeptides. The growth factor VEGF-C, as well as native human, non-human
mammalian, and avian polynucleotide sequences encoding VEGF-C, and VEGF-C
variants and analogs, have been described in detail in International Patent
Application
Number PCT/LTS98/01973, filed 02 February 1998 and published on 06 August 1998
as International Publication Number WO 98/33917; in Joukov et al., J. Biol.
Chem.,
273(12): 6599-6602 (1998); and in Joukov et al., EMBO J., 16(13): 3898-3911
(1997), all of which are incorporated herein by reference in the entirety. As
explained
therein in detail, human VEGF-C is initially produced in human cells as a
prepro-
VEGF-C polypeptide of 419 amino acids. A cDNA and deduced amino acid sequence
for human prepro-VEGF-C are set forth in SEQ 117 NOs: 3 and 4, respectively,
and a
cDNA encoding human VEGF-C has been deposited with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 (USA),
pursuant to the provisions of the Budapest Treaty (Deposit date of 24 July
1995 and
ATCC Accession Number 97231). VEGF-C sequences from other species have also
been reported. See Genbank Accession Nos. MMU73620 (Mus musculus); and
CCY15837 (Coturnix coturnix) for example, incorporated herein by reference.
The prepro-VEGF-C polypeptide is processed in multiple stages to
produce a mature and most active VEGF-C polypeptide of about 21-23 kD (as
CA 02283470 1999-09-29
-26-
assessed by SDS-PAGE under reducing conditions). Such processing includes
cleavage of a signal peptide (SEQ 117 NO: 4, residues 1-31); cleavage of a
carboxyl-
terminal peptide (corresponding approximately to amino acids 228-419 of SEQ ID
NO: 4 and having a pattern of spaced cysteine residues reminiscent of a
Balbiani ring 3
protein (BR3P) sequence [Dignam et al., Gene, 88:133-40 (1990); Paulsson et
al., J.
Mol. Biol., 211:331-49 (1990)]) to produce a partially-processed form of about
29
kD; and cleavage (apparently extracellularly) of an amino-terminal peptide
(corresponding approximately to amino acids 32-103 of SEQ 1D NO: 4) to produce
a
fully-processed mature form of about 21-23 kD. Experimental evidence
demonstrates
that partially-processed forms of VEGF-C (e.g., the 29 kD form) are able to
bind the
VEGFR-3 receptor, whereas high affinity binding to VEGFR-2 occurs only with
the
fully processed forms of VEGF-C.
Moreover, it has been demonstrated that amino acids 103-227 of SEQ
ID NO: 4 are not all critical for maintaining VEGF-C functions. A polypeptide
consisting of amino acids 113-213 (and lacking residues 103-112 and 214-227)
of
SEQ ID NO: 2 retains the ability to bind and stimulate VEGFR-3, and it is
expected
that a polypeptide spanning from about residue 131 to about residue 211 will
retain
VEGF-C biological activity. The cysteine residue at position 156 has been
shown to
be important for VEGFR-2 binding ability. However, VEGF-C ~Cls6 polypeptides
(i.e., analogs that lack this cysteine due to deletion or substitution) remain
potent
activators of VEGFR-3, and are therefore considered to be among the preferred
candidates for treatment of lymphedema. (It has been shown that a VEGF-C C
156S
serine substitution analog promotes lymphatic growth when over-expressed in
the skin
of transgenic mice behind the K14 promotee, in a manner analogous to what was
described in Jeltsch et al., Science, 276:1423 (1997), incorporated herein by
reference.) The cysteine at position 165 of SEQ B7 NO: 4 is essential for
binding to
either receptor, whereas analogs lacking the cysteines at positions 83 or 137
compete
with native VEGF-C for binding with both receptors and are able to stimulate
both
receptors.
An alignment of human VEGF-C with VEGF-C from other species
(performed using any generally accepted alignment algorithm) suggests
additional
residues wherein modifications can be introduced (e.g., insertions,
substitutions, and/or
deletions) without destroying VEGF-C biological activity. Any position at
which
CA 02283470 1999-09-29
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aligned VEGF-C polypeptides of two or more species have different amino acids,
especially different amino acids with side chains of different chemical
character, is a
likely position susceptible to modification without concomitant elimination of
function.
An exemplary alignment of human, murine, and quail VEGF-C is set forth in
Figure 5
of PCT/US98/01973.
Apart from the foregoing considerations, it will be understood that
innumerable conservative amino acid substitutions can be performed to a
wildtype
VEGF-C sequence which are likely to result in a polypeptide that retains VEGF-
C
biological activities, especially if the number of such substitutions is
small. By
"conservative amino acid substitution" is meant substitution of an amino acid
with an
amino acid having a side chain of a similar chemical character. Similar amino
acids for
making conservative substitutions include those having an acidic side chain
(glutamic
acid, aspartic acid); a basic side chain (arginine, lysine, histidine); a
polar amide side
chain (glutamine, asparagine); a hydrophobic, aliphatic side chain (leucine,
isoleucine,
1 S valine, alanine, glycine); an aromatic side chain (phenylalanine,
tryptophan, tyrosine); a
small side chain (glycine, alanine, serine, threonine, methionine); or an
aliphatic
hydroxyl side chain (serine, threonine). Addition or deletion of one or a few
internal
amino acids without destroying VEGF-C biological activities also is
contemplated.
Without intending to be limited to a particular theory, the mechanism
behind the ei~icacy of VEGF-C in treating or preventing lymphedema is believed
to
relate to the ability of VEGF-C to stimulate VEGFR-3 signaling. Administration
of
VEGF-C in quantities exceeding those usually found in interstitial fluids is
expected to
stimulate VEGFR-3 in human subjects who, by virtue of a dominant negative
heterozygous mutation, have insufficient VEGFR-3 signaling.
The growth factor named Vascular Endothelial Growth Factor D
(VEGF-D), as well as human sequences encoding VEGF-D, and VEGF-D variants and
analogs, have been described in detail in International Patent Application
Number
PCT/LJS97/14696, filed 21 August 1997 and published on 26 February 1998 as
International Publication Number WO 98/07832; and in Achen, et al., Proc.
Nat'l
Acad. Sci. U.S.A., 95(2): 548-553 (1998), both incorporated herein by
reference in the
entirety. As explained therein in detail, human VEGF-D is initially produced
in human
cells as a prepro-VEGF-D polypeptide of 354 amino acids. A cDNA and deduced
amino acid sequence for human prepro-VEGF-D are set forth in SEQ m Nos: 5 and
6,
CA 02283470 1999-09-29
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respectively. VEGF-D sequences from other species also have been reported. See
Genbank Accession Nos. D89628 (Mus musculus); and AF014827 (Rattus
norvegicus), for example, incorporated herein by reference.
The prepro-VEGF-D polypeptide has a putative signal peptide of 21
amino acids and is apparently proteolytically processed in a manner analogous
to the
processing of prepro-VEGF-C. A "recombinantly matured" VEGF-D lacking residues
1-92 and 202-354 of SEQ ID NO: 6 retains the ability to activate receptors
VEGFR-2
and VEGFR-3, and appears to associate as non-covalently linked dimers. Thus,
preferred VEGF-D polynucleotides include those polynucleotides that comprise a
nucleotide sequence encoding amino acids 93-201 of SEQ ID NO: 6.
The subject matter of the invention is further described and
demonstrated with reference to the following examples.
EXAMPLE 1
Demonstration that hereditary lymphedema is linked to the VEGFR-3 locus
The following experiments, conducted to identify a gene or genes
contributing to susceptibility to develop lymphedema, demonstrated that
hereditary
lymphedema correlates, in at least some families, to the chromosomal locus for
the
VEGFR-3 gene.
O VERVIEW
Families with inherited lymphedema were identified for the purpose of
conducting a linkage and positional candidate gene analysis. Thirteen distinct
families
from the United States and Canada were identified through referrals from
lymphedema
treatment centers, lymphedema support groups, and from Internet correspondence
(worldwide web site at www.pitt.edu/~genetics/lymph~. The study protocol was
approved by the Institutional Review Board of the University of Pittsburgh and
participants gave written informed consent. All members of the families were
of
western European ancestry. Forty members of one family ("Family 101") were
examined during a family reunion by a physiatrist experienced in lymphedema
treatment. Family members were considered affected with hereditary lymphedema
if
they exhibited asymmetry or obvious swelling of one or both legs. Members of
the
other 12 families were scored as affected if they had received a medical
diagnosis of
lymphedema, or if there were personal and family reports of extremity swelling
or
CA 02283470 1999-09-29
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asymmetry. Medical records were obtained to verify status whenever possible.
For
the purpose of linkage analysis, individuals with very mild or intermittent
swelling,
heavyset legs, obesity, or a history of leg infections as the only symptom
were
considered to have indeterminate disease status.
In the 13 families, 105 individuals were classified as affected, with a
male:female ratio of 1:2.3. The age of onset of lymphedema symptoms ranged
from
prenatal (diagnosed by ultrasound) to age SS. When affected by normal matings
were
analyzed, 76 of 191 children were affected, yielding a penetrance of 80%.
First degree
relatives of affected individuals were considered at risk.
Biological samples were obtained from members of the thirteen families
to conduct the genetic analyses. DNA was isolated from the EDTA-anticoagulated
whole blood by the method of Miller et al., Nucleic Acids Res., 16: 1215
(1998), and
from cytobrush specimens using the Puregene DNA isolation kit (Gentra Systems,
Minneapolis, MN). Analysis of the markers used in the genome scan were
performed
1 S by methods recognized in the art. [See Browman et al., Am. J. Hum.
Genetic., 63:861-
869 (1998); see also the NHLBI Mammalian Genotyping Service world-wide web
sites
(www.marshmed.org/genetics/methods/pcr.htm; and
www.marshmed.org/genetics/methods/gel.htm).
Two-point linkage analysis was conducted using an autosomal
dominant model predicting 80% penetrance in the heterozygous state, 99%
penetrance
in the homozygous state, and a 1% phenocopy rate. The frequency of the disease
allele was set at 1/10,000. Microsatellite marker allele frequencies were
calculated by
counting founder alleles, with the addition of counts of non-transmitted
alleles.
Multipoint analysis was carried out using distances obtained from the Location
Database (LDB-http://cedar.genetics.soton.ac.uk/public html). Multipoint and 2-
point
analyses were facilitated using the VITESSE (vl. l) program. [O'Connell, J.R.
and
Weeks, D.E., (1995), Nature Genet., 11:402-408].
DETAILED DESCRIPTION OF METHODS AND RESULTS
The first family studied, Family 101, was a large, mufti-generational
family demonstrating early onset lymphedema. (See Fig. 1.) Forty individuals
of this
family were examined and DNA sampled. In addition, blood was obtained from
another 11 members from mailing kits. Linkage simulation was performed using
CA 02283470 1999-09-29
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SLINK [Weeks et al., Am. J. Hum. Genet. 47: A204 (1990)] and linkage was
analyzed
using MSIM [Ott, J., Proc. Nat. Acad. Sci. USA, 86:4175-4178 (1989)] to
estimate
the potential power of two point linkage analysis in the family. Marker
genotypes
were simulated for a marker with heterozygosity of 0.875 under a linked (8=0)
and
unlinked (8=0.5) model using the 51 available individuals. The simulation
showed that
the power to detect linkage was greater than 90% for a LOD score threshold of
Z(8)
2Ø The false positive rate was less than 5%.
Shortly thereafter, two additional families (designated Families 106 and
111) segregating for autosomal dominant lymphedema were identified. These
three
families (Figures lA-1C, Families 101, 106 and 111) were genotyped for 366
autosomal markers by the NHLBI Mammalian Genotyping Service
(www.marshmed.org/genetics). Genotypes were checked for consistency using
Pedcheck [O'Connell, J.R. and Weeks, D.E., Am. J. Hum. Genet., 61:A288
(1997)].
Two point linkage analysis was performed using VITESSE [O'Connell, J.R. and
Weeks, D.E., Nature Genet., 11:402-408 (1995)). The model for linkage assumed
an
autosomal dominant model of inheritance, a disease allele frequency of 0.0001
and a
penetrance of 0.80.
The results from the genomic scan can be briefly summarized as
follows. A summed LOD score of greater than 4.0 was observed from distal
chromosome 5, markers DSS1456, DSS817 and DSS488. The markers on distal
chromosome Sq were the only markers having Z>3.0, the criteria established for
statistical significance. LOD scores greater than 2.0 (8=0-0.15) were also
detected for
chromosome 12 (D12S391 Z=2.03, all families), and chromosome 21 (D21SI440
Z=2.62, all families). The largest two-point LOD (Z=4.3; 8=0) was observed for
marker DSS408, localized to chromosome Sq34-q35.
