U.S. patent application number 10/418445 was filed with the patent office on 2003-10-09 for novel hepatoma-derived growth factor-like proteins, polynucleotides encoding them and methods of use.
This patent application is currently assigned to CuraGen Corporation. Invention is credited to Boldog, Ferenc L., Burgess, Catherine, La Rochelle, William J., Minskoff, Stacey, Shimkets, Richard A., Vernet, Corine, Yang, Meiji.
Application Number | 20030190708 10/418445 |
Document ID | / |
Family ID | 22561852 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030190708 |
Kind Code |
A1 |
Burgess, Catherine ; et
al. |
October 9, 2003 |
Novel hepatoma-derived growth factor-like proteins, polynucleotides
encoding them and methods of use
Abstract
The present invention provides HDGFX, a novel isolated
polypeptide, as well as a polynucleotide encoding HDGFX and
antibodies that immunospecifically bind to HDGFX or any derivative,
variant, mutant, or fragment of the HDGFX polypeptide,
polynucleotide or antibody. The invention additionally provides
methods in which the HDGFX polypeptide, polynucleotide and antibody
are used in detection and treatment of a broad range of
pathological states, as well as to other uses.
Inventors: |
Burgess, Catherine;
(Wethersfield, CT) ; Shimkets, Richard A.; (West
Haven, CT) ; Vernet, Corine; (North Branford, CT)
; Boldog, Ferenc L.; (North Haven, CT) ; Yang,
Meiji; (East Lyme, CT) ; La Rochelle, William J.;
(Madison, CT) ; Minskoff, Stacey; (Stamford,
CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
CuraGen Corporation
|
Family ID: |
22561852 |
Appl. No.: |
10/418445 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10418445 |
Apr 17, 2003 |
|
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09675016 |
Sep 28, 2000 |
|
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60156975 |
Oct 1, 1999 |
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Current U.S.
Class: |
435/69.1 ;
424/145.1; 435/320.1; 435/325; 514/14.4; 514/14.7; 514/7.6;
514/9.6; 530/399; 536/23.5 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 38/00 20130101; A61P 19/02 20180101; A61P 17/06 20180101; A61P
9/00 20180101; A61P 15/00 20180101; C07K 14/503 20130101; A61P
29/00 20180101; A61P 35/00 20180101; A61P 17/02 20180101; A61P 3/10
20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/399; 514/12; 424/145.1; 536/23.5 |
International
Class: |
A61K 038/18; C07K
014/475; C07H 021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule encoding HDGFX, said molecule
comprising a nucleotide sequence encoding a polypeptide having an
amino acid sequence that is at least 85% identical to SEQ ID NO:2,
or the complement of said nucleic acid molecule.
2. The nucleic acid molecule of claim 1, wherein said nucleotide
sequence encodes a polypeptide of SEQ ID NO:2, or the complement of
said nucleic acid molecule.
3-27. Canceled.
28. The isolated nucleic acid molecule of claim 1 encoding a mature
form of the polypeptide having an amino acid sequence of SEQ ID
NO:2.
29. The nucleic acid molecule of claim 1, wherein the nucleic acid
molecule is naturally occurring.
30. The nucleic acid molecule of claim 1, wherein the nucleic acid
molecule differs by a single nucleotide from a nucleic acid
sequence of SEQ ID NO1.
31. The isolated nucleic acid molecule of claim 1, said molecule
hybridizing under stringent conditions to a nucleic acid sequence
complementary to a nucleic acid molecule comprising the sequence of
nucleotides of SEQ ID NO:1, or the complement of said nucleic acid
molecule.
32. The isolated nucleic acid molecule of claim 1, said molecule
encoding a polypeptide comprising the amino acid sequence of SEQ ID
NO:2, or an amino acid sequence comprising one or more conservative
substitutions in the amino acid sequence of SEQ ID NO:2.
33. An oligonucleotide of the nucleic acid molecule of claim 1,
said nucleic acid molecule less than 100 nucleotides in length and
comprising at least 6 contiguous nucleotides of SEQ ID NO:1, or a
complement thereof.
34. A nucleic acid vector comprising the nucleic acid molecule of
claim 1.
35. The nucleic acid vector of claim 34, wherein said vector is an
expression vector.
36. The vector of claim 34, further comprising at least one
regulatory element operably linked to said nucleic acid
molecule.
37. A host cell comprising the isolated nucleic acid molecule of
claim 1.
38. A host cell comprising the vector of claim 34.
39. A method of producing an isolated polypeptide at least 80%
identical to a polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO:2;
b) a fragment of a polypeptide comprising an amino acid sequence of
SEQ ID NO:2,wherein the fragment comprises at least 6 contiguous
amino acids of SEQ ID NO:2; c) a derivative of a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; d) an analog of a
polypeptide comprising an amino acid sequence of SEQ ID NO:2; c) a
homolog of a polypeptide comprising an amino acid sequence of SEQ
ID NO:2; and f) a naturally occurring allelic variant of a
polypeptide comprising an amino acid sequence of SEQ ID NO:2;
wherein the polypeptide is encoded by a nucleic acid molecule that
hybridizes to a nucleic acid molecule of SEQ ID NO:1 under
stringent conditions, said method comprising the step of culturing
the host cell comprising the isolated nucleic acid molecule of
claim 1 under conditions in which the nucleic acid molecule is
expressed.
40. The method of claim 39 wherein the cell is a bacterial
cell.
41. The method of claim 39 wherein the cell is an insect cell.
42. The method of claim 39 wherein the cell is a yeast cell.
43. The method of claim 39 wherein the cell is a mammalian
cell.
44. A composition comprising a therapeutically or prophylactically
effective amount of a composition selected from the group
consisting of: a) an isolated nucleic acid molecule encoding HDGFX,
said molecule comprising a nucleotide sequence encoding a
polypeptide having a sequence that is at least 85% identical to SEQ
ID NO:2, or the complement of said nucleic acid molecule; b) an
isolated polypeptide at least 80% identical to a polypeptide
selected from the group consisting of: i) a polypeptide comprising
an amino acid sequence of SEQ ID NO:2; ii) a fragment of a
polypeptide comprising an amino acid sequence of SEQ ID NO:2,
wherein the fragment comprises at least 6 contiguous amino acids of
SEQ ID NO:2; iii) a derivative of a polypeptide comprising an amino
acid sequence of SEQ ID NO:2; iv) an analog of a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; v) a homolog of a
polypeptide comprising an amino acid sequence of SEQ ID NO:2; and
vi) a naturally occurring allelic variant of a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; wherein the
polypeptide is encoded by a nucleic acid molecule that hybridizes
to a nucleic acid molecule of SEQ ID NO:1 under stringent
conditions; and c) an antibody that selectively binds to the
polypeptide of step (b), and fragments, homologs, analogs, and
derivatives of said antibody; and a pharmaceutically acceptable
carrier.
45. A kit comprising in one or more containers, a therapeutically
or prophylactically effective amount of the composition of claim
19.
46. The use of a therapeutic in the manufacture of a medicament for
treating a syndrome associated with a human disease, the disease
selected from a proliferative disorder, a differentiative disorder,
and a tumorigenic disorder, wherein said therapeutic is selected
from the group consisting of: a) an isolated nucleic acid molecule
encoding HDGFX, said molecule comprising a nucleotide sequence
encoding a polypeptide having a sequence that is at least 85%
identical to SEQ ID NO:2, or the complement of said nucleic acid
molecule; b) an isolated polypeptide at least 80% identical to a
polypeptide selected from the group consisting of: i) a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; ii) a fragment of
a polypeptide comprising an amino acid sequence of SEQ ID NO:2,
wherein the fragment comprises at least 6 contiguous amino acids of
SEQ ID NO:2; iii) a derivative of a polypeptide comprising an amino
acid sequence of SEQ ID NO:2; iv) an analog of a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; v) a homolog of a
polypeptide comprising an amino acid sequence of SEQ ID NO:2; and
vi) a naturally occurring allelic variant of a polypeptide
comprising an amino acid sequence of SEQ ID NO:2; wherein the
polypeptide is encoded by a nucleic acid molecule that hybridizes
to a nucleic acid molecule of SEQ ID NO:1 under stringent
conditions; and c) an antibody that selectively binds to the
polypeptide of step (b), and fragments, homologs, analogs, and
derivatives of said antibody.
Description
RELATED APPLICATIONS
[0001] This application claims priority to provisional application
U.S. Ser. No. 60/156,975, filed Oct. 1, 1999, and non-provisional
application U.S. Ser. No. 09/675,019, filed Sep. 28, 2000, the
contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to novel nucleic acids and
polypeptides and more particularly to novel nucleic acids encoding
polypeptides related to growth factors.
BACKGROUND OF THE INVENTION
[0003] Hepatoma-derived growth factor (HDGF) and HDGF-related
proteins (HRP) belong to a gene family with a well-conserved amino
acid sequence at the N-terminus. Hepatoma-derived growth factor
HDGF was the first member identified in this new family of secreted
heparin-binding growth factors that are highly expressed in the
fetal aorta. Like other heparin binding proteins, HDGF is an acidic
polypeptide with mitogenic activity for fibroblasts
[0004] The biologic role of HDGF in vascular growth is unknown.
However, HDGF colocalizes with the proliferating cell nuclear
antigen (PCNA) in smooth muscles cells in human atherosclerotic
carotid arteries, suggesting that HDGF helps regulate smooth muscle
growth during development and in response to vascular injury.
SUMMARY OF THE INVENTION
[0005] The present invention is based, in part, upon the discovery
of a nucleic acid encoding a novel polypeptide having homology to
hepatoma-derived growth factor (HDGF) protein. The novel
hepatoma-derived growth factor X (HDGFX) polynucleotide sequences
and the HDGFX polypeptides encoded by these nucleic acid sequences,
and fragments, homologs, analogs, and derivatives thereof, are
claimed in the invention.
[0006] In one aspect, the invention provides an isolated HDGFX
nucleic acid (SEQ ID NO:1, as shown in Table 1), that encodes a
HDGFX polypeptide, or a fragment, homolog, analog or derivative
thereof. The nucleic acid can include, e.g., nucleic acid sequence
encoding a polypeptide at least 85% identical to a polypeptide
comprising the amino acid sequence of Table 1 (SEQ ID NO:2). The
nucleic acid can be, e.g., a genomic DNA fragment, or it can be a
cDNA molecule. In another aspect, the invention provides a
complement to the HDGFX nucleic acid shown in Table 1, or a
fragment, homolog, analog or derivative thereof.
[0007] Also included in the invention is a vector containing one or
more of the nucleic acids described herein, and a cell containing
the vectors or nucleic acids described herein.
[0008] The present invention is also directed to host cells
transformed with a recombinant expression vector comprising any of
the nucleic acid molecules described above.
[0009] In one aspect, the invention includes a pharmaceutical
composition that includes a HDGFX nucleic acid and a
pharmaceutically acceptable carrier or diluent. In a further
aspect, the invention includes a substantially purified HDGFX
polypeptide, e.g., any of the HDGFX polypeptides encoded by a HDGFX
nucleic acid, and fragments, homologs, analogs, and derivatives
thereof. The invention also includes a pharmaceutical composition
that includes a HDGFX polypeptide and a pharmaceutically acceptable
carrier or diluent.
[0010] In a further aspect, the invention provides an antibody that
binds specifically to a HDGFX polypeptide. The antibody can be,
e.g., a monoclonal or polyclonal antibody, and fragments, homologs,
analogs, and derivatives thereof. The invention also includes a
pharmaceutical composition including HDGFX antibody and a
pharmaceutically acceptable carrier or diluent. The present
invention is also directed to isolated antibodies that bind to an
epitope on a polypeptide encoded by any of the nucleic acid
molecules described above.
[0011] The present invention is further directed to kits comprising
antibodies that bind to a polypeptide encoded by any of the nucleic
acid molecules described above and a negative control antibody.
[0012] The invention further provides a method for producing a
HDGFX polypeptide. The method includes providing a cell containing
a HDGFX nucleic acid, e.g., a vector that includes a HDGFX nucleic
acid, and culturing the cell under conditions sufficient to express
the HDGFX polypeptide encoded by the nucleic acid. The expressed
HDGFX polypeptide is then recovered from the cell. Preferably, the
cell produces little or no endogenous HDGFX polypeptide. The cell
can be, e.g., a prokaryotic cell or eukaryotic cell.
[0013] The present invention provides a method of inducing an
immune response in a mammal against a polypeptide encoded by any of
the nucleic acid molecules disclosed above. In one embodiment, the
method includes administering to the mammal an amount of the
polypeptide sufficient to induce the immune response. In another
embodiment, the method includes administering to the mammal a
nucleic acid encoding a HDGFX polypeptide in an amount sufficient
to produce enough HDGFX polypeptide to induce the immune
response.
[0014] The present invention is also directed to methods of
identifying a compound that binds to HDGFX polypeptide by
contacting the HDGFX polypeptide with a compound and determining
whether the compound binds to the HDGFX polypeptide.
[0015] The invention further provides methods of identifying a
compound that modulates the activity of a HDGFX polypeptide by
contacting HDGFX polypeptide with a compound and determining
whether the HDGFX polypeptide activity is modified.
[0016] The present invention is also directed to compounds that
modulate HDGFX polypeptide activity identified by contacting a
HDGFX polypeptide with the compound and determining whether the
compound modifies activity of the HDGFX polypeptide, binds to the
HDGFX polypeptide, or binds to a nucleic acid molecule encoding a
HDGFX polypeptide.
[0017] In a further aspect, the invention includes a method of
diagnosing a tissue proliferation-associated disorder, such as
tumors, restenosis, psoriasis, diabetic and post-surgery
complications, and rheumatoid arthritis, in a subject. The method
includes providing a nucleic acid sample, e.g., RNA or DNA, or
both, from the subject and measuring the amount of the HDGFX
nucleic acid in the subject nucleic acid sample. The amount of
HDGFX nucleic acid sample in the subject nucleic acid is then
compared to the amount of HDGFX nucleic acid in a control sample.
An alteration in the amount of HDGFX nucleic acid in the sample
relative to the amount of HDGFX in the control sample indicates the
subject has a tissue proliferation-associated disorder.
[0018] In a still further aspect, the invention provides method of
treating or preventing or delaying a tissue
proliferation-associated disorder. The method includes
administering to a subject in which such treatment or prevention or
delay is desired a HDGFX nucleic acid, a HDGFX polypeptide, or a
HDGFX antibody in an amount sufficient to treat, prevent, or delay
a tissue proliferation-associated disorder in the subject.
[0019] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0020] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Western blot of the HDGFX (also referred to as
AL033539) polypeptide secreted by 293 cells.
[0022] FIG. 2. Western blot of HDGFX polypeptide expressed in E.
coli cells.
[0023] FIG. 3. Activation of monocytes in response to treatment
with HDGFX as measured by secretion of tumor necrosis factor
alpha.
[0024] FIG. 4. Activation of monocytes in response to treatment
with HDGFX as measured by secretion of interleukin-6.
[0025] FIG. 5. Proliferation of human pancreatic duct epithelial
cells in response to treatment with HDGFX.
[0026] FIG. 6. Proliferation of human pancreatic duct epithelial
cells in response to treatment with HDGFX.
[0027] FIG. 7. Northern blot analysis of c-myc and c-fos genes in
human pancreatic duct epithelial cells treated with HDGFX.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Included in the invention is HDGFX (also referred to as
AL033539_A), a novel hepatoma-derived growth factor ("HDGF")
related polypeptide, and nucleic acids encoding the polypeptide.
Antibodies that bind specifically to HDGFX polypeptides, or
fragments thereof, are included in the invention. The invention
further includes fragments, homologs, analogs, and derivatives of
HDGFX nucleic acids, polypeptides, and antibodies.
[0029] A DNA sequence of a human HDGFX gene (880 nucleotides; SEQ
ID NO:1), and its encoded amino acid sequence (SEQ ID NO:2), are
shown in Table 1. The translated protein is encoded from nucleotide
79 to 831. The HDGFX protein product (SEQ ID NO:2) is 251 amino
acids in length. The predicted molecular weight of the HDGFX
polypeptide is 27,233.3 daltons. The protein of SEQ ID NO:2 is
predicted by the PSORT software program to localize in the nucleus
with a certainty of 0.8800.
[0030] The protein of the invention encoded by clone HDGFX_A
includes the full protein disclosed as being encoded by the ORF
described herein, as well as any mature protein arising therefrom
as a result of posttranslational modifications. Thus, the proteins
of the invention encompass both a precursor and any active forms of
the HDGFX_A protein.
[0031] The software programs PSORT and SignalP predicts that HDGFX
contains no signal peptide.
1TABLE 1 Hepatoma-Derived Growth Factor Homolog Poly- nucleotide
(SEQ ID NO:1) and Amino Acid (SEQ ID NO:2) Sequences 1
GCAGCCGCCCTTACTGCGCGCGCGCAGACTTCGGCGTC- TACTTCC 46
GGTGTGGCCCAGGCGGGGTCCGCAGAACCAGCTATGTCGGCCTAC MetSerAlaTyr 91
GGCATGCCCATGTACAAGAGCGGGGACCTGGTGTTTGCCAAGTTA
GlyMetProMetTyrLysSerGlyAspLeuValPheAlaLysLeu 136
AAGGGCTATGCCCACTGGCCGGCGAGGATAGAGCACATGACCCAG
LysGlyTyrAlaHisTrpProAlaArglleGluHisMetThrGln 181
CCCAACCGCTACCAGGTGTTTTTCTTCGGGACCCACGAGACGGCC
ProAsnArgTyrGlnValPhePhePheGlyThrHisGluThrAla 226
TTCCTGAGTCCCAAACGCCTGTTCCCGTACAAGGAGTGCAAGGAG
PheLeuSerProLysArgLeuPheProTyrLysGluCysLysGlu 271
AAGTTCGGCAAGCCCAACAAGAGGCGCGGCTTCAGCGCGGGGCTG
LysPheGlyLysProAsnLysArgArgGlyPheSerAlaGlyLeu 316
TGGGAAATCGAGAACAACCCCACGGTCCAGGCCTCCGACTGCCCA
TrpGluIleGluAsnAsnProThrValGlnAlaSerAspCysPro 361
TTAGCCTCAGAGAAGGGCAGCGGAGACGGGCCTTGGCCGGAGCCC
LeuAlaSerGluLysGlySerGlyAspGlyProTrpProGluPro 406
GAGGCCGCAGAGGGCGACGAGGACAAGCCGACCCACGCTGGTGGC
GluAlaAlaGluGlyAspGluAspLysProThrHisAlaGlyGly 451
GGCGGCGACGAATTGGGGAAGCCGGACGACGACAAGCCCACTGAG
GlyGlyAspGluLeuGlyLysProAspAspAspLysProThrGlu 496
GAGGAGAAGGGGCCGCTGAAGAGGAGCGCGGGGGACCCGCCGGAG
GluGluLysGlyProLeuLysArgSerAlaGlyAspProProGlu 541
GACGCCCCCAAACGACCCAAGGAGGCAGCCCCCGACCAAGAGGAG
AspAlaProLysArgProLysGluAlaAlaProAspGlnGluGlu 586
GAGGCGGAGGCGGAGAGGGCGGCGGAAGCGGAGAGGGCGGCGGCG
GluAlaGluAlaGluArgAlaAlaGluAlaGluArgAlaAlaAla 631
GCGGCGGCGGCGACGGCCGTCGACGAGGAGAGTCCGTTCCTCGTG
AlaAlaAlaAlaThrAlaValAspGluGluSerProPheLeuVal 676
GCGGTGGAGAACGGCAGCGCCCCTAGCGAGCCGGGCCTGGTCTGC
AlaValGluAsnGlySerAlaProSerGluProGlyLeuValCys 721
GAGCCGCCTCAGCCAGAGGAGGAGGAGCTCCGGGAGGAAGAAGTC
GluProProGlnProGluGluGluGluLeuArgGluGluGluVal 766
GCGGACGAGGAGGCCTCCCAGGAGTGGCATGCCGAGGCACCGGGC
AlaAspGluGluAlaSerGlnGluTrpHisAlaGluAlaProGly 811
GGCGGAGATCGCGACAGCCTGTAGTTACCAGCGTTTCCAGAAGAG GlyGlyAspArgAspSerLeu
856 CCCCTGCCCCGTTCCTGCTGCGGCC
[0032] A search of the sequence databases using BLASTP and BLASTX
programs reveals that the HDGFX_A protein product (SEQ ID NO:2) has
143 of 234 residues (61%) identical to, and 155 of 243 residues
(66%) positive with, the 235 residue bovine hepatoma derived growth
factor related protein 3 (HRP-3) (TREMBLNEW-ACC:CAB40348).
Alignment results are shown in Table 2.
2TABLE 2 BLAST Results showing HDGFX vs. HRP-3 (Query = HDGFX;
Sbjct = HRP-3 (SEQ ID NO:3)) ptnr:TREMBLNEW-ACC:CAB40348 HEPATOMA
DERIVED GROWTH FACTOR RELATED PROTEIN 3 (HRP-3) - Bos
taurus(Bovine). 235 aa. Plus Strand HSPs: Score = 640 (225.3 bits),
Expect = 6.2e-62, P = 6.2e-62 Identities = 143/234 (61%), Positives
= 155/234 (66%), Frame = +1 Query: 79
MSAYGMPMYKSGDLVFAKLKGYAHWPARIEHMTQPNRYQVFFFGTHETAFLSPKRLFPYK 258
.vertline..vertline.+.vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline.+.vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline.+.vertline..vertline..vertli-
ne..vertline.+ Sbjct: 1
MSRFYRRKYKCGDLVFAKLKGYAHWPARIEQTAEANRYQVFFF- GTHIETAFLGPRHLFPYE 60
Query: 259 ECKEKFGKPNKRRGFSAGLWEIENN-
PTVQASDCPLASEKGSGDGPWPEPEAAEGDEDKPT 438 .vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline.+-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline.+ Sbjct: 61
ESKEKFGKPNKRRGFSEGLWEIENNPTVQASDYQCALEKSCPEEP--EP- EVAEGGEDPKS 118
Query: 439 HAGGGGDE-LGKPDDDKPTEEE--KGPLKRS-
AGDPPEDAPKRPKEAAPDQEEEAEAERAA 609 .vertline..vertline..vertline..-
vertline.+.vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline.+.vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline. Sbjct: 119
HTNGGDDDDQGKLGVDLPAEEENKKETLKRTAEDPPEDIPKRPKEADP---EEGE 170 Query:
610 EAERAAAAAAATAVDEESPFLVAVENGSAPSEPGLVCEPPQPEEEELREEEVADEEA 780
.vertline..vertline..vertline..vertline..vertline..vertline.-
++.vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline.+.vertline..vertline..vertline..vertline..vert-
line.+.vertline..vertline. Sbjct: 171
--ERKEAAAVAEEAEDARPLLVEVENDPA- ASVLGLAWGLPVMEQEP--EEESAEREA 223
[0033] HDGFX protein product (SEQ ID NO:2) was also found to have
135 of 228 residues (59%) identical to, and 156 of 228 residues
(68%) positive with the 240 residue human hepatoma-derived growth
factor (HDGF) protein (SWISSPROT-ACC:P51858).Alignment results are
shown in TABLE 3, sections A and B.
3TABLE 3 BLAST Results showing HDGFX vs. HDGF A. Query = HDGFX;
Sbjct = HDGF (SEQ ID NO:4) ptnr:SWISSPROT-ACC:P51858
HEPATOMA-DERIVED GROWTH FACTOR (HDOF) - Homo sapiens, 240 as.
Length = 240 Score = 608 (214.0 bits), Expect = 2.6e-59, P =
2.6e-59 Identities = 135/228 (59%), Positives = 156/228 (68%)
Query: 9 YKSGDLVFAKLKGYAIIWPARIEHMTQP----
---NRYQVFFFGTHETAFLSPKRLFPYKEC 62 .vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline.+.vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e.+.vertline.+.vertline.+.vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne.+.vertline. Sbjct: 10
YKCGDLVFAKMKGYPHWPARIDEMPEAAVKSTANKYQVFFFG- TNETAFLGPKDLFPYEES 69
Query: 63 KEKFGKPNKRRGFSAGLWEIENNPTV-
QASDCPLASEKGSGDGPWPEPEAAEGDEDKPTHA 122 .vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
+.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne.+.vertline..vertline.++.vertline.+.vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline.+
Sbjct: 70 KEKFGKPNKRKGFSEGLWEIENNPTVKASGYQSSQKKSCVEEPEPEPEAAEGDGDK-
KGNA 129 Query: 123 GGGGDELGKPDDDKPTEE--EKGPLKRSAGDPPEDAPK-
RPKEAAPDQEEEAEAERAAEAE 180 .vertline..vertline..vertline..vertlin-
e..vertline..vertline.+.vertline.+.vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline.+.vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne.+.vertline..vertline..vertline..vertline. Sbjct: 130
EGSSDEEGKLVIDEPAKEKNEKGALKRRAGDLLEDSPKRPKEAENPEGEEKEA 182 Query:
181 RAAAAAAATAVDEESPFLVAVENGSAPSEPGLVCEPPQPEEEELREEEVADEEASQ 236
.vertline..vertline.++.vertline..vertline.+.vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline.+.vertline.++ Sbjct: 183
--------AT-LEVERPLPMEVEKNSTPSEPGSGRGPPQEEEEEEDEEEEATKEDAE 230 B.