This initial chromosomal mapping was further refined by genotyping the
three affected families for eight additional markers localized to region Sq34-
q35. Six
of these were informative for linkage (DSS653, DSS498, DSS408, DSS2006,
DSS1353
and DSS1354). Linkage analysis of these markers using VITESSE yielded a 2-
point
LOD score of 6.1 at B=0 for marker DSS1354 (Table 1) and a maximum multipoint
LOD score of 8.8 at marker DSSI354 (Fig. 2). These findings supported the
localization of a gene within chromosome band Sq34-q35 that is a predisposing
factor
in hereditary lymphedema.
CA 02283470 1999-09-29
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TABLE 1
LOD scores for individual families estimated over
the interval defined by markers DSS498 and DSS2006.
Z(8) Z(8) 0.01Z(6)0.05 Z(8) Z(6) 0.2
0.0 0.1
Locus DSS498
Family 101 -3.18 -2.33 -0.45 0.42 0.88
Family 106 1.08 1.07 1.05 0.99 0.81
Family 111 -0.85 -0.77 -0.53 -0.34 -0.13
Family 105 1.22 1.20 1.11 0.98 0.72
Family 135 -2.48 -1.85 -1.12 -0.75 -0.38
Locus DSSI353
Family 101 -2.99 -2.48 -1.21 -0.63 -0.18
Family 106 0.28 0.29 0.3 5 0. 3 0. 3 8
8
Family 111 -1.06 -1.02 -0.88 -0.72 -0.42
Family 105 0.72 0.71 0.65 0.56 0.39
Family 135 -8.03 -4.18 -2.09 -1.13 -0.30
Locus DSS1354
Family 101 6.09 6.02 5.69 5.21 4.07
Family 106 1.42 1.40 1.32 1.20 0.96
Family 111 0.21 0.22 0.23 0.24 0.22
Family 105 0.43 0.42 0.40 0.36 0.28
Family 135 -6.88 -4.91 -3.20 -2.16 -1.07
Locus DSS408
Family 101 2.80 2.74 2.50 2.20 1.56
Family 106 0.66 0.68 0.73 0.76 0.71
Family 111 -1.70 -1.40 -0.80 -0.44 -0.10
Family 105 0.42 0.41 0.38 0.35 0.27
Family 135 -5.22 -4.24 -2.58 -1.67 -0.80
CA 02283470 1999-09-29
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Z(8) Z(8) 0.01 Z(8)0.05 Z(8) 0.1 Z(8) 0.2
0.0
Locus DSS2006
Family 101 4.51 4.70 4.85 4.66 3.80
Family 106 1.17 1.16 1.11 1.03 0.83
Family 111 -1.32 -1.18 -0.82 -0.56 -0.25
Family 105 0.43 0.42 0.40 0.36 0.28
Family 135 -3.86 -3.20 -2.11 -1.45 -0.73
During the completion of the genome scan, an additional ten
lymphedema families were ascertained. Two of these families (Families 105 and
135,
see Figures lE and 1D), were potentially informative for linkage and were
genotyped
for markers in the linked region. Examination of the two point LOD scores for
the five
informative families for markers in the linked region (Table 1) shows that
four of the
families (101, 105, 106 and 111) are consistent with linkage to chromosome 5q
while
family 135 excluded linkage across the entire region with LOD scores Z =< -2.0
for all
markers. Multipoint linkage analysis of Families 101, 105, 106 and 111 (Fig.
2)
yielded a peak LOD score of Z = 10 at marker DSS1354. These findings support
the
existence of at least two loci which predispose to hereditary lymphedema.
The order of markers DSS1353, DSSI354 and DSS408 with respect to
each other was uncertain. Multipoint linkage analysis using alternative orders
for these
markers gave similar results. Marker DSS498 is a framework marker and marker
DSS408 is mapped 11.2 centimorgans distal to DSS498, based on the CHIC
chromosome 5 sex averaged, recombination minimized map, version 3
(www.chlc.org). The physical distance between DSS498 and DSS408 is estimated
as
1.45 megabases based on the Genetic Location Database (LDB) chromosome 5
summary map (cedar.genetics.soton.ac.uk/public html~.
Database analysis identified sixteen genes within this region. Two of
these genes have been identified as having roles in development (MSX2 and
VEGFR-
3). MSX2 was considered an unlikely candidate gene for lymphedema because of
its
known involvement in craniofacial development [Jabs et al., Cell, 75: 443-450
(1993)]. VEGFR-3, the gene encoding a receptor for VEGF-C, was selected as a
better candidate gene for initial fi.~rther study for the following reasons.
CA 02283470 1999-09-29
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(1) VEGFR-3 is expressed in developing lymphatic endothelium in the
mouse [Kukk et al., Development, 122: 3829-3837 (1996); and
Kaipainen et al., Proc. Nat. Acad. Sci. USA, 92: 3566-3570 (1995)];
(2) expression of VEGFR-3 is induced in differentiating avian
S chorioallantoic membrane [Oh et al., Dev. Biol., 188:96-109 (1997)];
and
(3) overexpression of VEGF-C, a ligand of VEGFR-3, leads to hyperplasia
of the lymphatic vessels in transgenic mice [Jeltsch et al., Science, 276:
1423-1425 (1997)].
To explore the potential role of VEGFR-3 in lymphedema, probands
from the thirteen lymphedema families were screened for variation by direct
sequencing of portions of the VEGFR-3 gene. The sequencing strategy used
amplification primers generated based upon the VEGFR-3 cDNA sequence (SEQ ID
NO: 1) and information on the genomic organization of the related vascular
endothelial
growth factor receptor-2 (VEGFR-2/KDRlflk-I) [Yin et al., Mammalian Genome, 9:
408-410 (1998)]. Variable positions (single nucleotide polymorphisms), the
unique
sequence primers used to amplify sequences flanking each variable site, and
the method
of detecting each variant are summarized in Table 2.
CA 02283470 1999-09-29
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TABLE 2
Location, amplification primer sequences, amplification conditions, and
detection
mPthndc fnr fve intrauenic cinule nucleotide nolvmornhisms in the human VEGFR-
3 gene
Position Primer 1 sequencePrimer 2 Ann. [MgClz]Base Detection
sequence
in VEGFR-3 temp. changeMethod
gene
Exon 12, tcaccatcgatccaagcagttctgegtgagccgag56 1.o CT sequencing
amino C mM
acid 641 (SEQ m NO: (SEQ m NO:
7) 8)
Exon 24, caggacggggtgacttgagcccaggectgtctactg56 1.0 CT Sequencing
amino C mM
acid 1114
(SEQ m NO: (SEQ m NO:
9) 10)
Exon 3, ccagctcctacgtgttcgggcaacagctggatgtca56 1.0 C-T Hhal
amino C mM
acid 175 (SEQ m NO:
11 ) (SEQ m NO:
12)
656p 3' ctgtgagggcgtgggagtgtcctttgagccactgga54 1.5 GA Styl
to Exon C mM
6
(SEQ m NO: (SEQ 117
13) NO: 14)
SSbp 3' cacacgtcatcgacaccggtgggcaacagctggatgtca56 1.5 CT Apal
to Exon C mM
2
(SEQ m NO: (SEQ m NO:
15) 16)
All amplifications were done for 35 cycles with denaturation at 94° for
30 seconds,
annealing as above for 30 seconds, and extension at 72° for 30 seconds.
Amplification and sequencing primers were synthesized by the DNA Synthesis
Facility,
University of Pittsburgh. Amplification primers were tagged at the S' end with
the
forward or reverse M13 universal sequence to facilitate direct sequencing.
Amplimers
were subjected to cycle sequencing using the dRhodamine terminator ready
reaction
kit or the Dye Primer ready reaction kit for -M13 and M13 Rev primers (Perkin
Elmer)
and analyzed on the Prism ABI 377 fluorescent sequencer. Sequences were
aligned
for further analysis using SEQUENCHER 3.0 (Gene Codes).
Genomic sequence from approximately 50% of the VEGFR-3 gene was
determined in this manner, and five single nucleotide variants were observed.
Two of
the variants occurred in introns, and a third was a silent substitution in
predicted exon
3. These intragenic polymorphisms were used to map the VEGFR-3 gene. As shown
in Figure 2, VEGFR-3 maps within the region of chromosome Sq linked to the
lymphedema phenotype, consistent with it being selected as a candidate gene.
In two
families, (Family 127, pedigree not shown, and Family 135), a C~T transition
was
identified at nucleotide position 1940 of the VEGFR-3 cDNA (SEQ m NO: 1). This
nucleotide substitution is predicted to lead to a non-conservative
substitution of serine
(codon TCC) for proline (codon CCC) at residue 641 (putative exon 12, within
the
sixth immunoglobulin-like region of the receptor's extracellular domain) of
the amino
CA 02283470 1999-09-29
- 35 -
acid sequence of the receptor (SEQ ID NO: 2). However, this sequence change
was
observed in 2 of 120 randomly selected individuals from the general population
(240
alleles). Also, in one of the two families in which this variant was initially
detected,
family 135, linkage between lymphedema and chromosome 5q markers was excluded
(Table 1 and Figure 2). In probands from the other ten families, wild type
sequence
was observed at nucleotide position 1940. Collectively, these results suggest
that this
P641 S variant is not causative.
In one nuclear family (Family 127, pedigree shown in Figure 1F) a C~T
transition was observed at nucleotide position 3360 (SEQ ID NO: 1) of the
IfEGFR-3
cDNA. This nucleotide substitution is predicted to lead to a non-conservative
substitution of leucine (codon CTG) for proline (codon CCG) at residue 1114 of
the
amino acid sequence of the receptor (SEQ ID NO: 2). This P1114L mutation is
predicted to lie in the intracellular tyrosine kinase domain II involved in
intracellular
signaling [Pajusola et al., Cancer Res., 52:5738- 5743 (1992)]. Direct
sequencing of
predicted exon 24 of the VEGFR-3 gene alleles from members of this family
identified
this substitution only in affected and at-risk family members. This sequence
change
was not observed in 120 randomly selected individuals of mixed European
ancestry
from the general population (240 alleles). In probands from the other 11
families, wild
type sequence was observed at nucleotide position 3360.
Collectively, this data demonstrates that a missense mutation that
causes a non-conservative substitution in a kinase domain of the VEGFR-3
protein
correlates strongly with a heritable lymphedema in one family, and suggests
that other
mutations in the same gene may exist that correlate with heritable lymphedema
in other
families. As explained above, only a portion of the VEGFR-3 gene sequence was
analyzed to identify this first mutant of interest. Additional sequencing,
using standard
techniques and using the known VEGFR-3 gene sequence for guidance, is expected
to
identify additional mutations of interest that are observed in affected and at-
risk
members of other families studied.
CA 02283470 1999-09-29
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EXAMPLE 2
Demonstration that a C--T missense mutation at position 3360
in the VEGFR-3 coding sequence results in a tyrosine kinase negative mutant
The results set forth in Example 1 identified two missense mutations in
the VEGFR-3 coding sequence, one of which (C~T at position 3360) appeared to
correlate with heritable lymphedema and one of which (C~T transition at
position
1940) did not. The following experiments were conducted to determine the
biochemical significance of these mutations on VEGFR-3 biological activity.
To analyze how the two single amino acid substitutions affect the
VEGFR-3-mediated signaling, the corresponding mutant receptor expression
vectors
were generated using site-directed mutagenesis procedures and expressed in
293T cells
by transient transfection. The long form of human VEGFR-3 cDNA (SEQ 1D NO: 1)
was cloned as a Hind III-Bam HI fragment from the LTR-FLT41 plasmid [Pajusola
et
al., Oncogene 8: 2931-2937 (1993)] into pcDNA3.1/Z(+) (Invitrogen). The P641S
and P 1114L mutants of VEGFR-3 were generated from this construct with the
GeneEditor~ in vitro Site-Directed Mutagenesis System (Promega) using the
following oligonucleotides (the C~T mutations are indicated with bold
letters):
5'-CCTGAGTATCTCCCGCGTCGC-3' (SEQ ID NO: 17) for P641 S
mutation; and
5'-GGTGCCTCCCTGTACCCTGGG-3' (SEQ 117 NO: 18) for P 1114L
mutation.