Query = HDGFX; Sbjct = IIDGF (SEQ ID NO:5)
ptnr:SWISSPROT-ACC:p51858 HEPATOMA-DERIVED GROWTH FACTOR (HDGF) -
Homo sapiens, 240 aa. Score = 161 (56.7 bits), Expect = 1.2e-10, P
= 1.2e-10 Identities = 54/153 (35%), Positives = 73/153 (47%)
Query: 99
KGSGDGPWPEPEAAEGDEDKPTHAGGGGDELGKPDDDKPTEEEKGPLKRSAGDPPEDAPK 158
.vertline..vertline..vertline.+.vertline..vertline.+.vertlin-
e..vertline..vertline..vertline..vertline.+.vertline..vertline..vertline.+-
.vertline.+.vertline. Sbjct: 96
KASGYQSSQKKSCVEEPEPEPEAAEGDGDKKGNAE- GSSD-EEGKLVIDEPAKEKNEKGAL 154
Query: 159
RPKEAAPDQEEEAEAERAAEAERAAAAAAATAVDEESPFLVAVENGSAPSEPGLVCEPPQ 218
++.vertline..vertline.+++.vertline..vertline..vertline..vertline..vertlin-
e.++.vertline..vertline.+.vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline. Sbjct: 155
K-RRAGDLLEDSPKRPKEAENPEGEEKEAAT-LEVERPLPMEVEKNSTPSEPGSGRGPPQ 212
Query: 219 PEEEELREEEVADEEASQEWHAEAPGGGDRDSL 251
.vertline..vertline..vertline..vertline..vertline.+.vertline.+.vertline..-
vertline..vertline.++.vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline.+.vertline..vertline. Sbjct: 213
EEEEE--EDE--EEEATKE-DAEAPGIRDHESL 240
[0034] A search of the PRODOM database (E-value cutoff of 0.000001)
revealed that the HDGFX polypeptide sequence is homologous to a
family of growth factors (TABLE 4, black shading indicates identity
and gray shading indicates conservative substitution). The length
and complexity of the homology between the family members in Table
4 have a probability of less than 7e.sup.-27 that the conservation
between these sequences occurred by chance alone. See, Altschul et
al., 1997 Nucl. Acids Res. 25: 3389-3402. Proteins found to be
homologous include amino acids 10-117 of mouse HDGF (SEQ ID NO:6)
(SWISS-PROT: P51859), amino acids 1-123 of another mouse HDGFRP2
(SEQ ID NO:7) (SWISS-PROT:035540), amino acids 1-127 of human LEDGF
(SEQ ID NO:8) (SWISS-PROT: O75475), amino acids 1-127 of another
human LEDGF (SEQ ID NO:9) (SWISS:PROT: O75475), and amino acids
9-117 of mouse HDGFRP1 (SEQ ID NO:10) (SWISS-PROT:035539).
4TABLE 4 10 20 30 40 50 60
.....vertline......vertline......vertline...-
...vertline......vertline......vertline......vertline......vertline......v-
ertline......vertline......vertline......vertline. HDGFX(9-113)
-----Y S L A EHMTQP------ R Q S R 50 HDGF_MOUSE(10-117) ----Y C
MPEA STA Q 56 035540_MOUSE(1-123) MPHA DIADG P P I 60
075475_HUMAN(1-127) MTRD I V VPDG P T L I I 60 095368_HUMAN(1-127)
MTRD I V VPDG P T L I I 60 035539_MOUSE(9-117) ----Y T L A
EHVAEA------- R Q L RH 50 70 80 90 100 110 120
.....vertline......vertline......vertline......vert-
line......vertline......vertline......vertline......vertline......vertline-
......vertline......vertline......vertline. HDGFX(9-113) K C F R SA
E T QA DCPLASEKGSGDGPWPEP AAE----- 105 HDGF_MOUSE(10-117) E S F S E
T KA GYQSSQKKSCAAEPEVEP------- 108 035540_MOUSE(1-123) DKC D Y Q
HASY APPPVS-----SS SEAP ADLGCGSD 115 075475_HUMAN(1-127) S N Y D K
KF SQQAATKQSNASS VEVE KETSVSKE 120 095368_HUMAN(1-127) S N Y D K KF
SQQAATKQSNASS VEVE KETSVSKE 120 035539_MOUSE(9-117) E S F R S EHD M
EA SSLCSEEDQSYTE PGLA -EPELGQE 109 .....vertline..... HDGFX(9-113)
---------- 105 HDGF_MOUSE(10-117) -------- 108 035540_MOUSE(1-123)
VDKDKESR 123 075475_HUMAN(1-127) -DTDHEEK 127 095368_HUMAN(1-127)
-DTDHEEK 127 035539_MOUSE(9-117) --------- 109
[0035] HDGFX nucleic acids, and their encoded polypeptides,
according to the invention are useful in a variety of applications
and contexts. For example, HDGFX nucleic acids and polypeptides can
be used to identify proteins that are members of the hepatoma
derived growth factor family. The HDGFX nucleic acids and
polypeptides can also be used to screen for molecules which inhibit
or enhance HDGF activity or function. Specifically, the nucleic
acids and polypeptides according to the invention may be used as
targets for the identification of small molecules that modulate or
inhibit, e.g., angiogenesis neuronal development or
spermatogenesis.. These molecules can be used to treat, e.g.,
cancer, neurological disorders or infertility in mammals, e.g.
humans.
[0036] In addition, various HDGFX nucleic acids and polypeptides
according to the invention are useful, inter alia, as novel members
of the protein families according to the presence of domains and
sequence relatedness to previously described proteins. For example,
the HDGFX nucleic acids and their encoded polypeptides include
structural motifs that are characteristic of proteins belonging to
the hepatoma derived growth factor. Proteins belonging to this
family of proteins have been implicated in modulating and
inhibiting angiogenesis. Angiogenesis is an important normal
physiologic process in embryogenesis, wound repair and the female
reproductive cycle. However, as a pathological process, it plays a
central role in chronic inflammation, fibroproliferative disorders
and tumorigenesis. Thus, HDGFX nucleic acids and polypeptides,
antibodies and related compounds according to the invention will be
useful in therapeutic applications implicated in various cancers,
coronary artery disease, arthritis, and diabetic retinopathy.
[0037] Immunofluorescence data indicate that HDGF is localized in
the cytoplasm of hepatoma cells and is expressed ubiquitously in
normal tissues and tumor cell lines. See, e.g., Nakamura et al.,
1994 J. Biol. Chem. 269: 25143-25149. Therefore, in an alternative
embodiment, HDGFX, including the human hepatoma-derived growth
factor homolog according to the invention, may be a cytosolic
protein with a synthetic signal peptide. Nakamura et al. suggests
that HDGF is a likely novel heparin-binding protein with mitogenic
activity for fibroblasts performed outside the cells, despite the
presence of a putative nuclear localization signal. Therefore, in
another embodiment, HDGFX may serve as novel growth-modulating
factor to which various cells and tissues in the human body
respond. (see, EXAMPLE 8).
[0038] In addition, HDGFX nucleic acids, polypeptides, antibodies
and related compounds of the invention may be used to modulate
spermatogenesis, stimulate smooth muscle growth and modulate
neuronal development.
[0039] In-vivo studies with purified HDGFX proteins, as shown in
EXAMPLE 7 below, demonstrate an increase of splenic extramedullary
hematopoiesis and lymphoid hyperplasia. This indicates potential
therapeutic and diagnostic applications of HDGFX nucleic acids and
polypeptides antibodies and related compounds of the invention in
treating blood related disorders by, e.g., modulating hematopoiesis
and immunological related disorders by, e.g., stimulating the
immune system.
[0040] Additional utilities for HDGFX nucleic acids and
polypeptides according to the invention are disclosed herein.
[0041] HDGFX Nucleic Acids
[0042] The novel nucleic acids of the invention include those that
encode a HDGFX, HDGFX-like polypeptide or biologically active
portions thereof. Among these nucleic acids is the nucleic acid
whose sequence is provided in SEQ ID NO:1, or a fragment,
derivative, or homolog thereof. Additionally, the invention
includes mutant or variant nucleic acids of SEQ ID NO:1, or a
fragment thereof, any of whose bases may be changed from the
corresponding base shown in SEQ ID NO:1 while still encoding a
protein that maintains its HDGFX-like activities and physiological
functions. The invention further includes the complement of the
nucleic acid sequence of SEQ ID NOs:1, including fragments,
derivatives, analogs and homolog thereof. Examples of the
complementary strand of portions of HDGFX are shown as
oligonucleotide primers in the EXAMPLES section. The invention
additionally includes nucleic acids or nucleic acid fragments, or
complements thereto, whose structures include chemical
modifications.
[0043] Additional nucleic acids include nucleic acids encoding
HDGFX polypeptides that include the amino acid sequence of SEQ ID
NO:2. In some embodiments, a nucleic acid encoding a polypeptide
having the amino acid sequence of SEQ ID NO:2 includes the nucleic
acid sequence of SEQ ID NO:1, or a fragment thereof.
[0044] Additionally, a HDGFX nucleic acid of the invention includes
mutant or variant nucleic acids of SEQ ID NO:1, or a fragment
thereof, any of whose bases may be changed from the disclosed
sequence while still encoding a protein that maintains its
HDGFX-like activities and physiological functions. The invention
further includes the complement of the nucleic acid sequence of SEQ
ID NO:1, including fragments, derivatives, analogs and homolog
thereof. The invention additionally includes nucleic acids or
nucleic acid fragments, or complements thereto, whose structures
include chemical modifications.
[0045] A HDGFX nucleic acid of the invention can encode a mature
form of a HDGFX polypeptide. As used herein, a "mature" form of a
polypeptide or protein is the product of a naturally occurring
polypeptide or precursor form or proprotein. The naturally
occurring polypeptide, precursor or proprotein includes, by way of
nonlimiting example, the full length gene product, encoded by the
corresponding gene. Alternatively, it may be defined as the
polypeptide, precursor or proprotein encoded by an open reading
frame described herein. The product "mature" form arises, again by
way of nonlimiting example, as a result of one or more naturally
occurring processing steps as they may take place within the cell,
or host cell, in which the gene product arises. Examples of such
processing steps leading to a "mature" form of a polypeptide or
protein include the cleavage of the N-terminal methionine residue
encoded by the initiation codon of an open reading frame, or the
proteolytic cleavage of a signal peptide or leader sequence. Thus a
mature form arising from a precursor polypeptide or protein that
has residues 1 to N, where residue 1 is the N-terminal methionine,
would have residues 2 through N remaining after removal of the
N-terminal methionine. Alternatively, a mature form arising from a
precursor polypeptide or protein having residues 1 to N, in which
an N-terminal signal sequence from residue 1 to residue M is
cleaved, would have the residues from residue M+1 to residue N
remaining. Additionally, a "mature" protein or fragment may arise
from a cleavage event other than removal of an initiating
methionine or removal of a signal peptide. Further as used herein,
a "mature" form of a polypeptide or protein may arise from a step
of post-translational modification other than a proteolytic
cleavage event. Such additional processes include, by way of
non-limiting example, glycosylation, myristylation or
phosphorylation. In general, a mature polypeptide or protein may
result from the operation of only one of these processes, or a
combination of any of them.
[0046] Also included are nucleic acid fragments sufficient for use
as hybridization probes to identify nucleic acids encoding HDGFX
polypeptides (e.g., a HDGFX mRNA encoding SEQ ID NO:2) and
fragments for use as polymerase chain reaction (PCR) primers for
the amplification or mutation of HDGFX nucleic acid molecules. As
used herein, the term "nucleic acid molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules
(e.g., mRNA), analogs of the DNA or RNA generated using nucleotide
analogs, and derivatives, fragments and homologs thereof. The
nucleic acid molecule can be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[0047] "Probes" refer to nucleic acid sequences of variable length,
preferably between at least about 10 nucleotides (nt), 20 nt, 30
nt, 50 nt, 100 nt, 500 nt, 1000 nt, or as many as about, e.g.,
6,000 nt, depending on use. Probes are used in the detection of
identical, similar, or complementary nucleic acid sequences. Longer
length probes are usually obtained from a natural or recombinant
source (although they may be prepared by chemical synthesis as
well), are highly specific and much slower to hybridize than
oligomers. Probes may be single- or double-stranded and designed to
have specificity in PCR, membrane-based hybridization technologies,
or ELISA-like technologies.
[0048] An "isolated" nucleic acid molecule is one that is separated
from other nucleic acid molecules that are present in the natural
source of the nucleic acid. Examples of isolated nucleic acid
molecules include, but are not limited to, recombinant DNA
molecules contained in a vector, recombinant DNA molecules
maintained in a heterologous host cell, partially or substantially
purified nucleic acid molecules, and synthetic DNA or RNA
molecules. Preferably, an "isolated" nucleic acid is free of
sequences which naturally flank the nucleic acid (i.e., sequences
located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the organism from which the nucleic acid is derived. For
example, in various embodiments, the isolated HDGFX nucleic acid
molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3
kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material or culture medium
when produced by recombinant techniques, or of chemical precursors
or other chemicals when chemically synthesized.
[0049] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, or a complement of this nucleotide sequence, can be isolated
using standard molecular biology techniques and the sequence
information provided herein. Using all or a portion of the nucleic
acid sequence of SEQ ID NO:1 as a hybridization probe, HDGFX
nucleic acid sequences can be isolated using standard hybridization
and cloning techniques (e.g., as described in Sambrook et al.,
eds., Molecular Cloning: A Laboratory Manual 2.sup.nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and
Ausubel, et al., eds., Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., 1993.)
[0050] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to HDGFX nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0051] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues, which oligonucleotide has a
sufficient number of nucleotide bases to be used in a PCR reaction.
A short oligonucleotide sequence may be based on, or designed from,
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having about 10 nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one
embodiment, an oligonucleotide comprising a nucleic acid molecule
less than 100 nt in length would further comprise at lease 6
contiguous nucleotides of SEQ ID NO:1, or a complement thereof.
Oligonucleotides may be chemically synthesized and may be used as
probes.
[0052] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule that is a
complement of the nucleotide sequence shown in any of SEQ ID NO:1.
In another embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule that is a complement of
the nucleotide sequence shown in any of SEQ ID NO:1, or a portion
of this nucleotide sequence. A nucleic acid molecule that is
complementary to the nucleotide sequence shown in SEQ ID NO:1 is
one that is sufficiently complementary to the nucleotide sequence
shown in of SEQ ID NO:1 that it can hydrogen bond with little or no
mismatches to the nucleotide sequence shown in of SEQ ID NO:1,
thereby forming a stable duplex.
[0053] As used herein, the term "complementary" refers to
Watson-Crick or Hoogsteen base pairing between nucleotides units of
a nucleic acid molecule, and the term "binding" means the physical
or chemical interaction between two polypeptides or compounds or
associated polypeptides or compounds or combinations thereof.
Binding includes ionic, non-ionic, van der Waals, hydrophobic
interactions, etc. A physical interaction can be either direct or
indirect. Indirect interactions may be through or due to the
effects of another polypeptide or compound. Direct binding refers
to interactions that do not take place through, or due to, the
effect of another polypeptide or compound, but instead are without
other substantial chemical intermediates.
[0054] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID
NO:1, e.g., a fragment that can be used as a probe or primer, or a
fragment encoding a biologically active portion of HDGFX.
"Fragments" provided herein are defined as sequences of at least 6
(contiguous) nucleic acids or at least 4 (contiguous) amino acids,
a length sufficient to allow for specific hybridization in the case
of nucleic acids or for specific recognition of an epitope in the
case of amino acids, respectively, and are at most some portion
less than a full length sequence. Fragments may be derived from any
contiguous portion of a nucleic acid or amino acid sequence of
choice. "Derivatives" are nucleic acid sequences or amino acid
sequences formed from the native compounds either directly or by
modification or partial substitution. "Analogs" are nucleic acid
sequences or amino acid sequences that have a structure similar to,
but not identical to, the native compound but differs from it in
respect to certain components or side chains. Analogs may be
synthetic or from a different evolutionary origin and may have a
similar or opposite metabolic activity compared to wild type.
[0055] Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid, as described below. Derivatives or
analogs of the nucleic acids or proteins of the invention include,
but are not limited to, molecules comprising regions that are
substantially homologous to the nucleic acids or proteins of the
invention, in various embodiments, by at least about 70%, 80%, 85%,
90%, 95%, 98%, or even 99% identity (with a preferred identity of
80-99%) over a nucleic acid or amino acid sequence of identical
size or when compared to an aligned sequence in which the alignment
is done by a computer homology program known in the art, or whose
encoding nucleic acid is capable of hybridizing to the complement
of a sequence encoding the aforementioned proteins under stringent,
moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, N.Y., 1993, and below. An exemplary program
is the Gap program (Wisconsin Sequence Analysis Package, Version 8
for UNIX, Genetics Computer Group, University Research Park,
Madison, Wis.) using the default settings, which uses the algorithm
of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is
incorporated herein by reference in its entirety).
[0056] A "homologous nucleic acid sequence" or "homologous amino
acid sequence," or variations thereof, refer to sequences
characterized by a homology at the nucleotide level or amino acid
level as discussed above. Homologous nucleotide sequences encode
those sequences coding for isoforms of a HDGFX polypeptide.
Isoforms can be expressed in different tissues of the same organism
as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the
present invention, homologous nucleotide sequences include
nucleotide sequences encoding for a HDGFX polypeptide of species
other than humans, including, but not limited to, mammals, and thus
can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and
other organisms. Homologous nucleotide sequences also include, but
are not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the
nucleotide sequence encoding human HDGFX protein. Homologous
nucleic acid sequences include those nucleic acid sequences that
encode conservative amino acid substitutions (see below) in any of
SEQ ID NO:2 as well as a polypeptide having HDGFX activity.
Biological activities of the HDGFX proteins are described
herein.
[0057] As used herein, "identical" residues correspond to those
residues in a comparison between two sequences where the equivalent
nucleotide base or amino acid residue in an alignment of two
sequences is the same residue. Residues are alternatively described
as "similar" or "positive" when the comparisons between two
sequences in an alignment show that residues in an equivalent
position in a comparison are either the same amino acid or a
conserved amino acid as defined below.
[0058] The nucleotide sequence determined from the cloning of the
human HDGFX gene allows for the generation of probes and primers
designed for use in identifying the cell types disclosed and/or
cloning HDGFX protein homologues in other cell types, e.g., from
other tissues, as well as HDGFX homologues from other mammals. The
probe/primer typically comprises a substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400
or more consecutive sense strand nucleotide sequence of SEQ ID
NO:1; or an anti-sense strand nucleotide sequence of SEQ ID NO:1;
or of a naturally occurring mutant of SEQ ID NO:1.
[0059] Probes based on a human HDGFX nucleotide sequence can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. In various embodiments, the probe further
comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a HDGFX
protein, such as by measuring a level of a HDGFX protein-encoding
nucleic acid in a sample of cells from a subject e.g., detecting
mRNA levels or determining whether a genomic HDGFX gene has been
mutated or deleted.
[0060] "A polypeptide having a biologically active portion of a
HDGFX" refers to polypeptides exhibiting activity similar, but not
necessarily identical to, an activity of a polypeptide of the
present invention, including mature forms, as measured in a
particular biological assay, with or without dose dependency. A
nucleic acid fragment encoding a "biologically active portion of a
HDGFX polypeptide" can be prepared by isolating a portion of SEQ ID
NO:1 that encodes a polypeptide having a HDGFX polypeptide
biological activity such as those disclosed herein, expressing the
encoded portion of HDGFX protein (e.g., by recombinant expression
in vitro) and assessing the activity of the encoded portion of the
HDGFX polypeptide.
[0061] HDGFX Variants
[0062] The invention further encompasses nucleic acid molecules
that differ from the disclosed HDGFX nucleotide sequences due to
degeneracy of the genetic code. These nucleic acids thus encode the
same HDGFX protein as that encoded by the nucleotide sequence shown
in SEQ ID NO:1. In another embodiment, an isolated nucleic acid
molecule of the invention has a nucleotide sequence encoding a
protein having an amino acid sequence shown in any of SEQ ID
NO:2.
[0063] In addition to the human HDGFX nucleotide sequence shown in
any of SEQ ID NO:1, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences of a HDGFX may exist within a population
(e.g., the human population). Such genetic polymorphism in the
HDGFX gene may exist among individuals within a population due to
natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding a HDGFX protein, preferably a mammalian
HDGFX protein. Such natural allelic variations can typically result
in 1-5% variance in the nucleotide sequence of the HDGFX gene. Any
and all such nucleotide variations and resulting amino acid
polymorphisms in the HDGFX gene that are the result of natural
allelic variation and that do not alter the functional activity of
the HDGFX polypeptide are intended to be within the scope of the
invention.
[0064] Moreover, nucleic acid molecules encoding HDGFX proteins
from other species, and thus that have a nucleotide sequence that
differs from the human sequence of SEQ ID NO:1, are intended to be
within the scope of the invention. Nucleic acid molecules
corresponding to natural allelic variants and homologues of the
HDGFX cDNAs of the invention can be isolated based on their
homology to the human HDGFX nucleic acids disclosed herein using
the human cDNAs, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions.
[0065] In another embodiment, an isolated nucleic acid molecule of
the invention is at least 6 nucleotides in length and hybridizes
under stringent conditions to the nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO:1. In another embodiment, the
nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750
nucleotides in length. In another embodiment, an isolated nucleic
acid molecule of the invention hybridizes to the coding region. As
used herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under
which nucleotide sequences that exceed a minimum degree of
similarity to each other typically remain hybridized to each other.
For example, depending on the degree of stringency imposed,
nucleotide sequences at least about 60% similar to each other may
hybridize.
[0066] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a probe, primer or
oligonucleotide will hybridize to a target sequence; optimally the
probe will hybridize to no other sequences, and more generally will
not hybridize to sequences below a specified degree of similarity
to the probe. Stringent conditions are sequence-dependent and will
be different in different circumstances. Longer sequences hybridize
specifically at higher temperatures than shorter sequences.
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (T.sub.m) for the specific
sequence at a defined ionic strength and pH. The T.sub.m is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at
T.sub.m, 50% of the probes are occupied at equilibrium. Typically,
stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is
at least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60.degree. C. for longer probes, primers and oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0067] Stringent conditions such as described above are known to
those skilled in the art and can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
Preferably, the conditions are such that sequences at least about
65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% identical to each other
typically remain hybridized to each other. A non-limiting example
of stringent hybridization conditions is hybridization in a high
salt buffer comprising 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured
salmon sperm DNA at 65.degree. C. This hybridization is followed by
one or more washes in 0.2.times.SSC, 0.01% BSA at 50.degree. C. An
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO:1
corresponds to a naturally occurring nucleic acid molecule. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein).
[0068] Homologs (i.e., nucleic acids encoding HDGFX proteins
derived from species other than human) or other related sequences
(e.g., paralogs) can be obtained by low, moderate or high
stringency hybridization with all or a portion of the particular
human sequence as a probe using methods well known in the art for
nucleic acid hybridization and cloning.
[0069] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:1, or fragments, analogs or derivatives
thereof, under conditions of moderate stringency is provided. A
non-limiting example of moderate stringency hybridization
conditions are hybridization in 6.times.SSC, 5.times. Denhardt's
solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at
55.degree. C., followed by one or more washes in 1.times.SSC, 0.1%
SDS at 37.degree. C. Other conditions of moderate stringency that
may be used are well known in the art. See, e.g., Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley
& Sons, NY, and Kriegler, 1990, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY.
[0070] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NO:1, or fragments, analogs or derivatives thereof, under
conditions of low stringency, is provided. A non-limiting example
of low stringency hybridization conditions are hybridization in 35%
formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA,
10% (wt/vol) dextran sulfate at 40.degree. C., followed by one or
more washes in 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and
0.1% SDS at 50.degree. C. Other conditions of low stringency that
may be used are well known in the art (e.g., as employed for
cross-species hybridizations). See, e.g., Ausubel et al. (eds.),
1993, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, and Kriegler, 1990, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY; Shilo and Weinberg, 1981,
Proc Natl Acad Sci USA 78: 6789-6792.