For the transient expression studies, 293T cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum (GIBCO BRL, Life Technologies, Gaithersburg, MD), glutamine, and
antibiotics. Cells were transfected with 20 pg of plasmid encoding the wild
type or
mutant VEGFR-3 forms using the calcium phosphate method, and harvested 36
hours
after transfection for immunoprecipitation and Western blotting. Under these
conditions, RTK overexpression results in ligand-independent activation, thus
allowing
the receptor phosphorylation to be studied. An empty vector was used for mock
(control) transfections. (It will be appreciated that ligand stimulation
assays of
VEGFR-3 forms also can be employed, e.g., as described in U. S. Patent No.
5,776,755, incorporated herein by reference, using VEGF-C or VEGF-D ligands.)
CA 02283470 1999-09-29
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In order to investigate the effect of the two VEGFR-3 mutants on the
tyrosine phosphorylation of the VEGFR-3, Western blotting analysis was
performed
using anti-phosphotyrosine antibodies. The cell monolayers were washed three
times
with cold phosphate-buffered saline (PBS, containing 2 mM vanadate and 2 mM
PMSF) and scraped into RIPA buffer (150 mM NaCI, 1 % Nonidet P40, 0.5
deoxycholic acid sodium salt, 0.1 % SDS, 50 mM Tris-HCI, pH 8.0) containing 2
mM
Vanadate, 2 mM PMSF, and 0.07 U/ml Aprotinin.
The cell lysates were sonicated and centrifuged for 10 minutes at
19,000 X g, and the supernatants were incubated for 2 hours on ice with 2
pg/ml of
monoclonal anti-VEGFR-3 antibodies (9D9f~) [Jussila ei al., Cancer Res., 58:
1599-604 (1998)]. Thereafter, Protein A sepharose (Pharmacia) beads were added
and incubation was continued for 45 minutes with rotation at +4°C. The
sepharose
beads were then washed three times with ice-cold RIPA buffer and twice with
PBS
(both containing 2 mM vanadate, 2 mM PMSF), analyzed by 7.5 % SDS-PAGE and
transferred to a nitrocellulose filter (Protran Nitrocellulose, Schleicher &
Schuell, No.
401196) using semi-dry transfer apparatus. After blocking the filter with 5 %
BSA in
TBS-T buffer (10 mM Tris, pH 7.5, 150 mM NaCI, 0.05 % Tween 20), the filters
were
incubated with the phosphotyrosine-specific primary antibodies (Upstate
Biotechnology, #OS-321), followed by biotinylated goat-anti-mouse
immunoglobulins
(Dako, E0433) and Biotin-Streptavidin HRP complex (Amersham, RPN1051). The
bands were visualized by the enhanced chemiluminescence (ECL) method.
After analysis for phosphotyrine-containing proteins, the filters were
stripped by washing for 30 minutes at +50°C in 100 mM 2-
mercaptoethanol, 2 % SDS,
62.5 mM Tris-HCI, pH 6.7, with occasional agitation. The filters were washed
with
TBS-T, blocked again with BSA as described above, and analyzed for the
presence of
VEGFR-3 using the 9D9f~ antibodies and HRP-conjugated rabbit-anti-mouse
immunoglobulins (Dako, P0161).
The Western analyses revealed that the P641 S mutant receptor was
phosphorylated normally, i.e., in a manner similar to the wild type control.
However,
the proteolytic processing of the P641 S receptor protein may be affected, as
the 175
kD and 125 kD polypeptides seemed to have a higher relative density when
compared
to the 195 kD form.
CA 02283470 1999-09-29
-38-
In contrast, no phosphorylated P 1114L mutant protein was detected
using the phosphotyrosine antibodies. The expression of similar amounts of the
VEGFR-3 protein (normal and both mutants) was confirmed using the monoclonal
9D9f~ antibody, which is directed towards the extracellular domain of the
VEGFR-3.
Both the P641 S and the P 1114L mutant VEGFR-3 migrated slightly faster than
the
wild type VEGFR-3 in the gel electrophoresis.
In order to analyze the possible dominant negative effect of the Pl 114L
mutant on the wild-type receptor, a second, similar set of experiments were
performed
wherein the 293T yells were transfected with an increasing amount of the
P1114L
expression vector in combination with decreasing amounts of the wild type
vector.
Wild type to mutant ratios of 1:0, 3:1, 1:1, 1:3 and 0:1 were used. The cells
were
lysed 48 hours after transfection and the lysates were analyzed by
immunoprecipitation
and Western blotting as described above. These experiments permitted
evaluation of
whether the mutant protein interferes with wild type protein phosphorylation
and
estimation of the minimal amount of the WT protein needed for observable
tyrosyl
autophosphorylation. Immunoprecipitates from cells transfected with only the
WT
plasmid revealed WT protein that was strongly phosphorylated in this
experiment (lane
2), whereas immunoprecipitates from cells transfected with only the mutant
plasmid
were again inactive (unphosphorylated).
Interestingly, when transfection was made using 75% of WT and 25
of mutant plasmid, the phosphorylation of the receptors was decreased by about
90%.
This result strongly suggests that the P1114L mutant receptor forms
heterodimers with
the WT receptor, but cannot phosphorylate the WT receptor, thus failing to
activate it.
Under this theory, the WT receptor monomers in the heterodimers would also
remain
inactive, causing a disproportionate decrease of the total amount of activated
receptor,
when co-transfected with the mutant. Wildtype-wildtype homodimers would remain
active and be responsible for the observed signaling. When the wild type and
mutant
receptor expression vectors were transfected at a 1:1 ratio, the VEGFR-3
phosphorylation was about 4% of the wild type alone, whereas at a 1:3 ratio,
no
tyrosine phosphorylation of VEGFR-3 was observed.
The foregoing results are consistent with the linkage analyses in
Example 1: the mutation at position 641 that did not appear to correlate with
lymphedema also did not appear to be disfunctional, whereas the mutation at
position
CA 02283470 1999-09-29
-39-
1114 appeared to cause a dominant negative mutation that shows no tyrosine
phosphorylation alone and that drastically reduces VEGFR-3 signaling in cells
expressing both the mutant and wild type VEGFR-3 genes.
Collectively, these data indicate that the P1114L VEGFR-3 mutant is
unable to act as a part of the signaling cascade, and also acts in a dominant
negative
manner, thus possibly interfering partially with the activation of the wild
type
VEGFR-3. Such effects of the mutation may eventually lead to lymphedema.
EXAMPLE 3
Treatment of lymphedema with a VEGFR-3 ligand
The data from Examples 1 and 2 collectively indicate a causative role in
heritable lymphedema for a mutation in the VEGFR-3 gene that interferes with
VEGFR-3 signaling. Such a mutation behaves in an autosomal dominant pattern,
due
to the apparent necessity for receptor dimerization in the signaling process.
However,
the data from Example 2 suggests that some residual signaling may still occur
in
1 S heterozygous affected individuals, presumably through pairing of VEGFR-3
proteins
expressed from the wild type allele. The following experiments are designed to
demonstrate the efficacy of VEGFR-3 ligand treatment in such affected
individuals, to
raise VEGFR-3 signaling to levels approaching normal and thereby
ameliorate/palliate
the symptoms of hereditary lymphedema.
Initially, an appropriate animal model is selected. Several potential
animal models have been described in the literature. [See, e.g., Lyon et al.,
Mouse
News Lett. 71: 26 (1984), Mouse News Lett. 74: 96 (1986), and Genetic variants
and
strains of the laboratory mouse, 2nd ed., New York: Oxford University Press
(1989),
p. 70 (Chylous ascites mouse); Dumont et al., Science, 282: 946-949 (1998)
(heterozygous VEGFR-3 knockout mouse); Patterson et al., "Hereditary
Lymphedema," Comparative Pathology Bulletin, 3: 2 (1971) (canine hereditary
lymphedema model); van der Putte, "Congenital Hereditary Lymphedema in the
Pig,"
Lympho, ll: 1-9 (1978); and Campbell-Beggs et al., "Chyloabdomen in a neonatal
foal," Veterinary Record, 137: 96-98 (1995).] Those models which are
determined to
have analogous mutations to the VEGFR-3 gene are preferred. Analogous
mutations
would include mutations affecting corresponding residues and also mutations
affecting
different residues but causing similar functional alterations. The Chylous
ascites
CA 02283470 1999-09-29
-40-
mouse VEGFR-3 gene contains a missense mutation at a position corresponding to
residue 1053 of SEQ ID No. 2, which maps to the catalytic pocket region of the
tyrosine kinase catalytic domain. Thus, the "Chy" mouse is expected to display
similar
functional alterations to human mutations affecting tyrosine kinase activity,
a
prediction which can be confirmed by functional assays such as those described
in
Example 2. In a preferred embodiment, "knock in" homologous recombination
genetic
engineering strategies are used to create an animal model (e.g., a mouse
model) having
a VEGFR-3 allelic variation analogous to the human variations described
herein. [See,
e.g., Partanen et al., Genes & Development, 12: 2332-2344 (1998) (gene
targeting to
introduce mutations into another receptor protein (FGFR-1) in mice).] For
example,
the P 1114L mutation in human VEGFR-3 occurs in a VEGFR-3 region having highly
conserved amino acid identity with murine VEGFR-3 (Genbank Accession No.
L07296). Thus, a corresponding P1114L can be introduced into the murine VEGFR-
3
by "knock-in" homologous recombination. Optionally, such mice can be bred to
the
heterozygous VEGFR-3 knockout mice or Chy mice described above to further
modify
the phenotypic severity of the lymphedema disease.
The mice as described above are treated with a candidate therapeutic,
e.g., a recombinant mature form of VEGF-C, at various dosing schedules, e.g.,
once
daily by intravenous (IV) or intramuscular (IM) injection at a dose of 1-1000
ng/g
body weight, preferably 10-100 ng/g, which should result in a peak level
saturating
VEGFR-3 (Kd about 150 pM) but not VEGFR-2 (Kd around 400 pM). For VEGFR-
3-specific forms, such as VEGF-CoClss, even higher dosing is contemplated, to
sustain
VEGFR-3-saturating physiological concentrations for longer periods. Direct IM
injection at multiple sites in the muscles of affected extremities is a
preferred route of
administration. The dosing is adjusted according to the efficacy of the
treatment and
the presence of possible side effects due to the lowering of blood pressure,
which has
been observed in response to VEGF administration IV. The efficacy of treatment
is
measured via IVMRI imaging of the water content and volume of swelling of the
abdomen and the extremities of the animals. The amount of fluid in the
abdominal
cavity is estimated and the animals are weighed during the follow-up.
In studies using VEGFR-3 -l+ x Chy mice progeny, the animals will
also have the 13-galactosidase marker in their lymphatic endothelium. After a
successful treatment, the treated and non-treated experimental animals and
VEGFR-3
CA 02283470 1999-09-29
-41 -
-/+ controls are killed and their lymphatic vessels are visualized by f3-gal
and antibody
staining. The staining patterns of experimental and control animals are
compared for
vessel diameter, numbers of endothelial cells, density of blood and lymphatic
vessels,
and nuclear density/section surface area for the estimation of tissue oedema.
S Such experiments are repeated with various candidate therapeutics
(e.g., VEGF-C or VEGF-D recombinant polypeptides; VEGF-C and VEGF-D gene
therapy vectors; and combinations thereof) at various dosing schedules to
determine an
optimum treatment regimen.
EXAMPLE 4
Chromosomal structure of the human VEGFR-3 gene
Sequencing and mapping of human DNA corresponding
to the VEGFR-
3 locus icated that this gene consists of thirty exons
has ind separated by twenty-nine
introns
of varying
size. The
exon intron
organization
is summarized
as follows:
EXON Bp of SEQ ID NO: 1
NUMBER size
(bpl INTRON
SIZE
1 20-77
58 by unknown
2 78-174
97 by > 1 kb
3 175-419
245 by 218 by
4 420-532
113 by 120 by
5 533-695
163 by 107 by
6 696-835
140 by 269 by
7 836-1004
169 by 261 by
8 1005-1122
118 by >1 kb
9 1123-1277
CA 02283470 1999-09-29
-42-
155 by unknown
10 1278-1440
163 by > 1 kb
11 1441-1567
127 by unknown
12 1568-1676
109 by unknown
13 1677-2039
363 by 293 by
14 2040-2186
147 by 99 by
15 2187-2318
132 by approx. 160 by
16 2319-2425
107 by 301 by
17 2426-2561
139 by >464 by
18 2562-2666
105 by unknown
19 2667-2780
114 by 143 by
20 2781-2869
89 by >1 kb
21 2870-3020
151 by unknown
22 3021-3115
95 by unknown
23 3116-3 23 8
123 by unknown
24 3239-3350
112 by 974 by
25 3351-3450
100 by 400 by
CA 02283470 1999-09-29
- 43 -
26 3451-3557
107 by unknown
27 3558-3705
148 by > 1 kb
28 3706-3826
121 by unknown
29 3 827-3 912
86 by unknown
30a (Flt4 short) 3913-4111
199 by 3.7 kb
30b (Flt4long) 3913-4416
>504 by (CDS 504 bp)
The foregoing information permits rapid design of oligonucleotides for
amplifying
select portions of the VEGFR-3 gene from genomic DNA, or RNA, or cDNA, to
facilitate rapid analysis of an individual's VEGFR-3 coding sequence, to
determine
whether the individual possesses a mutation that correlates with a lymphedema
phenotype.