[0071] Conservative Mutations
[0072] In addition to naturally-occurring allelic variants of a
HDGFX nucleotide sequence, e.g., a gene sequence, that may exist in
the population, the skilled artisan will further appreciate that
changes can be introduced by mutation into the nucleotide sequence
of SEQ ID NO:1, thereby leading to changes in the amino acid
sequence of the encoded HDGFX protein, without altering the
functional ability of the HDGFX protein. For example, nucleotide
substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the sequence of
SEQ ID NO:1. A "non-essential" amino acid residue is a residue at a
position in the sequence that can be altered from the wild-type
sequence of the HDGFX polypeptide without altering the biological
activity, whereas an "essential" amino acid residue is a residue at
a position that is required for biological activity. For example,
amino acid residues that are conserved among members of a family of
HDGFX proteins, of which the HDGFX proteins of the present
invention are members, are predicted to be particularly unamenable
to alteration.
[0073] For example, a HDGFX protein according to the present
invention can contain at least one domain that is a typically
conserved region in a HDGFX protein family member. As such, these
conserved domains are not likely to be amenable to mutation. Other
amino acid residues, however, (e.g., those that are poorly
conserved among members of the HDGFX protein family) may not be as
essential for activity and thus are more likely to be amenable to
alteration.
[0074] Another aspect of the invention pertains to nucleic acid
molecules encoding HDGFX proteins that contain changes in amino
acid residues relative to the amino acid sequence of SEQ IDNO:2
that are not essential for activity. In one embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence
encoding a protein, wherein the protein comprises an amino acid
sequence at least about 75% similar to the amino acid sequence of
SEQ ID NO:2. Preferably, the protein encoded by the nucleic acid is
at least about 80% identical to any of SEQ ID NO:2, more preferably
at least about 90%, 95%, 98%, and most preferably at least about
99% identical to SEQ ID NO:2.
[0075] An isolated nucleic acid molecule encoding a protein
homologous to the protein of SEQ ID NO:2 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the corresponding nucleotide sequence, such that one
or more amino acid substitutions, additions or deletions are
introduced into the encoded protein.
[0076] Mutations can be introduced into SEQ ID NO:1 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. Certain amino acids have
side chains with more than one classifiable characteristic. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, tryptophan,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tyrosine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in a HDGFX polypeptide is replaced
with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a HDGFX coding sequence, such as by
saturation mutagenesis, and the resultant mutants can be screened
for HDGFX polypeptide biological activity to identify mutants that
retain activity. Following mutagenesis of SEQ ID NO:1 the encoded
protein can be expressed by any recombinant technology known in the
art and the activity of the protein can be determined.
[0077] The relatedness of amino acid families may also be
determined based on side chain interactions. Substituted amino
acids may be fully conserved "strong" residues or fully conserved
"weak" residues. The "strong" group of conserved amino acid
residues may be any one of the following groups: STA, NEQK, NHQK,
NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino
acid codes are grouped by those amino acids that may be substituted
for each other. Likewise, the "weak" group of conserved residues
may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND,
SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.
[0078] Antisense HDGFX Nucleic Acids
[0079] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that are hybridizable to or
complementary to a HDGFX nucleic acid, e.g., the antisense nucleic
acid can be complementary to a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1, or fragments, analogs or
derivatives thereof. An "antisense" nucleic acid includes a
nucleotide sequence that is complementary to a "sense" nucleic acid
encoding a protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
In specific aspects, antisense nucleic acid molecules are provided
that comprise a sequence complementary to at least about 10, 25,
50, 100, 250 or 500 nucleotides or an entire HDGFX coding strand,
or to only a portion thereof. Nucleic acid molecules encoding
fragments, homologs, derivatives and analogs of a HDGFX protein of
SEQ ID NO:2 or antisense nucleic acids complementary to a HDGFX
nucleic acid sequence of SEQ ID NO:1 are additionally provided.
[0080] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding a HDGFX polypeptide. The term "coding region"
refers to the region of the nucleotide sequence comprising codons
which are translated into amino acid residues (e.g., the protein
coding region of a HDGFX polypeptide that corresponds to any of SEQ
ID NO:2). In another embodiment, the antisense nucleic acid
molecule is antisense to a "noncoding region" of the coding strand
of a nucleotide sequence encoding a HDGFX polypeptide. The term
"noncoding region" refers to 5' and 3' sequences which flank the
coding region that are not translated into amino acids (i.e., also
referred to as 5' and 3' untranslated regions).
[0081] The HDGFX coding strand sequences disclosed herein (e.g.,
SEQ ID NO:1) allow for antisense nucleic acids to be designed
according to the rules of Watson and Crick or Hoogsteen base
pairing. The antisense nucleic acid molecule can be complementary
to the entire coding region of a HDGFX mRNA. Alternatively, the
antisense nucleic acid molecule can be an oligonucleotide that is
antisense to only a portion of the coding or noncoding region of a
HDGFX mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of the HDGFX mRNA. An antisense oligonucleotide can be, for
example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides
in length.
[0082] An antisense nucleic acid of the invention can be
constructed using chemical synthesis or enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used.
[0083] Examples of modified nucleotides that can be used to
generate the antisense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0084] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a HDGFX protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule that binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies that
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of antisense molecules, vector constructs in which
the antisense nucleic acid molecule is placed under the control of
a strong pol II or pol III promoter are generally preferred.
[0085] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-O-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res
15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett 215: 327-330).
[0086] Such modifications include, by way of nonlimiting example,
modified bases, and nucleic acids whose sugar phosphate backbones
are modified or derivatized. These modifications are carried out at
least in part to enhance the chemical stability of the modified
nucleic acid, such that they may be used, for example, as antisense
binding nucleic acids in therapeutic applications in a subject.
[0087] Also within the invention is a HDGFX ribozymes. Ribozymes
are catalytic RNA molecules with ribonuclease activity that are
capable of cleaving a single-stranded nucleic acid, such as a HDGFX
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes (described in Haselhoff and Gerlach
(1988) Nature 334:585-591)) can be used to catalytically cleave the
HDGFX mRNA transcripts to thereby inhibit translation of the HDGFX
mRNA. A ribozyme having specificity for a HDGFX-encoding nucleic
acid can be designed based upon the nucleotide sequence of a HDGFX
nucleic acid disclosed herein (i.e., SEQ ID NO:1). For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a HDGFX-encoding mRNA.
See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, a HDGFX mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science
261:1411-1418.
[0088] Alternatively, HDGFX gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of a HDGFX gene (e.g., the HDGFX gene promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the HDGFX gene in target cells. See generally,
Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al.
(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays
14: 807-15.
[0089] In various embodiments, the HDGFX nucleic acids can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribosephosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein,
the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid
mimics, e.g., DNA mimics, in which the deoxyribosephosphate
backbone is replaced by a pseudopeptide backbone and only the four
natural nucleobases are retained. The neutral backbone of PNAs has
been shown to allow for specific hybridization to DNA and RNA under
conditions of low ionic strength. The synthesis of PNA oligomers
can be performed using standard solid phase peptide synthesis
protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe
et al. (1996) Proc. Nat. Acad. Sci. (USA) 93: 14670-675.
[0090] PNAs based on HDGFX nucleic acids can be used in therapeutic
and diagnostic applications. For example, PNAs can be used as
antisense or antigene agents for sequence-specific modulation of
gene expression by, e.g., inducing transcription or translation
arrest or inhibiting replication. PNA based on HDGFX nucleic acids
can also be used, e.g., in the analysis of single base pair
mutations in a gene by, e.g., PNA directed PCR clamping; as
artificial restriction enzymes when used in combination with other
enzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes
or primers for DNA sequence and hybridization (Hyrup et al. (1996),
above; Perry-O'Keefe (1996), above).
[0091] In a further embodiment, PNAs of HDGFX nucleic acids can be
modified, e.g., to enhance their stability or cellular uptake, by
attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug delivery known in the art. For example, PNA-DNA
chimeras of the nucleic acids can be generated that may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact
with the DNA portion while the PNA portion would provide high
binding affinity and specificity. PNA-DNA chimeras can be linked
using linkers of appropriate lengths selected in terms of base
stacking, number of bonds between the nucleobases, and orientation
(Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be
performed as described in Hyrup (1996) above and Finn et al. (1996)
Nucl Acids Res 24: 3357-63. For example, a DNA chain can be
synthesized on a solid support using standard phosphoramidite
coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thy- midine phosphoramidite, can
be used between the PNA and the 5' end of DNA (Mag et al. (1989)
Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a
stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al. (1996) above).
Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg
Med Chem Lett 5: 1119-11124.
[0092] In other embodiments, a HDGFX nucleic acid or antisense
nucleic acid may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. W088/09810) or the blood-brain barrier (see, e.g.,
PCT Publication No. W089/10134). In addition, oligonucleotides can
be modified with hybridization triggered cleavage agents (See,
e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating
agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this
end, the oligonucleotide may be conjugated to another molecule,
e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a hybridization-triggered cleavage agent, etc.
[0093] HDGFX Polypeptides
[0094] A HDGFX polypeptide of the invention includes a protein
whose sequence is provided in SEQ ID NO:2. The invention also
includes a mature form of a HDGFX polypeptide, as well as a mutant
or variant form of a HDGFX polypeptide. In some embodiments, a
mutant or variant HDGFX includes a protein in which any residues
may be changed from the corresponding residue shown in Table 1,
while still encoding a protein that maintains its HDGFX-like
activities and physiological functions, or a functional fragment
thereof. The invention includes the polypeptides encoded by the
variant HDGFX nucleic acids described above. In the mutant or
variant protein, up to 20% or more of the residues may be so
changed.
[0095] In general, a HDGFX polypeptide variant that preserves HDGFX
function includes any HDGFX polypeptide variant in which residues
at a particular position in the sequence have been substituted by
other amino acids. A HDGFX variant polypeptide also includes a
HDGFX polypeptide in which an additional residue or residues has
been inserted between two residues of the parent protein as well as
a protein in which one or more residues have been deleted from a
reference HDGFX polypeptide sequence (e.g., SEQ ID NO:2, or a
mature form of SEQ ID NO:2). Thus, any amino acid substitution,
insertion, or deletion with respect to a reference HDGFX
polypeptide sequence (e.g., SEQ ID NO:2, or a mature form of SEQ ID
NO:2) is encompassed by the invention. In some embodiments, a
mutant or variant proteins may include one or more substitutions,
insertions, or deletions with respect to a reference HDGFX
sequence.
[0096] The invention also includes isolated HDGFX proteins, and
biologically active portions thereof, or derivatives, fragments,
analogs or homologs thereof. Also provided are polypeptide
fragments suitable for use as immunogens to raise anti-HDGFX
antibodies. In one embodiment, native HDGFX proteins can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, HDGFX proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, a HDGFX
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[0097] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the HDGFX protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of a HDGFX protein in which the protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
a HDGFX protein having less than about 30% (by dry weight) of
non-HDGFX protein (also referred to herein as a "contaminating
protein"), more preferably less than about 20% of non-HDGFX
protein, still more preferably less than about 10% of non-HDGFX
protein, and most preferably less than about 5% non-HDGFX protein.
When the HDGFX protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation.
[0098] The language "substantially free of chemical precursors or
other chemicals" includes preparations of a HDGFX protein in which
the protein is separated from chemical precursors or other
chemicals that are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of a HDGFX protein having
less than about 30% (by dry weight) of chemical precursors or non
HDGFX polypeptides, more preferably less than about 20% chemical
precursors or non-HDGFX polypeptides, still more preferably less
than about 10% chemical precursors or non-HDGFX polypeptides, and
most preferably less than about 5% chemical precursors or non-HDGFX
polypeptides.
[0099] Biologically active portions of a HDGFX protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the HDGFX protein, e.g.,
the amino acid sequence shown in SEQ ID NO:2 that include fewer
amino acids than the full length HDGFX proteins, and exhibit at
least one activity of a HDGFX protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the HDGFX protein. A biologically active portion of a
HDGFX protein can be a polypeptide which is, for example, 10, 25,
50, 100 or more amino acids in length.
[0100] A biologically active portion of a HDGFX of the present
invention may contain at least one of the above-identified domains
conserved among the HDGFX family of proteins. Moreover, other
biologically active portions, in which other regions of the protein
are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
HDGFX protein.
[0101] In some embodiments, the HDGFX protein is substantially
homologous to any of SEQ ID NO:2 and retains the functional
activity of the protein of SEQ ID NO:2, yet differs in amino acid
sequence due to natural allelic variation or mutagenesis, as
described in detail below. Accordingly, in another embodiment, the
HDGFX protein is a protein that comprises an amino acid sequence at
least about 45% homologous, and more preferably about 55, 65, 70,
75, 80, 85, 90, 95, 98 or even 99% homologous to the amino acid
sequence of SEQ ID NO:2 and retains the functional activity of the
HDGFX proteins of the corresponding polypeptide having the sequence
of SEQ ID NO:2.
[0102] Determining Homology Between two or More Sequences
[0103] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in either
of the sequences being compared for optimal alignment between the
sequences). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are homologous at that position
(i.e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino acid or nucleic acid "identity").
[0104] The nucleic acid sequence homology may be determined as the
degree of identity between two sequences. The homology may be
determined using computer programs known in the art, such as GAP
software provided in the GCG program package. See, Needleman and
Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the
following settings for nucleic acid sequence comparison: GAP
creation penalty of 5.0 and GAP extension penalty of 0.3, the
coding region of the analogous nucleic acid sequences referred to
above exhibits a degree of identity preferably of at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part
of the DNA sequence shown in SEQ ID NO:1. Equivalent software
procedures for determining the extent of sequence identity are
widely known in the art may be used in the present context.
[0105] The term "sequence identity" refers to the degree to which
two polynucleotide or polypeptide sequences are identical on a
residue-by-residue basis over a particular region of comparison.
The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over that region of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T or U, C, G, or I, in the
case of nucleic acids) occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the region of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The term "substantial identity" as
used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least
80 percent sequence identity, preferably at least 85 percent
identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference
sequence over a comparison region. The term "percentage of positive
residues" is calculated by comparing two optimally aligned
sequences over that region of comparison, determining the number of
positions at which the identical and conservative amino acid
substitutions, as defined above, occur in both sequences to yield
the number of matched positions, dividing the number of matched
positions by the total number of positions in the region of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of positive residues.
[0106] Chimeric and Fusion HDGFX Proteins
[0107] The invention also provides HDGFX chimeric or fusion
proteins. As used herein, a HDGFX "chimeric protein" or "fusion
protein" includes a HDGFX polypeptide operatively linked to a
non-HDGFX polypeptide. A "HDGFX polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a HDGFX
polypeptide, or a fragment, variant or derivative thereof, whereas
a "non-HDGFX polypeptide" refers to a polypeptide having an amino
acid sequence corresponding to a protein that is not substantially
homologous to the HDGFX protein, e.g., a protein that is different
from the HDGFX protein and that is derived from the same or a
different organism. Thus, within a HDGFX fusion protein, the HDGFX
polypeptide can correspond to all or a portion of a HDGFX protein.
In one embodiment, a HDGFX fusion protein comprises at least one
biologically active portion of a HDGFX protein. In another
embodiment, a HDGFX fusion protein comprises at least two
biologically active portions of a HDGFX protein. Within the fusion
protein, the term "operatively linked" is intended to indicate that
the HDGFX polypeptide and the non-HDGFX polypeptide are fused
in-frame to each other. The non-HDGFX polypeptide can be fused to
the N-terminus or C-terminus of the HDGFX polypeptide.
[0108] For example, in one embodiment a HDGFX fusion protein
comprises a HDGFX polypeptide operably linked to the extracellular
domain of a second protein. Such fusion proteins can be further
utilized in screening assays for compounds that modulate HDGFX
activity (such assays are described in detail below).
[0109] In another embodiment, the fusion protein is a GST-HDGFX
fusion protein in which the HDGFX sequences are fused to the
C-terminus of the GST (i.e., glutathione S-transferase) sequences.
Such fusion proteins can facilitate the purification of recombinant
HDGFX.
[0110] In yet another embodiment, the fusion protein is a HDGFX
protein containing a heterologous signal sequence at its
N-terminus. For example, the native HDGFX signal sequence can be
removed and replaced with a signal sequence from another protein.
In certain host cells (e.g., mammalian host cells), expression
and/or secretion of the HDGFX can be increased through use of a
heterologous signal sequence.
[0111] In a further embodiment, the fusion protein is a
HDGFX-immunoglobulin fusion protein in which the HDGFX sequences
comprising one or more domains are fused to sequences derived from
a member of the immunoglobulin protein family. The
HDGFX-immunoglobulin fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject to inhibit an interaction between a HDGFX ligand and a
HDGFX protein on the surface of a cell, to thereby suppress
HDGFX-mediated signal transduction in vivo. In one example, a
contemplated HDGFX ligand of the invention is a HDGFX receptor. The
HDGFX-immunoglobulin fusion proteins can be used to modulate the
bioavailability of a HDGFX cognate ligand. Inhibition of the HDGFX
ligand/HDGFX interaction may be useful therapeutically for both the
treatment of proliferative and differentiative disorders, as well
as modulating (e.g., promoting or inhibiting) cell survival.
Moreover, the HDGFX-immunoglobulin fusion proteins of the invention
can be used as immunogens to produce anti-HDGFX antibodies in a
subject, to purify HDGFX ligands, and in screening assays to
identify molecules that inhibit the interaction of a HDGFX with a
HDGFX ligand. A HDGFX chimeric or fusion protein of the invention
can be produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, e.g., by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers that give
rise to complementary overhangs between two consecutive gene
fragments that can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Ausubel et al.
(eds.) Current Protocols in Molecular Biology, John Wiley &
Sons, 1992). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST
polypeptide). A HDGFX-encoding nucleic acid can be cloned into such
an expression vector such that the fusion moiety is linked in-frame
to the HDGFX protein.
[0112] HDGFX Agonists and Antagonists
[0113] The present invention also pertains to variants of a HDGFX
protein that function as either HDGFX agonists (mimetics) or as
HDGFX antagonists. Variants of a HDGFX protein can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the
HDGFX protein. An agonist of the HDGFX protein can retain
substantially the same, or a subset of, the biological activities
of the naturally occurring form of the HDGFX protein. An antagonist
of the HDGFX protein can inhibit one or more of the activities of
the naturally occurring form of the HDGFX protein by, for example,
competitively binding to a downstream or upstream member of a
cellular signaling cascade which includes the HDGFX protein. Thus,
specific biological effects can be elicited by treatment with a
variant of limited function. In one embodiment, treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein has fewer side
effects in a subject relative to treatment with the naturally
occurring form of the HDGFX protein.
[0114] Variants of the HDGFX protein that function as either HDGFX
agonists (mimetics) or as HDGFX antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the HDGFX protein for HDGFX protein agonist or
antagonist activity. In one embodiment, a variegated library of
HDGFX variants is generated by combinatorial mutagenesis at the
nucleic acid level and is encoded by a variegated gene library. A
variegated library of HDGFX variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential HDGFX sequences is expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins
(e.g., for phage display) containing the set of HDGFX sequences
therein. There are a variety of methods which can be used to
produce libraries of potential HDGFX variants from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence can be performed in an automatic DNA synthesizer, and the
synthetic gene then ligated into an appropriate expression vector.
Use of a degenerate set of genes allows for the provision, in one
mixture, of all of the sequences encoding the desired set of
potential HDGFX variant sequences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see, e.g., Narang
(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem
53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)
Nucl Acid Res 11:477.
[0115] Polypeptide Libraries
[0116] In addition, libraries of fragments of the HDGFX protein
coding sequence can be used to generate a variegated population of
growth promoter fragments for screening and subsequent selection of
variants of a HDGFX protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double stranded
PCR fragment of a HDGFX coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA that can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal and internal fragments of various sizes of the HDGFX
protein.
[0117] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of HDGFX proteins. The most widely used techniques,
which are amenable to high throughput analysis, for screening large
gene libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
HDGFX variants (Arkin and Yourvan (1992) PNAS 89:7811-7815;
Delgrave et al. (1993) Protein Engineering 6:327-331).
[0118] Anti-HDGFX Antibodies
[0119] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single chain, F.sub.ab, F.sub.ab' and F.sub.(ab')2
fragments, and an F.sub.ab expression library. In general, antibody
molecules obtained from humans relates to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain. Reference herein to antibodies includes a reference
to all such classes, subclasses and types of human antibody
species.
[0120] An isolated protein of the invention intended to serve as an
antigen, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that immunospecifically bind the
antigen, using standard techniques for polyclonal and monoclonal
antibody preparation. The full-length protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of the antigen for use as immunogens. An antigenic peptide fragment
comprises at least 6 amino acid residues of the amino acid sequence
of the full length protein, such as an amino acid sequence shown in
SEQ ID NO:2, and encompasses an epitope thereof such that an
antibody raised against the peptide forms a specific immune complex
with the full length protein or with any fragment that contains the
epitope. Preferably, the antigenic peptide comprises at least 10
amino acid residues, or at least 15 amino acid residues, or at
least 20 amino acid residues, or at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions
of the protein that are located on its surface; commonly these are
hydrophilic regions.
[0121] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of the
HDGFX that is located on the surface of the protein, e.g., a
hydrophilic region. A hydrophobicity analysis of the human HDGFX
protein sequence will indicate which regions of a HDGFX polypeptide
are particularly hydrophilic and, therefore, are likely to encode
surface residues useful for targeting antibody production. As a
means for targeting antibody production, hydropathy plots showing
regions of hydrophilicity and hydrophobicity may be generated by
any method well known in the art, including, for example, the Kyte
Doolittle or the Hopp Woods methods, either with or without Fourier
transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad.
Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157:
105-142, each incorporated herein by reference in their entirety.
Antibodies that are specific for one or more domains within an
antigenic protein, or derivatives, fragments, analogs or homologs
thereof, are also provided herein.
[0122] As disclosed herein, HDGFX protein sequence of SEQ ID NO:2,
or derivatives, fragments, analogs or homologs thereof, may be
utilized as immunogens in the generation of antibodies that
immunospecifically-bind these protein components. The term
"antibody" as used herein refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that specifically
binds (immunoreacts with) an antigen, such as HDGFX. Such
antibodies include, but are not limited to, polyclonal, monoclonal,
chimeric, single chain, F.sub.ab and F.sub.(ab')2 fragments, and an
F.sub.ab expression library. In a specific embodiment, antibodies
to human HDGFX proteins are disclosed. Various procedures known
within the art may be used for the production of polyclonal or
monoclonal antibodies to a HDGFX protein sequence of SEQ ID NO:2,
or a derivative, fragment, analog or homolog thereof. Some of these
proteins are discussed below.
[0123] In one embodiment, methods for the screening of antibodies
that possess the desired specificity include, but are not limited
to, enzyme-linked immunosorbent assay (ELISA) and other
immunologically-mediated techniques known within the art. In a
specific embodiment, selection of antibodies that are specific to a
particular domain of a HDGFX protein is facilitated by generation
of hybridomas that bind to the fragment of a HDGFX protein
possessing such a domain. Antibodies that are specific for one or
more domains within a HDGFX protein, e.g., the carboxy-terminal
residues specific to HDGFX when compared to HGDF (see, e.g., Tables
3-4), or derivatives, fragments, analogs or homologs thereof, are
also provided herein.
[0124] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies directed against
a protein of the invention, or against derivatives, fragments,
analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Some of these antibodies are
discussed below.
[0125] Polyclonal Antibodies
[0126] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by one or more injections with the native protein,
a synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example, the
naturally occurring immunogenic protein, a chemically synthesized
polypeptide representing the immunogenic protein, or a
recombinantly expressed immunogenic protein. Furthermore, the
protein may be conjugated to a second protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. The preparation can further include an adjuvant.
Various adjuvants used to increase the immunological response
include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.),
adjuvants usable in humans such as Bacille Calmette-Guerin and
Corynebacterium parvum, or similar immunostimulatory agents.
Additional examples of adjuvants which can be employed include
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate).
[0127] The polyclonal antibody molecules directed against the
immunogenic protein can be isolated from the mammal (e.g., from the
blood) and further purified by well known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0128] Monoclonal Antibodies
[0129] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs thus contain an
antigen binding site capable of immunoreacting with a particular
epitope of the antigen characterized by a unique binding affinity
for it.
[0130] Monoclonal antibodies can be prepared using hybridoma
methods, such as those described in the art. See, e.g., Kohler and
Milstein, 1975 Nature, 256:495. In a hybridoma method, a mouse,
hamster, or other appropriate host animal, is typically immunized
with an immunizing agent to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
immunizing agent. Alternatively, the lymphocytes can be immunized
in vitro.