EXAMPLE 5
Identification of additional non-conservative missense mutants
Using procedures essentially as described in Example 1, the VEGFR-3
coding sequences from additional affected and unaffected individuals from
families
having members suffering from heritable lymphedema were studied. The analysis
focused on families with statistical linkage to chromosome 5q as described in
Example
1. The additional analysis included the PCR amplification and sequencing of
Exon 17,
Exon 22, and Exon 23 sequences with the following PCR primers:
Exon 17-1 S'-CATCAAGACGGGCTACCT-3' (SEQ B7 NO: 23)
Exon 17-2 5'-CCGCTGACCCCACACCTT-3' (SEQ ID NO: 24)
Exon 22-1 5'-GAGTTGACCTCCCAAGGT-3' (SEQ 1D NO: 25)
Exon 22-2 5'-TCTCCTGGACAGGCAGTC-3' (SEQ >D NO: 26)
Exon 23-1 5'-GAGTTGACCTCCCAAGGT-3' (SEQ m NO. 27)
Exon 23-2 5'-TCTCCTGGACAGGCAGTC-3' (SEQ 1D NO. 28)
CA 02283470 1999-09-29
-44-
These additional studies identified four additional non-conservative
missense mutations in evolutionarily conserved amino acids in kinase domains I
and II
of human VEGFR-3. Each mutation, shown in Table 3 below, was observed in a
single independently ascertained family, and in each family, the mutation co-
segregates
with individuals suffering from, or considered at risk for developing,
lymphedema.
None of these mutations were observed in the VEGFR-3 genes in a random sample
of
more than 300 chromosomes from individuals from families unai~licted with
heritable
lymphedema.
TABLE 3
Mutations in VEGFR-3 causing Hereditary Lymphedema*
Nucleotide Amino Acid Functional
Exon Substitution**Substitution Domain
24 C3360T P1114L Kinase 2
17 G2588A G857R Kinase 1
23 G3141C R1041P Kinase 2
23 T3150C L1044P Kinase 2
23 G3164A D 1049N Kinase 2
* Numbers indicate nucleotide or amino acid positions in SEQ ID NOs: 1 and 2.
**It will be appeciated that, since DNA is double-stranded, each mutation
could be
characterized in two equivalent ways, depending on whether reference is being
made to
the coding or the non-coding strand.
Referring to SEQ >D NO: 2, the kinase domains of VEGFR-3 comprise
approximately residues 843-943 and residues 1009-1165. Within these domains,
molecular modeling suggests that residues 6852, 6854, 6857, K879, E896, H1035,
D1037, N1042, D1055, F1056, 61057, E1084, D1096 and 81159 are of particular
importance in comprising or shaping the catalytic pocket within the kinase
domains.
See van Der Geer and Hunter, Ann. Rev. Cell. Biol., 10: 251-337 (1994); and
Mohammadi et al., Cell 86: 577-587 (1996). Thus, this data identifying
additional
mutations implicate missense mutations within a kinase domain of the VEGFR-3
protein as correlating strongly with a risk for developing a heritable
lymphedema
phenotype. Mutations which affect residues in and around the catalytic pocket
appear
particularly likely to correlate with lymphedema. The P 1114L mutation, though
not
CA 02283470 1999-09-29
-45-
situated within the catalytic pocket, is postulated to cause a conformational
alteration
that affects the catalytic pocket. The G857R mutation is postulated to block
the
catalytic pocket and/or the ATP binding site of the kinase domain.
EXAMPLE 6
Functional Analysis of Additional VEGFR-3 missense mutations
Using procedures essentially as described above in Example 2, the
functional state ofthe G857R, L1044P, and D1049N mutations were analyzed.
(PLCLB buffer, comprising 150 mm NaCI, 5% glycerol, 1%Triton X-100, 1.SM
MgCl2, 50 mm HEPES, pH 7.5, was substituted for RIPA buffer described in
Example
2 for immunoprecipitation and Western blotting protocols.) A VEGFR-3-encoding
construct comprising the G857R mutation was generated from the long form of
human
VEGFR-3 cDNA using the oligonucleotide:
5'-CGG CGC CTT CAG GAA GGT GGT-3' (SEQ ID NO: 20)
A construct comprising the L 1044P mutation was generated from the long form
of
human VEGFR-3 cDNA using the oligonucleotide:
5'-CGG AAC ATT CCG CTG TCG GAA-3' (SEQ ID NO: 21)
A construct comprising the D 1049N mutation was generated from the long form
of
human VEGFR-3 cDNA using the oligonucleotide:
5'-GTC GGA AAG CAA CGT GGT GAA-3' (SEQ ID NO: 22).
The constructs were transiently transfected into 293T cells and
harvested for Western blotting essentially as described in Example 2, except
for the
buffer substitution described above. In contrast to wild type VEGFR-3 and
VEGFR-3
containing the P641S mutation, no phosphorylated G857R or L1044P mutant
protein
was detected using the phosphotyrosine antibodies, consistent with the results
that had
been observed for P1114L. The expression of similar amounts of the VEGFR-3
protein was confirmed using the monoclonal 9D9f~7 antibody, which is directed
towards the extracellular domain of the VEGFR-3 in the Western blotting. This
data
suggested that these observed mutations did indeed affect VEGFR-3 kinase
function.
The D1049N mutant appeared to retain at least some tyrosine kinase activity.
It is also
noteworthy that VEGFR-1 and VEGFR-2 contain an asparagine residue at the
position
in their tyrosine kinase domains which corresponds to position 1049 of VEGFR-
3.
CA 02283470 1999-09-29
a
-46- i:
ata suggest that the D 1049N variation may only be an allelic variant '
that correlates with hereditary lymphedema, rather than a causative mutation.
To determine whether the VEGFR-3 mutants function in a dominant
negative manner, each construct was co-transfected at varying ratios with wild
type
receptor into 293T cells essentially as described in Example 2. Unlike the
results
observed for P 1114L and described in Example 2, neither the G857R mutant nor
the
L1044P mutant seemed to interfere with phosphorylation of the co-transfected
wild
type receptor.
The absence of a dominant negative effect in these experiments does
not foreclose a conclusion that the mutations described above are causative.
It has
been found that a significant fraction of ligand-activated receptor tyrosine
kinases
traffic to the lysosomal compartment after internalization, where they are
degraded.
However, receptors which are not ligand-activated preferentially recycle back
to the
cell surface after internalization. Thus, it is possible that the turnover
time of the
weakly phosphorylated mutant receptor is significantly longer than that of the
wild
type receptor protein. If this were true, the amount of the mutant receptor on
the
endothelial cell surface could be considerably higher than the amount of the
phosphorylated and rapidly internalized wild type receptor, and any available
ligand
would thus bind a disproportionally high number of mutant receptors. Both a
possible
dominant negative effect of the mutant receptor and an abnormally long half
life of the
tyrosine kinase negative mutant receptor could eventually lead to lymphedema.
Alternatively, a mutation that merely decreases (but does not eliminate) VEGFR-
3
tyrosine kinase activity may display a constitutive low level of
internalization and
degravdation that is insufficient to trigger sufficient downstream signalling,
but
decreases the effective concentration of VEGFR-3 on cell surfaces for ligand
binding
and effective activation, leading eventually to lymphedema.
While the present invention has been described in terms of specific
embodiments, it is understood that variations and modifications will occur to
those in
the art, all of which are intended as aspects of the present invention.
Accordingly, only
such limitations as appear in the claims should be placed on the invention.
CA 02283470 1999-12-23
' 47
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LUDWIG INSTITUTE FOR CANCER RESEARCH -AND- HELSINKI
UNIVERSITY LICENSING LTD. OY -AND- UNIVERSITY OF
PITTSBURG OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION
(ii) TITLE OF INVENTION: SCREENING AND THERAPY FOR LYMPHATIC DISORDERS
INVOLVING THE FLT4 RECEPTOR TYROSINE KINASE
(VEGFR-3 )
ZO (111) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
2 ~ (B) COMPUTER: IHM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,283,470
(B) FILING DATE: 29-SEPT-1999
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 09/375,248
(B) FILING DATE: 16-AUG-1999
30 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART 6 BIGGAR
CA 02283470 1999-12-23
48
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCI~T NUMBER: 64267-1017
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO.: 1:.
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 4111
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE
(A) NAME/ID;Y: CDS
(B) LOCATION: (20)..(4111)
2 (C) OTHER INFORMATION: Human Flt4 (VEGFR-3) long form
0 cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:
CCACGCGCAG CGGCCGGAG ATG CAG CGG GGC GCC GCG CTG TGC 52
CTG CGA CTG
Met Gln Arg Gly Ala Ala Leu Cys Leu Arg Leu
1 5 10
TGG CTC TGC CTG GGA CTC CTG GAC GGC CTG GTG AGT GGC 100
TAC TCC ATG
Trp Leu Cys Leu Gly Leu Leu Asp Gly Leu Val Ser Gly
Tyr Ser Met
20 25
30 ACC CCC CCG ACC TTG AAC ATC ACG GAG GAG TCA CAC GTC 148
ATC GAC ACC
Thr Pro Pro Thr Leu Asn Ile Thr Glu Glu Ser His Val
Ile Asp Thr
30 35 40
GGT GAC AGC CTG TCC ATC TCC TGC AGG GGA CAG CAC CCC 196
CTC GAG TGG
Gly Asp Ser Leu Ser Ile Ser Cys Arg Gly Gln His Pro
Leu Glu Trp
45 50 55
GCT TGG CCA GGA GCT CAG GAG GCG CCA GCC ACC GGA GAC 244
AAG GAC AGC
Ala Trp Pro Gly Ala Gln Glu Ala Pro Ala Thr Gly Asp
Lys Asp Ser
4 60 65 70 75
0
CA 02283470 1999-12-23
' 49
GAG GAC ACG GGG GTG GTG CGA GAC TGC GAG GGC ACA GAC GCC AGG CCC 292
Glu Asp Thr Gly Val Val Arg Asp Cys Glu Gly Thr Asp Ala Arg Pro
80 85 90
TAC TGC AAG GTG TTG CTG CTG CAC GAG GTA CAT GCC AAC GAC ACA GGC 340
Tyr Cys Lys Val Leu Leu Leu His Glu Val His Ala Asn Asp Thr Gly
95 100 105
AGC TAC GTC TGC TAC TAC AAG TAC ATC AAG GCA CGC ATC GAG GGC ACC 388
Ser Tyr Val Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr
110 115 120
ACG GCC GCC AGC TCC TAC GTG TTC GTG AGA GAC TTT GAG CAG CCA TTC 436
Thr Ala Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe
125 130 135
ATC AAC AAG CCT GAC ACG CTC TTG GTC AAC AGG AAG GAC GCC ATG TGG 484
2 0 Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp
140 145 150 155
GTG CCC TGT CTG GTG TCC ATC CCC GGC CTC AAT GTC ACG CTG CGC TCG 532
Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser
160 165 170
CAA AGC TCG GTG CTG TGG GCA GAC GGG CAG GAG GTG GTG TGG GAT GAC 580
Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp
175 180 185
CGG CGG GGC ATG CTC GTG TCC ACG CCA CTG CTG CAC GAT GCC CTG TAC 628
Arg Arg Gly Met Leu Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr
190 195 200
CTG CAG TGC GAG ACC ACC TGG GGA GAC CAG GAC TTC CTT TCC AAC CCC 676
Leu Gln Cys Glu Thr Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro
205 210 215
TTC CTG GTG CAC ATC ACA GGC AAC GAG CTC TAT GAC ATC CAG CTG TTG 724
4 0 Phe Leu Val His Ile Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu
220 225 230 235
CCC AGG AAG TCG CTG GAG CTG CTG GTA GGG GAG AAG CTG GTC CTG AAC 772
Pro Arg Lys Ser Leu Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn
240 245 250
TGC ACC GTG TGG GCT GAG TTT AAC TCA GGT GTC ACC TTT GAC TGG GAC 820
Cys Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp
255 260 265
TAC CCA GGG AAG GAG GCA GAG CGG GGT AAG TGG GTG CCC GAG CGA CGC 868
Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg
270 275 280
TCC CAG CAG ACC CAC ACA GAA CTC TCC AGC ATC CTG ACC ATC CAC AAC 916
Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn
285 290 295
GTC AGC CAG CAC GAC CTG GGC TCG TAT GTG TGC AAG GCC AAC AAC GGC 964
Val Ser Gln His Asp Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly
300 305 310 315
CA 02283470 1999-12-23
ATC CAG CGA TTT CGG GAG AGC ACC GAG GTC ATT GTG CAT GAA AAT CCC 1012
Ile Gln Arg Phe Arg Glu Ser Thr Glu Val Ile Val His Glu Asn Pro
320 325 330
TTC ATC AGC GTC GAG TGG CTC AAA GGA CCC ATC CTG GAG GCC ACG GCA 1060
Phe Ile Ser Val Glu Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala
335 340 345
10 GGA GAC GAG CTG GTG AAG CTG CCC GTG AAG CTG GCA GCG TAC CCC CCG 1108
Gly Asp Glu Leu Val Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro
350 355 360
CCC GAG TTC CAG TGG TAC AAG GAT GGA AAG GCA CTG TCC GGG CGC CAC 1156
Pro Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His
365 370 375
AGT CCA CAT GCC CTG GTG CTC AAG GAG GTG ACA GAG GCC AGC ACA GGC 1204
Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly
2 0 380 385 390 395
ACC TAC ACC CTC GCC CTG TGG AAC TCC GCT GCT GGC CTG AGG CGC AAC 1252
Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn
400 405 410
ATC AGC CTG GAG CTG GTG GTG AAT GTG CCC CCC CAG ATA CAT GAG AAG 1300
Ile Ser Leu Glu Leu Val Val Asn Val Pro Pro Gln Ile His Glu Lys
415 420 425
30 GAG GCC TCC TCC CCC AGC ATC TAC TCG CGT CAC AGC CGC CAG GCC CTC 1348
Glu Ala Ser Ser Pro Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu
430 435 440
ACC TGC ACG GCC TAC GGG GTG CCC CTG CCT CTC AGC ATC CAG TGG CAC 1396
Thr Cys Thr Ala Tyr Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His
445 450 455
TGG CGG CCC TGG ACA CCC TGC AAG ATG TTT GCC CAG CGT AGT CTC CGG 1444
Trp Arg Pro Trp Thr Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg
40 460 465 470 475
CGG CGG CAG CAG CAA GAC CTC ATG CCA CAG TGC CGT GAC TGG AGG GCG 1492
Arg Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala
480 485 490
GTG ACC ACG CAG GAT GCC GTG AAC CCC ATC GAG AGC CTG GAC ACC TGG 1540
Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp
495 500 505
5O ACC GAG TTT GTG GAG GGA AAG AAT AAG ACT GTG AGC AAG CTG GTG ATC 1588
Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile
510 515 520
CAG AAT GCC AAC GTG TCT GCC ATG TAC AAG TGT GTG GTC TCC AAC AAG 1636
Gln Asn Ala Asn Val Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys
525 530 535
GTG GGC CAG GAT GAG CGG CTC ATC TAC TTC TAT GTG ACC ACC ATC CCC 1684
Val Gly Gln Asp Glu Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro
540 545 550 555
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GAC GGC TTC ACC ATC GAA TCC AAG CCA TCC GAG GAG CTA CTA GAG GGC 1732
Asp Gly Phe Thr Ile Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly
560 565 57p
CAG CCG GTG CTC CTG AGC TGC CAA GCC GAC AGC TAC AAG TAC GAG CAT 1780
Gln Pro Val Leu Leu Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His
575 580 585
CTG CGC TGG TAC CGC CTC AAC CTG TCC ACG CTG CAC GAT GCG CAC GGG 1828
Leu Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp Ala His Gly
590 595 600
AAC CCG CTT CTG CTC GAC TGC AAG AAC GTG CAT CTG TTC GCC ACC CCT 1876
Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro
605 610 615
CTG GCC GCC AGC CTG GAG GAG GTG GCA CCT GGG GCG CGC CAC GCC ACG 1924
Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr
2 0 620 625 630 635
CTC AGC CTG AGT ATC CCC CGC GTC GCG CCC GAG CAC GAG GGC CAC TAT 1972
Leu Ser Leu Ser Ile Pro Arg Val Ala Pro Glu His Glu Gly His Tyr
640 645 650
GTG TGC GAA GTG CAA GAC CGG CGC AGC CAT GAC AAG CAC TGC CAC AAG 2020
Val Cys Glu Val Gln Asp Arg Arg Ser His Asp Lys His Cys His Lys
655 660 665
30 AAG TAC CTG TCG GTG CAG GCC CTG GAA GCC CCT CGG CTC ACG CAG AAC 2068
Lys Tyr Leu Ser Val Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn
670 675 680
TTG ACC GAC CTC CTG GTG AAC GTG AGC GAC TCG CTG GAG ATG CAG TGC 2116
Leu Thr Asp Leu Leu Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys
685 690 695
TTG GTG GCC GGA GCG CAC GCG CCC AGC ATC GTG TGG TAC AAA GAC GAG 2164
Leu Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu
4 0 700 705 710 715
AGG CTG CTG GAG GAA AAG TCT GGA GTC GAC TTG GCG GAC TCC AAC CAG 2212
Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln
720 725 730
AAG CTG AGC ATC CAG CGC GTG CGC GAG GAG GAT GCG GGA CGC TAT CTG 2260
Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu
735 740 745
50 TGC AGC GTG TGC AAC GCC AAG GGC TGC GTC AAC TCC TCC GCC AGC GTG 2308
Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val
750 755 760
GCC GTG GAA GGC TCC GAG GAT AAG GGC AGC ATG GAG ATC GTG ATC CTT 2356
Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu
765 770 775
GTC GGT ACC GGC GTC ATC GCT GTC TTC TTC TGG GTC CTC CTC CTC CTC 2404
Val Gly Thr Gly Val Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu
60 780 785 790 795
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ATC TTC TGT AAC ATG AGG AGG CCG GCC CAC GCA GAC ATC AAG ACG GGC 2452
Ile Phe Cys Asn Met Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly
800 805 810
TAC CTG TCC ATC ATC ATG GAC CCC GGG GAG GTG CCT CTG GAG GAG CAA 2500
Tyr Leu Ser Ile Ile Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln
815 820 825
lO TGC GAA TAC CTG TCC TAC GAT GCC AGC CAG TGG GAA TTC CCC CGA GAG 2548
Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu
830 835 840
CGG CTG CAC CTG GGG AGA GTG CTC GGC TAC GGC GCC TTC GGG AAG GTG 2596
Arg Leu His Leu Gly Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val
845 850 855
GTG GAA GCC TCC GCT TTC GGC ATC CAC AAG GGC AGC AGC TGT GAC ACC 2644
Val Glu Ala Ser Ala Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr
2 0 860 865 870 875
GTG GCC GTG AAA ATG CTG AAA GAG GGC GCC ACG GCC AGC GAG CAC CGC 2692
Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg
880 885 890
GCG CTG ATG TCG GAG CTC AAG ATC CTC ATT CAC ATC GGC AAC CAC CTC 2740
Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn His Leu
895 900 905
30 AAC GTG GTC AAC CTC CTC GGG GCG TGC ACC AAG CCG CAG GGC CCC CTC 2788
Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu
910 915 920
ATG GTG ATC GTG GAG TTC TGC AAG TAC GGC AAC CTC TCC AAC TTC CTG 2836
Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu
925 930 935
CGC GCC AAG CGG GAC GCC TTC AGC CCC TGC GCG GAG AAG TCT CCC GAG 2884
Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu
4 0 940 945 950 955
CAG CGC GGA CGC TTC CGC GCC ATG GTG GAG CTC GCC AGG CTG GAT CGG 2932
Gln Arg Gly Arg Phe Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg
960 965 970
AGG CGG CCG GGG AGC AGC GAC AGG GTC CTC TTC GCG CGG TTC TCG AAG 2980
Arg Arg Pro Gly Ser Ser Asp Arg Val Leu Phe Ala Arg Phe Ser Lys
975 980 985
50 ACC GAG GGC GGA GCG AGG CGG GCT TCT CCA GAC CAA GAA GCT GAG GAC 3028
Thr Glu Gly Gly Ala Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp
990 995 1000
CTG TGG CTG AGC CCG CTG ACC ATG GAA GAT CTT GTC TGC TAC AGC TTC 3076
Leu Trp Leu Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe
1005 1010 1015
CAG GTG GCC AGA GGG ATG GAG TTC CTG GCT TCC CGA AAG TGC ATC CAC 3124
Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His
60 1020 1025 1030 1035
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AGA GAC CTG GCT GCT CGG AAC ATT CTG CTG TCG GAA AGC GAC GTG GTG 3172
Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val
1040 1045 1050
AAG ATC TGT GAC TTT GGC CTT GCC CGG GAC ATC TAC AAA GAC CCT GAC 3220
Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp
1055 1060 1065
TAC GTC CGC AAG GGC AGT GCC CGG CTG CCC CTG AAG TGG ATG GCC CCT 3268
Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro
1070 1075 1080
GAA AGC ATC TTC GAC AAG GTG TAC ACC ACG CAG AGT GAC GTG TGG TCC 3316
Glu Ser Ile Phe Asp Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser
1085 1090 1095
TTT GGG GTG CTT CTC TGG GAG ATC TTC TCT CTG GGG GCC TCC CCG TAC 3364
Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr
1100 1105 1110 1115
CCT GGG GTG CAG ATC AAT GAG GAG TTC TGC CAG CGG CTG AGA GAC GGC 3412
Pro Gly Val Gln Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly
1120 1125 1130
ACA AGG ATG AGG GCC CCG GAG CTG GCC ACT CCC GCC ATA CGC CGC ATG 3460
Thr Arg Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile
1135 1140 1145
ATG CTG AAC TGC TGG TCC GGA GAC CCC AAG GCG AGA CCT GCA TTC TCG 3508
Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser
1150 1155 1160
GAG CTG GTG GAG ATC CTG GGG GAC CTG CTC CAG GGC AGG GGC CTG CAA 3556
Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln
1165 1170 ~ 1175
GAG GAA GAG GAG GTC TGC ATG GCC CCG CGC AGC TCT CAG AGC TCA GAA 3604
Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu
4 0 1180 1185 1190 1195
GAG GGC AGC TTC TCG CAG GTG TCC ACC ATG GCC CTA CAC ATC GCC CAG 3652
Glu Gly Ser Phe Ser Gln Val Ser Thr Met Ala Leu His Ile Ala Gln
1200 1205 1210
GCT GAC GCT GAG GAC AGC CCG CCA AGC CTG CAG CGC CAC AGC CTG GCC 3700
Ala Asp Ala Glu Asp Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala
1215 1220 1225
5O GCC AGG TAT TAC AAC TGG GTG TCC TTT CCC GGG TGC CTG GCC AGA GGG 3748
Ala Arg Tyr Tyr Asn Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly
1230 1235 1240
GCT GAG ACC CGT GGT TCC TCC AGG ATG AAG ACA TTT GAG GAA TTC CCC 3796
Ala Glu Thr Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro
1245 1250 1255
ATG ACC CCA ACG ACC TAC AAA GGC TCT GTG GAC AAC CAG ACA GAC AGT 3844
Met Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser
60 1260 1265 1270 1275
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GGG ATG GTG CTG GCC TCG GAG TTTGAG CAG ATA GAG AGC AGG 3892
GAG CAT
Gly Met Val Leu Ala Ser Glu PheGlu Gln Ile Glu Ser Arg
Glu His
1280 1285 1290
AGA CAA GAA AGC GGC TTC AGC AAAGGA CCT GGC CAG AAT GTG 3940
TGT GCT
Arg Gln Glu Ser Gly Phe Ser LysGly Pro Gly Gln Asn Val
Cys Ala
1295 1300 1305
GTG ACC AGG GCA CAC CCT GAC CAAGGG AGG CGG CGG CGG CCT 3988
TCC GAG
Val Thr Arg Ala His Pro Asp GlnGly Arg Arg Arg Arg Pro
Ser Glu
1310 1315 1320
CGG GGG GCC CGA GGA GGC CAG TTTTAC AAC AGC GAG TAT GGG 4036
GTG GAG
Arg Gly Ala Arg Gly Gly Gln PheTyr Asn Ser Glu Tyr Gly