[0131] The immunizing agent will typically include the protein
antigen, a fragment thereof or a fusion protein thereof. Generally,
either peripheral blood lymphocytes are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103). Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells can be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0132] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies. See,
e.g. Kozbor 1984 J. Immunol., 133:3001; Brodeur et al. Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, (1987) pp. 51-63.
[0133] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the antigen. Preferably, the binding specificity
of monoclonal antibodies produced by the hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis. See, e.g. Munson and Pollard
1980 Anal. Biochem. 107: 220. It is an objective, especially
important in therapeutic applications of monoclonal antibodies, to
identify antibodies having a high degree of specificity and a high
binding affinity for the target antigen.
[0134] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding,1986). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be
grown in vivo as ascites in a mammal.
[0135] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0136] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the
constant domains of an antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody.
[0137] Humanized Antibodies
[0138] The antibodies directed against the protein antigens of the
invention can further comprise humanized antibodies or human
antibodies. These antibodies are suitable for administration to
humans without engendering an immune response by the human against
the administered immunoglobulin. Humanized forms of antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) that are principally
comprised of the sequence of a human immunoglobulin, and contain
minimal sequence derived from a non-human immunoglobulin.
Humanization can be performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. (See also U.S.
Pat. No. 5,225,539.) In some instances, Fv framework residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Humanized antibodies can also comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0139] Human Antibodies
[0140] Fully human antibodies essentially relate to antibody
molecules in which the entire sequence of both the light chain and
the heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0141] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by introducing human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks
et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature
368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild
et al,(Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol. 13 65-93 (1995)).
[0142] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. (See publication
WO 94/02602). The endogenous genes encoding the heavy and light
immunoglobulin chains in the nonhuman host have been incapacitated,
and active loci encoding human heavy and light chain
immunoglobulins are inserted into the host's genome. The human
genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. The preferred
embodiment of such a nonhuman animal is a mouse, and is termed the
Xenomouse.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells which secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0143] An example of a method of producing a nonhuman host,
exemplified as a mouse, lacking expression of an endogenous
immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598.
It can be obtained by a method including deleting the J segment
genes from at least one endogenous heavy chain locus in an
embryonic stem cell to prevent rearrangement of the locus and to
prevent formation of a transcript of a rearranged immunoglobulin
heavy chain locus, the deletion being effected by a targeting
vector containing a gene encoding a selectable marker; and
producing from the embryonic stem cell a transgenic mouse whose
somatic and germ cells contain the gene encoding the selectable
marker.
[0144] A method for producing an antibody of interest, such as a
human antibody, is disclosed in U.S. Pat. No. 5,916,771. It
includes introducing an expression vector that contains a
nucleotide sequence encoding a heavy chain into one mammalian host
cell in culture, introducing an expression vector containing a
nucleotide sequence encoding a light chain into another mammalian
host cell, and fusing the two cells to form a hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and
the light chain.
[0145] In a further improvement on this procedure, a method for
identifying a clinically relevant epitope on an immunogen, and a
correlative method for selecting an antibody that binds
immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT publication WO 99/53049.
[0146] F.sub.ab Fragments and Single Chain Antibodies
[0147] Techniques can be adapted for the production of single-chain
antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted
for the construction of F.sub.ab expression libraries (see e.g.,
Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and
effective identification of monoclonal F.sub.ab fragments with the
desired specificity for a protein or derivatives, fragments,
analogs or homologs thereof. Antibody fragments that contain the
idiotypes to a protein antigen may be produced by techniques known
in the art including, but not limited to: (i) an F.sub.(ab')2
fragment produced by pepsin digestion of an antibody molecule; (ii)
an F.sub.ab fragment generated by reducing the disulfide bridges of
an F.sub.(ab')2 fragment; (iii) an F.sub.ab fragment generated by
the treatment of the antibody molecule with papain and a reducing
agent and (iv) F.sub.v fragments.
[0148] Bispecific Antibodies
[0149] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for an antigenic protein of the invention. The
second binding target is any other antigen, and advantageously is a
cell-surface protein or receptor or receptor subunit.
[0150] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983)). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0151] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0152] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0153] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent sodium arsenite to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0154] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab').sub.2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0155] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0156] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0157] Exemplary bispecific antibodies can bind to two different
epitopes, at least one of which originates in the protein antigen
of the invention. Alternatively, an anti-antigenic arm of an
immunoglobulin molecule can be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular antigen. Bispecific antibodies
can also be used to direct cytotoxic agents to cells which express
a particular antigen. These antibodies possess an antigen-binding
arm and an arm which binds a cytotoxic agent or a radionuclide
chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the protein antigen described herein and
further binds tissue factor (TF).
[0158] Heteroconjugate Antibodies
[0159] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0160] Effector Function Engineering
[0161] It can be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) can be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0162] Immunoconjugates
[0163] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0164] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0165] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0166] In another embodiment, the antibody can be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is in turn
conjugated to a cytotoxic agent.
[0167] Immunoliposomes
[0168] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0169] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0170] Diagnostic Applications of Antibodies Directed Against the
Proteins of the Invention
[0171] Antibodies directed against a protein of the invention may
be used in methods known within the art relating to the
localization and/or quantitation of the protein (e.g., for use in
measuring levels of the protein within appropriate physiological
samples, for use in diagnostic methods, for use in imaging the
protein, and the like). In a given embodiment, antibodies against
the proteins, or derivatives, fragments, analogs or homologs
thereof, that contain the antigen binding domain, are utilized as
pharmacologically-active compounds (see below).
[0172] An antibody specific for a protein of the invention can be
used to isolate the protein by standard techniques, such as
immunoaffinity chromatography or immunoprecipitation. Such an
antibody can facilitate the purification of the natural protein
antigen from cells and of recombinantly produced antigen expressed
in host cells. Moreover, such an antibody can be used to detect the
antigenic protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
antigenic protein. Antibodies directed against the protein can be
used diagnostically to monitor protein levels in tissue as part of
a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0173] Pharmaceutical Compositions of Antibodies
[0174] Antibodies specifically binding a protein of the invention,
as well as other molecules identified by the screening assays
disclosed herein, can be administered for the treatment of various
disorders in the form of pharmaceutical compositions. Principles
and considerations involved in preparing such compositions, as well
as guidance in the choice of components are provided, for example,
in Remington: The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.
1995; Drug Absorption Enhancement: Concepts, Possibilities,
Limitations, And Trends, Harwood Academic Publishers, Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[0175] If the antigenic protein is intracellular and whole
antibodies are used as inhibitors, internalizing antibodies are
preferred. However, liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,
90: 7889-7893 (1993). The formulation herein can also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Alternatively, or in addition,
the composition can comprise an agent that enhances its function,
such as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0176] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nanoparticles, and nanocapsules) or in macroemulsions.
[0177] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0178] Antibody Therapeutics
[0179] Antibodies of the invention, including polyclonal,
monoclonal, humanized and fully human antibodies, may used as
therapeutic agents. Such agents will generally be employed to treat
or prevent a disease or pathology in a subject. An antibody
preparation, preferably one having high specificity and high
affinity for its target antigen, is administered to the subject and
will generally have an effect due to its binding with the target.
Such an effect may be one of two kinds, depending on the specific
nature of the interaction between the given antibody molecule and
the target antigen in question. In the first instance,
administration of the antibody may abrogate or inhibit the binding
of the target with an endogenous ligand to which it naturally
binds. In this case, the antibody binds to the target and masks a
binding site of the naturally occurring ligand, wherein the ligand
serves as an effector molecule. Thus the receptor mediates a signal
transduction pathway for which ligand is responsible.
[0180] Alternatively, the effect may be one in which the antibody
elicits a physiological result by virtue of binding to an effector
binding site on the target molecule. In this case the target, a
receptor having an endogenous ligand which may be absent or
defective in the disease or pathology, binds the antibody as a
surrogate effector ligand, initiating a receptor-based signal
transduction event by the receptor.
[0181] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target, and
in other cases, promotes a physiological response. The amount
required to be administered will furthermore depend on the binding
affinity of the antibody for its specific antigen, and will also
depend on the rate at which an administered antibody is depleted
from the free volume other subject to which it is administered.
Common ranges for therapeutically effective dosing of an antibody
or antibody fragment of the invention may be, by way of nonlimiting
example, from about 0.1 mg/kg body weight to about 50 mg/kg body
weight. Common dosing frequencies may range, for example, from
twice daily to once a week.
[0182] HDGFX Recombinant Vectors and Host Cells
[0183] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
HDGFX protein, or derivatives, fragments, analogs or homologs
thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "expression vectors". In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" can be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0184] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner that
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence in many
types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., HDGFX proteins, mutant forms of the HDGFX, fusion proteins,
etc.).
[0185] The recombinant expression vectors of the invention can be
designed for expression of a HDGFX nucleic acid in prokaryotic or
eukaryotic cells. For example, the HDGFX can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example, using T7 promoter
regulatory sequences and T7 polymerase.
[0186] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: (1) to
increase expression of recombinant protein; (2) to increase the
solubility of the recombinant protein; and (3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often a proteolytic cleavage site is
introduced in fusion expression vectors at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0187] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0188] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, Gottesman, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128.
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., (1992) Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0189] In another embodiment, the HDGFX expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San
Diego, Calif.).
[0190] Alternatively, the HDGFX nucleic acid can be expressed in
insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect
cells (e.g., SF9 cells) include the pAc series (Smith et al. (1983)
Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow and Summers
(1989) Virology 170:31-39).
[0191] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO
J 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0192] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv Immunol 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, e.g., the murine hox promoters
(Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev
3:537-546).
[0193] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to a HDGFX mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0194] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0195] A host cell can be any prokaryotic or eukaryotic cell. For
example, the HDGFX protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0196] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0197] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding the growth promoter or can be introduced on a
separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0198] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) the HDGFX protein. Accordingly, the invention further
provides methods for producing the HDGFX protein using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant
expression vector encoding the HDGFX polypeptide has been
introduced) in a suitable medium such that the HDGFX protein is
produced. In another embodiment, the method further comprises
isolating the HDGFX from the medium or the host cell.
[0199] Transgenic Animals
[0200] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which HDGFX-coding sequences have been introduced. Such
host cells can then be used to create non-human transgenic animals
in which exogenous HDGFX sequences have been introduced into their
genome or homologous recombinant animals in which endogenous HDGFX
sequences have been altered. Such animals are useful for studying
the function and/or activity of the HDGFX sequences and for
identifying and/or evaluating modulators of HDGFX activity. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA that is integrated into the genome of a cell from
which a transgenic animal develops and that remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous HDGFX gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0201] A transgenic animal of the invention can be created by
introducing HDGFX-encoding nucleic acid into the male pronuclei of
a fertilized oocyte, e.g., by microinjection, retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The human HDGFX DNA sequence of SEQ ID NO:1 can be
introduced as a transgene into the genome of a non-human animal.
Alternatively, a nonhuman homologue of the human HDGFX gene, such
as a mouse HDGFX gene, can be isolated based on hybridization to
the human HDGFX cDNA (described further above) and used as a
transgene. Intronic sequences and polyadenylation signals can also
be included in the transgene to increase the efficiency of
expression of the transgene. A tissue-specific regulatory
sequence(s) can be operably linked to the HDGFX transgene to direct
expression of HDGFX protein to particular cells. Methods for
generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan 1986, In:
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. Similar methods are used for production of
other transgenic animals. A transgenic founder animal can be
identified based upon the presence of the HDGFX transgene in its
genome and/or expression of HDGFX mRNA in tissues or cells of the
animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene encoding a HDGFX can further be bred
to other transgenic animals carrying other transgenes.
[0202] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a HDGFX gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the HDGFX gene. The
HDGFX gene can be a human gene (e.g., SEQ ID NO:1), but more
preferably, is a non-human homologue of a human HDGFX gene. For
example, a mouse homologue of human HDGFX gene of SEQ ID NO:1 can
be used to construct a homologous recombination vector suitable for
altering an endogenous HDGFX gene in the mouse genome. In one
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous HDGFX gene is functionally disrupted
(i.e., no longer encodes a functional protein; also referred to as
a "knock out" vector).