Val Glu
1325 1330 1335
CTG TCG GAG CCA AGC GAG GAG CACTGC TCC CCG TCT GCC CGC 4084
GAC GTG
Leu Ser Glu Pro Ser Glu Glu HisCys Ser Pro Ser Ala Arg
Asp Val
2 0 1340 1345 1350 1355
ACT TTC TTC ACA GAC AAC AGC TAA 4111
TAC
Thr Phe Phe Thr Asp Asn Ser
Tyr
1360
(2) INFORMATION FOR SEQ ID NO.:2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1363
30 (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO.:
2:
Met Gln Arg Gly Ala Ala Leu
Cys Leu Arg Leu Trp Leu Cys
Leu Gly
1 5 10 15
4 0 Leu Leu Asp Gly Leu Val Ser Gly Tyr Ser Met Thr Pro Pro Thr Leu
25 30
Asn Ile Thr Glu Glu Ser His Val Ile Asp Thr Gly Asp Ser Leu Ser
35 40 45
Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Ala Trp Pro Gly Ala
50 55 60
Gln Glu Ala Pro Ala Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val
50 65 70 75 80
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Val Arg Aap Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu
85 90 95
Leu Leu His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr
100 105 110
Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Ser
115 120 125
Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn Lys Pro Asp
130 135 140
Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp Val Pro Cys Leu Val
145 150 155 160
Ser Ile Pro Gly Leu Asn Val Thr Leu Arg Ser Gln Ser Ser Val Leu
165 170 175
2 0 Trp Pro Asp Gly Gln Glu Val Val Trp Asp Asp Arg Arg Gly Met Leu
180 185 190
Val Ser Thr Pro Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr
195 200 205
Thr Trp Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile
210 215 220
Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser Leu
30 225 230 235 240
Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala
245 250 255
Glu Phe Asn Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln
260 265 270
Ala Glu Arg Gly Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His
275 280 285
Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln His Asp
290 295 300
Leu Gly Ser Tyr Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg
305 310 315 320
Glu Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu
325 330 335
Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu Val
340 345 350
Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp
355 360 365
Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His Ser Pro His Ala Leu
370 375 380
Val Leu Lys Glu Val Thr Glu Ala Ser Thr Gly Thr Tyr Thr Leu Ala
385 390 395 400
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Leu Trp Asn Ser Ala Ala Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu
405 410 415
Val Val Asn Val Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro
420 425 430
Ser Ile Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr
435 440 445
Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp Thr
450 455 460
Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Gln
465 470 475 480
Asp Leu Met Pro Gln Cys Arg Asp Trp Arg Ala Val Thr Thr Gln Asp
485 490 495
2 0 Ala Val Asn Pro Ile Glu Ser Leu Asp Thr Trp Thr Glu Phe Val Glu
500 505 510
Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asn Ala Asn Val
515 520 525
Ser Ala Met Tyr Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu
530 535 540
Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile
30 545 550 555 560
Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu Leu
565 570 575
Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg Trp Tyr Arg
580 585 590
Leu Asn Leu Ser Thr Leu His Asp Ala His Gly Asn Pro Leu Leu Leu
595 600 605
Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Ala Ala Ser Leu
610 615 620
Glu Glu Val Ala Pro Gly Ala Arg His Ala Thr Leu Ser Leu Ser Ile
625 630 635 640
Pro Arg Val Ala Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln
645 650 655
Asp Arg Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val
660 665 670
Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu
675 680 685
Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val Ala Gly Ala
690 695 700
His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Glu
705 710 715 720
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Lys Ser Gly Val Asp Leu Ala Asp Ser Asn Gln Lys Leu Ser Ile Gln
725 730 735
Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn
740 745 750
Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser
755 760 765
Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Val Gly Thr Gly Val
770 775 780
Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met
785 790 795 800
Arg Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile
805 810 815
2 0 Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser
820 825 830
Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly
835 840 845
Arg Val Leu Gly Tyr Gly Ala Phe Gly Lys Val Val Glu Ala S~r Ala
850 855 860
Phe Gly Ile His Lys Gly Ser Ser Cys Asp Thr Val Ala Val Lys Met
30 865 870 875 880
Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu
885 890 895
Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu
900 905 910
Leu Gly Ala Cys Thr Lys Pro Gln Gly Pro Leu Met Val Ile Val Glu
915 920 925
Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Ala Lys Arg Asp
930 935 940
Ala Phe Ser Pro Cys Ala Glu Lys Ser Pro Glu Gln Arg Gly Arg Phe
945 950 955 960
Arg Ala Met Val Glu Leu Ala Arg Leu Asp Arg Arg Arg pro Gly Ser
965 970 975
SO Ser Asp A~g Val Leu Phe Ala Arg Phe Ser Lys Thr Glu Gly Gly Ala
980 985 990
Arg Arg Ala Ser Pro Asp Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro
995 1000 1005
Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly
1010 1015 1020
Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala
60 025 1030 1035 1040
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Arg Asn Ile Leu Leu Ser Glu Ser Asp Val Val Lys Ile Cys Asp Phe
1045 1050 1055
Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly
1060 1065 1070
Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp
1075 1080 1085
Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu
1090 1095 1100
Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile
105 1110 1115 1120
Asn Glu Glu Phe Cys Gln Arg Leu Arg Asp Gly Thr Arg Met Arg Ala
1125 1130 1135
2 0 Pro Glu Leu Ala Thr Pro Ala Ile Arg Arg Ile Met Leu Asn Cys Trp
1140 1145 1150
Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Glu Leu Val Glu Ile
1155 1160 1165
Leu Gly Asp Leu Leu Gln Gly Arg Gly Leu Gln Glu Glu Glu Glu Val
1170 1175 1180
Cys Met Ala Pro Arg Ser Ser Gln Ser Ser Glu Glu Gly Ser Phe Ser
30 185 1190 1195 1200
Gln Val Ser Thr Met Ala Leu His Ile Ala Gln Ala Asp Ala Glu Asp
1205 1210 1215
Ser Pro Pro Ser Leu Gln Arg His Ser Leu Ala Ala Arg Tyr Tyr Asn
1220 1225 1230
Trp Val Ser Phe Pro Gly Cys Leu Ala Arg Gly Ala Glu Thr Arg Gly
1235 1240 1245
Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr Thr
1250 1255 1260
Tyr Lys Gly Ser Val Asp Asn Gln Thr Asp Ser Gly Met Val Leu Ala
265 1270 1275 1280
Ser Glu Glu Phe Glu Gln Ile Glu Ser Arg His Arg Gln Glu Ser Gly
1285 1290 1295
Phe Ser Cys Lys Gly Pro Gly Gln Asn Val Ala Val Thr Arg Ala His
1300 1305 1310
Pro Asp Ser Gln Gly Arg Arg Arg Arg pro Glu Arg Gly Ala Arg Gly
1315 1320 1325
Gly Gln Val Phe Tyr Asn Ser Glu Tyr Gly Glu Leu Ser Glu Pro Ser
1330 1335 1340
Glu Glu Asp His Cys Ser Pro Ser Ala Arg Val Thr Phe Phe Thr Asp
345 1350 1355 1360
Asn Ser Tyr
CA 02283470 1999-12-23
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(2) INFORMATION FOR SEQ ID NO.: 3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1997
(H) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (352)..(1611)
(C) OTHER INFORMATION: Human eDNA
for prepro-VEGF-C
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO.: 3:
CCCGCCCCGC CTCTCCAAAA AGCTACACCG ACGCGGACCGCGGCGGCGTCCTCCCTCGCC
60
CTCGCTTCAC CTCGCGGGCT CCGAATGCGG GGAGCTCGGATGTCCGGTTTCCTGTGAGGC
120
TTTTACCTGA CACCCGCCGC CTTTCCCCGG CACTGGCTGGGAGGGCGCCCTGCAAAGTTG
160
GGAACGCGGA GCCCCGGACC CGCTCCCGCC GCCTCCGGCTCGCCCAGGGGGGGTCGCCGG
240
2 GAGGAGCCCG GGGGAGAGGG ACCAGGAGGG GCCCGCGGCCTCGCAGGGGCGCCCGCGCCC
O 300
CCACCCCTGC CCCCGCCAGC GGACCGGTCC CCCACCCCCGGTCCTTCCACC ATG CAC
357
Met His
1
TTG CTG GGC TTC TTC TCT GTG GCG TGT CTC GCC GCG CTG 405
TCT CTG GCT
Leu Leu Gly Phe Phe Ser Val Ala Cys Leu Ala Ala Leu
Ser Leu Ala
5 10 15
CTC CCG GGT CCT CGC GAG GCG CCC GCC GCC GCC GAG TCC 453
GCC GCC TTC
30 Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Glu Ser
Ala Ala Phe
25 30
GGA CTC GAC CTC TCG GAC GCG GAG CCC GGC GAG ACG GCT 501
GAC GCG GCC
Gly Leu Asp Leu Ser Asp Ala Glu Pro Gly Glu Thr Ala
Asp Ala Ala
35 40 45 50
TAT GCA AGC AAA GAT CTG GAG GAG CAG TCT GTG AGT GTA 549
TTA CGG TCC
Tyr Ala Ser Lys Asp Leu Glu Glu Gln Ser Val Ser Val
Leu Arg Ser
55 60 65
40
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GAT GAA CTC ATG ACT GTA CTC TAC CCA GAA TAT TGG AAA ATG TAC AAG 597
Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys
75 80
TGT CAG CTA AGG AAA GGA TGG CAA
GGC CAT AAC
AGA GAA
CAG GCC
AAC 645
Cys Gln Leu Arg Lys Gly Trp Gln Asn Arg Glu Gln Ala Asn
Gly His
85 90 95
10
CTC AAC TCA AGG ACA GAA ACT ATA TTT GCT GCA GCA CAT TAT
GAG AAA 693
Leu Asn Ser Arg Thr Glu Thr Ile Phe Ala Ala Ala His Tyr
Glu Lys
100 105 110
AAT ACA GAG ATC TTG AAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA 741
Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln
115 120 125 130
TGC ATG CCA CGG GAG GTG TGT ATA GAT GTG GGG AAG GAG TTT GGA GTC 789
2 0 Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val
135 140 145
GCG ACA AAC ACC TTC TTT AAA CCT CCA TGT GTG TCC GTC TAC AGA TGT 837
Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys
150 155 160
GGG GGT TGC TGC AAT AGT GAG GGG CTG CAG TGC ATG AAC ACC AGC ACG 885
Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr
165 170 175
AGC TAC CTC AGC AAG ACG TTA TTT GAA ATT ACA GTG CCT CTC TCT CAA 933
Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln
180 185 190
GGC CCC AAA CCA GTA ACA ATC AGT TTT GCC AAT CAC ACT TCC TGC CGA 981
Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg
195 200 205 210
TGC ATG TCT AAA CTG GAT GTT TAC AGA CAA GTT CAT TCC ATT ATT AGA 1029
4 0 Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg
215 220 225
CGT TCC CTG CCA GCA ACA CTA CCA CAG TGT CAG GCA GCG AAC AAG ACC 1077
Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr
230 235 240
TGC CCC ACC AAT TAC ATG TGG AAT AAT CAC ATC TGC AGA TGC CTG GCT 1125
Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala
245 250 255
CAG GAA GAT TTT ATG TTT TCC TCG GAT GCT GGA GAT GAC TCA ACA GAT 1173
Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp
260 265 270
GGA TTC CAT GAC ATC TGT GGA CCA AAC AAG GAG CTG GAT GAA GAG ACC 1221
Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr
275 280 285 290
TGT CAG TGT GTC TGC AGA GCG GGG CTT CGG CCT GCC AGC TGT GGA CCC 1269
Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro
295 300 305
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CAC AAA GAA CTA GAC AGA AAC TCA TGC CAG TGT GTC TGT AAA AAC AAA 1317
His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys
310 315 320
CTC TTC CCC AGC CAA TGT GGG GCC AAC CGA GAA TTT GAT GAA AAC ACA 1365
Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr
325 330 335
TGC CAG TGT GTA TGT AAA AGA ACC TGC CCC AGA AAT CAA CCC CTA AAT 1413
Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn
340 345 350
CCT GGA AAA TGT GCC TGT GAA TGT ACA GAA AGT CCA CAG AAA TGC TTG 1461
Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu
355 360 365 370
TTA AAA GGA AAG AAG TTC CAC CAC CAA ACA TGC AGC TGT TAC AGA CGG 1509
Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg
2 0 375 380 385
CCA TGT ACG AAC CGC CAG AAG GCT TGT GAG CCA GGA TTT TCA TAT AGT 1557
Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser
390 395 400
GAA GAA GTG TGT CGT TGT GTC CCT TCA TAT TGG AAA AGA CCA CAA ATG 1605
Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met
405 410 415
30 AGC TAA GATTGTACTG TTTTCCAGTT CATCGATTTT CTATTATGGA AAACTGTGTT 1661
Ser
420
GCCACAGTAG AACTGTCTGT GAACAGAGAG ACCCTTGTGG GTCCATGCTA ACAAAGACAA 1721
AAGTCTGTCT TTCCTGAACC ATGTGGATAA CTTTACAGAA ATGGACTGGA GCTCATCTGC 1781
AAAAGGCCTC TTGTAAAGAC TGGTTTTCTG CGAATGACCA AACAGCCAAG ATTTTCCTCT 1841
TGTGATTTCT TTAAAAGAAT GACTATATAA TTTATTTCCA CTAAAAATAT TGTTTCTGCA 1901
TTCATTTTTA TAGCAACAAC AATTGGTAAA ACTCACTGTG ATCAATATTT TTATATCATG 1961
CAAAATATGT TTAAAATAAA ATGAAAATTG TATTAT 1997
(2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 419
(H) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
CA 02283470 1999-12-23
' 62
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 4:
Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala
1 5 10 15
Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe
20 25 30
Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala
35 40 45
Thr Tyr Ser Asp Glu Glu Gln Ser
Ala Ala Lys Leu Leu Arg
Ser Val
50 55 60
Ser Asp GluLeu Thr Leu Tyr Pro Tyr Trp Met
Val Met Val Glu Lys
65 70 75 80
Tyr Cys GlnLeu Lys Gly Trp Gln Asn Arg Gln
Lys Arg Gly His Glu
85 90 95
Ala Leu AsnSer Thr Glu Thr Ile Phe Ala Ala
Asn Arg Glu Lys Ala
100 105 110
His Asn ThrGlu Leu Ser Ile Asp Glu Trp Lys
Tyr Ile Lys Asn Arg
115 120 125
Thr Cys MetPro Glu Cys Ile Asp Gly Lys Phe
Gln Arg Val Val Glu
130 135 140
Gly Ala ThrAsn Phe Lys Pro Pro Val Ser Tyr
Val Thr Phe Cys Val
145 150 155 160
Arg Gly GlyCys Asn Glu Gly Leu Cys Met Thr
Cys Cys Ser Gln Asn
165 170 175
Ser Ser TyrLeu Lys Leu Phe Glu Thr Val Leu
Thr Ser Thr Ile Pro
4 180 185 190
0
Ser Gly ProLys Val Ile Ser Phe Asn His Ser
Gln Pro Thr Ala Thr
195 200 205
Cys Cys MetSer Leu Val Tyr Arg Val His Ile
Arg Lys Asp Gln Ser
210 215 220
Ile Arg SerLeu Ala Leu Pro Gln Gln Ala Asn
Arg Pro Thr Cys Ala
225 230 235 240
50
Lys Cys ProThr Tyr Trp Ile Cys Cys
Thr Asn Met Asn Arg
Asn
His
245 250 255
Leu Gln Glu Ser Ser Asp Gly Asp Ser
Ala Asp Ala Asp
Phe
Met
Phe
260 265 270
CA 02283470 1999-12-23
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Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu
275 280 285
Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys
290 295 300
Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys
305 310 315 320
Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu
325 330 335
Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro
340 345 350
Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys
355 360 365
2 0 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr
370 375 380
Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser
385 390 395 400
Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro
405 410 415
Gln Met Ser
(2) INFORMATION
FOR SEQ
ID NO.:
5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2029
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
4 0 (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(ix) FEATURE
(A) NAME/KEY: CDS
(8) LOCATION: (411)..(1475)
(C) OTHER INFORMATION: Human prepro-VEGF-D
eDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.:
5:
CA 02283470 1999-12-23
' 64
GTTGGGTTCC AGCTTTCTGT AGCTGTAAGC ATTGGTGGCC ACACCACCTC 60
CTTACAAAGC
AACTAGAACC TGCGGCATAC ATTGGAGAGA TTTTTTTAAT TTTCTGGACA 120
TGAAGTAAAT
TTAGAGTGCT TTCTAATTTC AGGTAGAAGA CATGTCCACC TTCTGATTAT 180
TTTTGGAGAA
CATTTTGATT TTTTTCATCT CTCTCTCCCC ACCCCTAAGA TTGTGCAAAA 240
AAAGCGTACC
TTGCCTAATT GAAATAATTT CATTGGATTT TGATCAGAAC TGATTATTTG 300
GTTTTCTGTG
TGAAGTTTTG AGGTTTCAAA CTTTCCTTCT GGAGAATGCC TTTTGAAACA 360
ATTTTCTCTA
GCTGCCTGAT GTCAACTGCT TAGTAATCAG TGGATATTGA AATATTCAAA ATG TAC 416
Met Tyr
1
AGA GAG TGG GTA GTG GTG AAT GTT TTC ATG ATG TTG TAC GTC CAG CTG 464
Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu
5 10 15
GTG CAG GGC TCC AGT AAT GAA CAT GGA CCA GTG AAG CGA TCA TCT CAG 512
Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln
25 30
2 O TCC ACA TTG GAA CGA TCT GAA CAG CAG ATC AGG GCT GCT TCT AGT TTG 560
Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu
35 40 45 50
GAG GAA CTA CTT CGA ATT ACT CAC TCT GAG GAC TGG AAG CTG TGG AGA 608
Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu Trp Arg
55 60 65
TGC AGG CTG AGG CTC AAA AGT TTT ACC AGT ATG GAC TCT CGC TCA GCA 656
Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg Ser Ala
70 75 eo
TCC CAT CGG TCC ACT AGG TTT GCG GCA ACT TTC TAT GAC ATT GAA ACA 704
Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile Glu Thr
85 90 95
CTA AAA GTT ATA GAT GAA GAA TGG CAA AGA ACT CAG TGC AGC CCT AGA 752
Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg
100 105 110
GAA ACG TGC GTG GAG GTG GCC AGT GAG CTG GGG AAG AGT ACC AAC ACA 800
Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr
115 120 125 130
TTC TTC AAG CCC CCT TGT GTG AAC GTG TTC CGA TGT GGT GGC TGT TGC 848
Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys
135 140 145
AAT GAA GAG AGC CTT ATC TGT ATG AAC ACC AGC ACC TCG TAC ATT TCC 896
Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser
5 0 150 155 160
AAA CAG CTC TTT GAG ATA TCA GTG CCT TTG ACA TCA GTA CCT GAA TTA 944
Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu
165 170 175
CA 02283470 1999-12-23
GTG CCT GTT AAA GTT GCC AAT CAT ACA GGT TGT AAG TGC TTG CCA ACA 992
Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr
180 185 190
GCC CCC CGC CAT CCA TAC TCA ATT ATC AGA AGA TCC ATC CAG ATC CCT 1040
Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro
195 200 205 210
10 GAA GAA GAT CGC TGT TCC CAT TCC AAG AAA CTC TGT CCT ATT GAC ATG 1088
Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met
215 220 225
CTA TGG GAT AGC AAC AAA TGT AAA TGT GTT TTG CAG GAG GAA AAT CCA 1136
Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro
230 235 240
CTT GCT GGA ACA GAA GAC CAC TCT CAT CTC CAG GAA CCA GCT CTC TGT 1184
Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala Leu Cys
2 0 245 250 255
GGG CCA CAC ATG ATG TTT GAC GAA GAT CGT TGC GAG TGT GTC TGT AAA 1232
Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys
260 265 270
ACA CCA TGT CCC AAA GAT CTA ATC CAG CAC CCC AAA AAC TGC AGT TGC 1280
Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys Ser Cys
275 280 285 290
30 TTT GAG TGC AAA GAA AGT CTG GAG ACC TGC TGC CAG AAG CAC AAG CTA 1328
Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His Lys Leu
295 300 305
TTT CAC CCA GAC ACC TGC AGC TGT GAG GAC AGA TGC CCC TTT CAT ACC 1376
Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr
310 315 320
AGA CCA TGT GCA AGT GGC AAA ACA GCA TGT GCA AAG CAT TGC CGC TTT 1424
Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe
4 0 325 330 335
CCA AAG GAG AAA AGG GCT GCC CAG GGG CCC CAC AGC CGA AAG AAT CCT 1472
Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro
340 345 350
TGA TTCAGCGTTC CAAGTTCCCC ATCCCTGTCA TTTTTAACAG CATGCTGCTT 1525
355
TGCCAAGTTG CTGTCACTGT TTTTTTCCCA GGTGTTAAAA AAAAAATCCA TTTTACACAG 1585
5O CACCACAGTG AATCCAGACC AACCTTCCAT TCACACCAGC TAAGGAGTCC CTGGTTCATT 1645
GATGGATGTC TTCTAGCTGC AGATGCCTCT GCGCACCAAG GAATGGAGAG GAGGGGACCC 1705
ATGTAATCCT TTTGTTTAGT TTTGTTTTTG TTTTTTGGTG AATGAGAAAG GTGTGCTGGT 1765
CATGGAATGG CAGGTGTCAT ATGACTGATT ACTCAGAGCA GATGAGGAAA ACTGTAGTCT 1825
CTGAGTCCTT TGCTAATCGC AACTCTTGTG AATTATTCTG ATTCTTTTTT ATGCAGAATT 1885
CA 02283470 1999-12-23
66
TGATTCGTAT GATCAGTACT GACTTTCTGA TTACTGTCCA GCTTATAGTC TTCCAGTTTA 1945
ATGAACTACC ATCTGATGTT TCATATTTAA GTGTATTTAA AGAAAATAAA CACCATTATT 2005
2029
(2) INFORMATION FOR SEQ 6:
ID NO.:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 354
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: ID 6:
SEQ NO.:
Met Tyr Arg Glu Trp Val AsnVal PheMet LeuTyrVal
Val Val Met
1 5 10 15
2 Gln Leu Val Gln Gly Ser GluHis GlyPro LysArgSer
0 Ser Asn Val
25 30
Ser Gln Ser Thr Leu Glu GluGln GlnIle AlaAlaSer
Arg Ser Arg
35 40 45
Ser Leu Glu Glu Leu Leu ThrHis SerGlu TrpLysLeu
Arg Ile Asp
50 55 60
Trp Arg Cys Arg Leu Arg SerPhe ThrSer AspSerArg
Leu Lys Met
3 65 70 75 80
0
Ser Ala Ser His Arg Ser PheAla AlaThr TyrAspIle
Thr Arg Phe
85 90 95
Glu Thr Leu Lys Val Ile GluTrp GlnArg GlnCysSer
Asp Glu Thr
100 105 110
Pro Arg Glu Thr Cys Val AlaSer GluLeu LysSerThr
Glu Val Gly
115 120 125
40
Asn Thr Phe Phe Lys Pro ValAsn ValPhe CysGlyGly
Pro Cys Arg
130 135 140
Cys Cys Asn Glu Glu Ser CysMet AsnThr ThrSerTyr
Leu Ile Ser
145 150 155 160
CA 02283470 1999-12-23
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Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro
165 170 175
Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu
180 185 190
Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln
195 200 205
Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile
210 215 220
Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu
225 230 235 240
Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala
245 250 255
2 0 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val
260 265 270
Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys
275 280 285
Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His
290 295 300
Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe
30 305 310 315 320
His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys
325 330 335
Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys
340 345 350
Asn Pro
(2) INFORMATION
FOR SEQ
ID NO.:
7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 17
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
CA 02283470 1999-12-23
68
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 7:
TCACCATCGA TCCAAGC 1~
(2) INFORMATION FOR SEQ ID NO.: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 8:
AGTTCTGCGT GAGCCGAG 18
(2) INFORMATION
FOR SEQ
ID NO.:
9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
CA 02283470 1999-12-23
' 69
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9:
CAGGACGGGG TGACTTGA 18
(2) INFORMATION FOR SEQ ID NO.: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 10:
GCCCAGGCCT GTCTACTG 18
(2) INFORMATION
FOR SEQ
ID NO.:
11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
CA 02283470 1999-12-23
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 11:
CCAGCTCCTA CGTGTTCG 18
(2) INFORMATION FOR SEQ ID NO.: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
10 (B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 12:
GGCAACAGCT GGATGTCA 18
(2) INFORMATION
FOR SEQ
ID NO.:
13:
(ij SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vij ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
CA 02283470 1999-12-23
71
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 13:
CTGTGAGGGC GTGGGAGT 18
(2) INFORMATION FOR SEQ ID NO.: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 14:
GTCCTTTGAG CCACTGGA 18
(2) INFORMATION
FOR SEQ
ID NO.:
15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
CA 02283470 1999-12-23
' 72
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 15:
GACACGTCAT CGACACCGGT G 21
(2) INFORMATION FOR SEQ ID NO.: 16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 16:
GGCAACAGCT GGATGTCA 18
(2) INFORMATION
FOR SEQ
ID NO.:
17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ixj FEATURE
CA 02283470 1999-12-23
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(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 17:
CCTGAGTATC TCCCGCGTCG C 21
(2) INFORMATION FOR SEQ ID NO.: 18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide
2 O (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 18:
GGTGCCTCCC TGTACCCTGG G 21
(2) INFORMATION
FOR SEQ
ID NO.:
19:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1363
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
30 (ii) MOLECULE TYPE; polypeptide
(vi) ORIGINAL SOURCE:
CA 02283470 1999-12-23
74
(A) ORGANISM: Mus musculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 19:
Met Gln Pro Gly Ala Ala Leu Asn Leu Arg Leu Trp Leu Cys Leu Gly
1 5 10 15
Leu Leu Gln Gly Leu Ala Asn Gly Tyr Ser Met Thr Pro Pro Thr Leu
20 25 30
Asn Ile Thr Glu Asp Ser Tyr Val Ile Asp Thr Gly Asp Ser Leu Ser
35 40 45
Ile Ser Cys Arg Gly Gln His Pro Leu Glu Trp Thr Trp Pro Gly Ala
50 55 60
Gln Glu Val Leu Thr Thr Gly Gly Lys Asp Ser Glu Asp Thr Arg Val
65 70 75 80
Val His Asp Cys Glu Gly Thr Glu Ala Arg Pro Tyr Cys Lys Val Leu
2 0 85 90 95
Leu Leu Ala Gln Thr His Ala Asn Asn Thr Gly Ser Tyr His Cys Tyr
100 105 110
Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser Thr
115 120 125
Tyr Val Phe Val Arg Asp Phe Lys His Pro Phe Ile Asn Lys Pro Asp
130 135 140
Thr Leu Leu Val Asn Arg Lys Asp Ser Met Trp Val Pro Cys Leu Val
145 150 155 160
Ser Ile Pro Gly Leu Asn Ile Thr Leu Arg Ser Gln Ser Ser Ala Leu
165 170 175
His Pro Asp Gly Gln Glu Val Leu Trp Asp Asp Arg Arg Gly Met Arg
180 185 190
Val Pro Thr Gln Leu Leu Arg Asp Ala Leu Tyr Leu Gln Cys Glu Thr
195 200 205
Thr Trp Gly Asp Gln Asn Phe Leu Ser Asn Leu Phe Val Val His Ile
210 215 220
Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Tyr Pro Lys Lys Ser Met
225 230 235 240
Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys Thr Val Trp Ala
245 250 255
Glu Phe Asp Ser Gly Val Thr Phe Asp Trp Asp Tyr Pro Gly Lys Gln
260 265 270
Ala Glu Arg Ala Lys Trp Val Pro Glu Arg Arg Ser Gln Gln Thr His
275 280 285
Thr Glu Leu Ser Ser Ile Leu Thr Ile His Asn Val Ser Gln Asn Asp
290 295 300
CA 02283470 1999-12-23
' 75
Leu Gly Pro Tyr Val Cys Glu Ala Asn Asn Gly Ile Gln Arg Phe Arg
305 310 315 320
Glu Ser Thr Glu Val Ile Val His Glu Lys Pro Phe Ile Ser Val Glu
325 330 335
Trp Leu Lys Gly Pro Val Leu Glu Ala Thr Ala Gly Asp Glu Leu Val
340 345 350
Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu Phe Gln Trp
355 360 365
Tyr Lys Asp Arg Lys Ala Val Thr Gly Arg His Asn Pro His Ala Leu
370 375 380
Val Leu Lys Glu Val Thr Glu Ala Ser Ala Gly Val Tyr Thr Leu Ala
385 390 395 400
2 0 Leu Trp Asn Ser Ala Ala Gly Leu Arg Gln Asn Ile Ser Leu Glu Leu
405 410 415
Val Val Asn Val Pro Pro His Ile His Glu Lys Glu Ala Ser Ser Pro
420 425 430
Ser Ile Tyr Ser Arg His Ser Arg Gln Thr Leu Thr Cys Thr Ala Tyr
435 440 445
Gly Val Pro Gln Pro Leu Ser Val Gln Trp His Trp Arg Pro Trp Thr
30 450 455 460
Pro Cys Lys Thr Phe Ala Gln Arg Ser Leu Arg Arg Arg Gln Gln Arg
465 470 475 480
Asp Gly Met Pro Gln Cys Arg Asp Trp Lys Glu Val Thr Thr Gln Asp
485 490 495
Ala Val Asn Pro Ile Glu Ser Leu Asp Ser Trp Thr Glu Phe Val Glu
500 505 510
Gly Lys Asn Lys Thr Val Ser Lys Leu Val Ile Gln Asp Ala Asn Val
515 520 525
Ser Ala Met Tyr Lys Cys Val Val Val Asn Lys Val Gly Gln Asp Glu
530 535 540
Arg Leu Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Ser Ile
545 550 555 560
Glu Ser Glu Pro Ser Glu Asp Pro Leu Glu Gly Gln Ser Val Arg Leu
565 570 575
Ser Cys Arg Ala Asp Asn Tyr Thr Tyr Glu His Leu Arg Trp Tyr Arg
580 585 590
Leu Asn Leu Ser Thr Leu His Asp Ala Gln Gly Asn Pro Leu Leu Leu
595 600 605
Asp Cys Lys Asn Val His Leu Phe Ala Thr Pro Leu Glu Ala Asn Leu
610 615 620
CA 02283470 1999-12-23
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Glu Glu Ala Glu Pro Gly Ala Arg His Ala Thr Leu Ser Leu Asn Ile
625 630 635 640
Pro Arg Val Ala Pro Glu Asp Glu Gly Asp Tyr Val Cys Glu Val Gln
645 650 655
Asp Arg Arg Ser Gln Asp Lys His Cys His Lys Lys Tyr Leu Ser Val
660 665 670
Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu Leu
675 680 685
Val Asn Val Ser Asp Ser Leu Glu Met Arg Cys Pro Val Ala Gly Ala
690 695 700
His Val Pro Ser Ile Val Trp Tyr Lys Asp Glu Arg Leu Leu Glu Lys
705 710 715 720
2 0 Glu Ser Gly Ile Asp Leu Ala Asp Ser Asn Gln Arg Leu Ser Ile Gln
725 730 735
Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr Leu Cys Ser Val Cys Asn
740 745 750
Ala Lys Gly Cys Val Asn Ser Ser Ala Ser Val Ala Val Glu Gly Ser
755 760 765
Glu Asp Lys Gly Ser Met Glu Ile Val Ile Leu Ile Gly Thr Gly Val
30 770 775 780
Ile Ala Val Phe Phe Trp Val Leu Leu Leu Leu Ile Phe Cys Asn Met
785 790 795 800
Lys Arg Pro Ala His Ala Asp Ile Lys Thr Gly Tyr Leu Ser Ile Ile
805 810 815
Met Asp Pro Gly Glu Val Pro Leu Glu Glu Gln Cys Glu Tyr Leu Ser
820 825 830
Tyr Asp Ala Ser Gln Trp Glu Phe Pro Arg Glu Arg Leu His Leu Gly
835 840 845
Arg Val Leu Gly His Gly Ala Phe Gly Lys Val Val Glu Ala Ser Ala
850 855 860
Phe Gly Ile Asn Lys Gly Ser Ser Gys Asp Thr Val Ala Val Lys Met
865 870 875 880
Leu Lys Glu Gly Ala Thr Ala Ser Glu His Arg Ala Leu Met Ser Glu
885 890 895
Leu Lys Ile Leu Ile His Ile Gly Asn His Leu Asn Val Val Asn Leu
900 905 910
Leu Gly Ala Cys Thr Lys Pro Asn Gly Pro Leu Met Val Ile Val Glu
915 920 925
Phe Cys Lys Tyr Gly Asn Leu Ser Asn Phe Leu Arg Val Lys Arg Asp
930 935 940
CA 02283470 1999-12-23
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Thr Phe Asn Pro Tyr Ala Glu Lys Ser Pro Glu Gln Arg Arg Arg Phe
945 950 955 960
Arg Ala Met Val Glu Gly Ala Lys Ala Asp Arg Arg Arg pro Gly Ser
965 970 975
Ser Asp Arg Ala Leu Phe Thr Arg Phe Leu Met Gly Lys Gly Ser Ala
980 985 990
Arg Arg Ala Pro Leu Val Gln Glu Ala Glu Asp Leu Trp Leu Ser Pro
995 1000 1005
Leu Thr Met Glu Asp Leu Val Cys Tyr Ser Phe Gln Val Ala Arg Gly
1010 1015 1020
Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala
1025 1030 1035 1040
2 0 Arg Asn Ile Leu Leu Ser Glu Ser Asp Ile Val Lys Ile Cys Asp Phe
1045 1050 1055
Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly
1060 1065 1070
Ser Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp
1075 1080 1085
Lys Val Tyr Thr Thr Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu
30 1090 1095 1100
Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Gln Ile
1105 1110 1115 1120
Asn Glu Glu Phe Cys Gln Arg Leu Lys Asp Gly Thr Arg Met Arg Ala
1125 1130 1135
Pro Glu Leu Ala Thr Pro Ala Ile Arg His Ile Met Gln Ser Cys Trp
1140 1145 1150
Ser Gly Asp Pro Lys Ala Arg Pro Ala Phe Ser Asp Leu Val Glu Ile
1155 1160 1165
Leu Gly Asp Leu Leu Gln Gly Gly Gly Trp Gln Glu Glu Glu Glu Glu
1170 1175 1180
Arg Met Ala Leu His Ser Ser Gln Ser Ser Glu Glu Asp Gly Phe Met
1185 1190 1195 1200
Gln Ala Ser Thr Thr Ala Leu His Ile Thr Glu Ala Asp Ala Asp Asp
1205 1210 1215
Ser Pro Pro Ser Met His Cys His Ser Leu Ala Ala Arg Tyr Tyr Asn
1220 1225 1230
Cys Val Ser Phe Pro Gly Arg Leu Ala Arg Gly Thr Lys Thr Pro Gly
1235 1240 1245
Ser Ser Arg Met Lys Thr Phe Glu Glu Leu Pro Met Thr Pro Thr Thr
1250 1255 1260
CA 02283470 1999-12-23
78
Tyr Lys AlaSer Gly Leu
Met Met Ala
Asp Val
Asn
Gln
Thr
Asp
Ser
1265 1270 1275 1280
Ser Glu GluPheGlu Glu Leu Glu Ser Arg ArgPro Gly
His Glu Ser
1285 1290 1295
Phe Ser CysLysGly Pro Gly Gln His Met IlePro Gly
Asp Arg His
1300 1305 1310
Pro Asp ProGlnGly Arg Arg Arg Arg pro GlnGly Gln
Thr Ala Gly
1315 1320 1325
Gly Lys ValPheTyr Asn Asn Glu Tyr Gly ValSer Pro
Glu Gln Cys
1330 1335 1340
Thr Glu GlyAspCys Cys pro Ser Ala Gly ThrPhe Ala
Ser Phe Asp
1345 1350 1355 1360
2 0 Ser Tyr
Ser
(2) INFORMATION FOR SEQ ID NO.: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
30 (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 20:
CGGCGCCTTC AGGAAGGTGG T 21
40 (2) INFORMATION FOR SEQ ID NO.: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
CA 02283470 1999-12-23
r
Y
' 79
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 21:
CGGAACATTC CGCTGTCGGA A 21
(2) INFORMATION FOR SEQ ID NO.: 22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
2 O (D ) TOPOLOGY
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 22:
GTCGGAAAGC AACGTGGTGA A 21
CA 02283470 1999-12-23
(2) INFORMATION FOR SEQ ID NO.: 23:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
10 (A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 23:
CATCAAGACG GGCTACCT 18
(2) INFORMATION FOR SEQ ID NO.: 24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
2 0 (B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 24:
CCGCTGACCC CACACCTT 18
CA 02283470 1999-12-23
81
(2) INFORMATION FOR SEQ ID NO.: 25:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 25:
GAGTTGACCT CCCAAGGT 18
(2) INFORMATION FOR SEQ ID NO.: 26:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
2 0 (B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 26:
TCTCCTGGAC AGGCAGTC 18
CA 02283470 1999-12-23
82
(2) INFORMATION FOR SEQ ID NO.: 27:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
(8) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 27:
GAGTTGACCT CCCAAGGT 18
(2) INFORMATION FOR SEQ ID NO.: 28:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18
2 0 (8) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 28:
TCTCCTGGAC AGGCAGTC 18