[0203] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous HDGFX gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous HDGFX protein). In the homologous
recombination vector, the altered portion of the HDGFX gene is
flanked at its 5' and 3' ends by additional nucleic acid of the
HDGFX gene to allow for homologous recombination to occur between
the exogenous HDGFX protein gene carried by the vector and an
endogenous HDGFX protein gene in an embryonic stem cell. The
additional flanking HDGFX protein nucleic acid is of sufficient
length for successful homologous recombination with the endogenous
gene. Typically, several kilobases of flanking DNA (both at the 5'
and 3' ends) are included in the vector. See e.g., Thomas et al.
(1987) Cell 51:503 for a description of homologous recombination
vectors. The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced HDGFX
protein gene has homologously recombined with the endogenous HDGFX
protein gene are selected (see e.g., Li et al. (1992) Cell
69:915).
[0204] The selected cells are then injected into a blastocyst of an
animal (e.g., a mouse) to form aggregation chimeras. See e.g.,
Bradley 1987, In: Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, Robertson, ed. IRL, Oxford, pp. 113-152. A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Curr Opin Biotechnol 2:823-829; PCT International
Publication Nos.: WO 90/1184; WO 91/01140; WO 92/0968; and WO
93/04169.
[0205] In another embodiment, transgenic non-humans animals can be
produced that contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:181-185. If a cre/loxP recombinase system is
used to regulate expression of the transgene, animals containing
transgenes encoding both the Cre recombinase and a selected protein
are required. Such animals can be provided through the construction
of "double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0206] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813. In brief, a cell, e.g., a somatic
cell, from the transgenic animal can be isolated and induced to
exit the growth cycle and enter G.sub.0 phase. The quiescent cell
can then be fused, e.g., through the use of electrical pulses, to
an enucleated oocyte from an animal of the same species from which
the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyte and then
transferred to pseudopregnant female foster animal. The offspring
borne of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0207] Pharmaceutical Compositions
[0208] The HDGFX nucleic acid molecules, HDGFX proteins, and
anti-HDGFX antibodies of the invention, and derivatives, fragments,
analogs and homologs thereof are designated "active compounds" or
"Therapeutics" herein. Additionally, low molecular weight compounds
which have the property that they either bind to the HDGFX nucleic
acid molecules, the HDGFX proteins, and the anti-HDGFX antibodies
of the invention, and derivatives, fragments, analogs and homologs
thereof, or induce pharmacological agonist or antagonist responses
commonly ascribed to a HDGFX nucleic acid molecule, a HDGFX
protein, and derivatives, fragments, analogs and homologs thereof,
are also termed "active compounds" or "Therapeutics" herein. These
Therapeutics can be incorporated into pharmaceutical compositions
suitable for administration to a subject. Such compositions
typically comprise the nucleic acid molecule, protein, or antibody
and a pharmaceutically acceptable carrier.
[0209] As used herein, "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, a
standard reference text in the field, which is incorporated herein
by reference. Preferred examples of such carriers or diluents
include, but are not limited to, water, saline, Ringer's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0210] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0211] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0212] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a HDGFX protein or
anti-HDGFX protein antibody) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0213] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding azgents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0214] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0215] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0216] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0217] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0218] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release
pharmaceutical active agents over shorter time periods.
[0219] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0220] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by any of a number of routes, e.g.,
as described in U.S. Pat. No. 5,703,055. Delivery can thus also
include, e.g., intravenous injection, local administration (see
U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen
et al. (1994) PNAS 91:3054-3057). The pharmaceutical preparation of
the gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells that produce the gene
delivery system.
[0221] The pharmaceutical compositions can be included in a kit,
e.g., in a container, pack, or dispenser together with instructions
for administration.
[0222] Also within the invention is the use of a therapeutic in the
manufacture of a medicament for treating a syndrome associated with
a human disease, the disease selected from a HDGFX-associated
disorder, wherein said therapeutic is selected from the group
consisting of a HDGFX polypeptide, a HDGFX nucleic acid, and an
anti-HDGFX antibody.
[0223] Additional Uses and Methods of the Invention
[0224] Various HDGF family members have been implicated in
angiogenesis, spermatogenesis, smooth muscle growth and neuronal
development. Accordingly, this suggests a role of HDGFX in treating
or diagnosing disease related to these functions. For example, HDGF
will be useful in therapeutic applications implicated in various
cancers, coronary artery disease, arthritis, diabetic retinopathy,
infertility and various neurological diseases, e.g., Parkinson's
Disease, Alzheimer's, amyotropic lateral sclerosis and psychiatric
disorders.
[0225] The potential role(s) of HDGFX in tumorigenesis may include
autocrine stimulation of tumor growth, hormone independence,
angiogenesis, metastatic progression, chemoresistance, radiotherapy
resistance, survival in trophic factor limited secondary tissue
site microenvironments, and stimulation of tumor cell matrix
degradation and tumor cell migration (i.e., tumor invasion).
[0226] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: (a) screening assays; (b) detection assays
(e.g., chromosomal mapping, cell and tissue typing, forensic
biology), (c) predictive medicine (e.g., diagnostic assays,
prognostic assays, monitoring clinical trials, and
pharmacogenomics); and (d) methods of treatment (e.g., therapeutic
and prophylactic).
[0227] The isolated nucleic acid molecules of the invention can be
used to express a HDGFX protein (e.g., via a recombinant expression
vector in a host cell in gene therapy applications), to detect a
HDGFX mRNA (e.g., in a biological sample) or a genetic lesion in a
HDGFX gene, and to modulate HDGFX activity, as described further
below. In addition, the HDGFX proteins can be used to screen drugs
or compounds that modulate the HDGFX activity or expression as well
as to treat disorders characterized by insufficient or excessive
production of the HDGFX protein, for example proliferative or
differentiative disorders, or production of the HDGFX protein forms
that have decreased or aberrant activity compared to the HDGFX wild
type protein. In addition, the anti-HDGFX antibodies of the
invention can be used to detect and isolate HDGFX proteins and
modulate HDGFX activity.
[0228] This invention further pertains to novel agents identified
by the above described screening assays and uses thereof for
treatments as described herein.
[0229] Screening Assays
[0230] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, polypeptides, nucleic
acids or polynucleotides, peptides, peptidomimetics, small
molecules including agonists or antagonists, or other drugs) that
bind to HDGFX proteins or have a stimulatory or inhibitory effect
on, for example, HDGFX expression or HDGFX activity. The candidate
or test compounds or agents that may bind to a HDGFX polypeptide
may have a molecular weight around 50 Da, 100 Da, 150 Da, 300 Da,
330 Da, 350 Da, 400 Da, 500 Da, 750 Da, 1000 Da, 1250 Da, 1500 Da,
1750 Da, 2000 Da, 5000 Da, 10,000 Da, 25,000 Da, 50,000 Da, 75,000
Da, 100,000 Da or more than 100,000 Da. In certain embodiments, the
candidate substance that binds to a HDGFX polypeptide has a
molecular weight not more than about 1500 Da.
[0231] Details of functional assays are provided herein further
below. Any of the assays described, as well as additional assays
known to practitioners in the fields of pharmacology, hematology,
internal medicine, oncology and the like, may be employed in order
to screen candidate substance for their properties as therapeutic
agents. As noted, the therapeutic agents of the invention encompass
proteins, polypeptides, nucleic acids or polynucleotides, peptides,
peptidomimetics, small molecules including agonists or antagonists,
or other drugs described herein.
[0232] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a HDGFX protein or polypeptide or biologically active
portion thereof. The test compounds of the present invention can be
obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
"one-bead one-compound" library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam (1997) Anticancer Drug
Des 12:145).
[0233] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc
Natl Acad Sci U.S.A. 90:6909; Erb et al. (1994) Proc Natl Acad Sci
U.S.A. 91:11422; Zuckermann et al. (1994) J Med Chem 37:2678; Cho
et al. (1993) Science 261:1303; Carrell et al. (1994) Angew Chem
Int Ed Engl 33:2059; Carell et al. (1994) Angew Chem Int Ed Engl
33:2061; and Gallop et al. (1994) J Med Chem 37:1233.
[0234] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc Natl Acad Sci U.S.A. 87:6378-6382; Felici (1991) J Mol
Biol 222:301-310; Ladner above.).
[0235] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a membrane-bound form of a HDGFX protein, or a
biologically active portion thereof, on the cell surface is
contacted with a test compound and the ability of the test compound
to bind to a HDGFX protein determined. The cell, for example, can
of mammalian origin or a yeast cell. Determining the ability of the
test compound to bind to the HDGFX protein can be accomplished, for
example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound to the HDGFX
protein or biologically active portion thereof can be determined by
detecting the labeled compound in a complex. For example, test
compounds can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, test compounds can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to product.
In one embodiment, the assay comprises contacting a cell which
expresses a membrane-bound form of a HDGFX protein, or a
biologically active portion thereof, on the cell surface with a
known compound which binds a HDGFX to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with a HDGFX protein,
wherein determining the ability of the test compound to interact
with a HDGFX protein comprises determining the ability of the test
compound to preferentially bind to a HDGFX or a biologically active
portion thereof as compared to the known compound.
[0236] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a membrane-bound form of a
HDGFX protein, or a biologically active portion thereof, on the
cell surface with a test compound and determining the ability of
the test compound to modulate (e.g., stimulate or inhibit) the
activity of the HDGFX protein or biologically active portion
thereof. Determining the ability of the test compound to modulate
the activity of a HDGFX polypeptide or a biologically active
portion thereof can be accomplished, for example, by determining
the ability of the HDGFX protein to bind to or interact with a
HDGFX target molecule. As used herein, a "target molecule" is a
molecule with which a HDGFX protein binds or interacts in nature,
for example, a molecule on the surface of a cell which expresses a
HDGFX interacting protein, a molecule on the surface of a second
cell, a molecule in the extracellular milieu, a molecule associated
with the internal surface of a cell membrane or a cytoplasmic
molecule. A HDGFX target molecule can be a non-HDGFX molecule or a
HDGFX protein or polypeptide of the present invention. In one
embodiment, a HDGFX target molecule is a component of a signal
transduction pathway that facilitates transduction of an
extracellular signal (e.g., a signal generated by binding of a
compound to a membrane-bound HDGFX molecule) through the cell
membrane and into the cell. The target, for example, can be a
second intercellular protein that has catalytic activity or a
protein that facilitates the association of downstream signaling
molecules with the HDGFX polypeptide.
[0237] Determining the ability of the HDGFX protein to bind to or
interact with a HDGFX target molecule can be accomplished by one of
the methods described above for determining direct binding. In one
embodiment, determining the ability of the HDGFX protein to bind to
or interact with a HDGFX target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e.
intracellular Ca.sup.2+, diacylglycerol, IP.sub.3, etc.), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising a
HDGFX-responsive regulatory element operatively linked to a nucleic
acid encoding a detectable marker, e.g., luciferase), or detecting
a cellular response, for example, cell survival, cellular
differentiation, or cell proliferation.
[0238] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a HDGFX protein or
biologically active portion thereof with a test compound and
determining the ability of the test compound to bind to the HDGFX
protein or biologically active portion thereof. Binding of the test
compound to the HDGFX protein can be determined either directly or
indirectly as described above. In one embodiment, the assay
comprises contacting the HDGFX protein or biologically active
portion thereof with a known compound which binds HDGFX to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with a
HDGFX protein, wherein determining the ability of the test compound
to interact with a HDGFX protein comprises determining the ability
of the test compound to preferentially bind to a HDGFX or
biologically active portion thereof as compared to the known
compound.
[0239] In another embodiment, an assay is a cell-free assay
comprising contacting a HDGFX protein or biologically active
portion thereof with a test compound and determining the ability of
the test compound to modulate (e.g., stimulate or inhibit) the
activity of the HDGFX protein or biologically active portion
thereof. Determining the ability of the test compound to modulate
the activity of a HDGFX polypeptide can be accomplished, for
example, by determining the ability of the HDGFX protein to bind to
a HDGFX target molecule by one of the methods described above for
determining direct binding. In an alternative embodiment,
determining the ability of the test compound to modulate the
activity of a HDGFX polypeptide can be accomplished by determining
the ability of the HDGFX protein further modulate a HDGFX target
molecule. For example, the catalytic/enzymatic activity of the
target molecule on an appropriate substrate can be determined as
previously described.
[0240] In yet another embodiment, the cell-free assay comprises
contacting the HDGFX protein or biologically active portion thereof
with a known compound which binds a HDGFX polypeptide to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with a
HDGFX protein, wherein determining the ability of the test compound
to interact with a HDGFX protein comprises determining the ability
of the HDGFX protein to preferentially bind to or modulate the
activity of a HDGFX target molecule.
[0241] The cell-free assays of the present invention are amenable
to use of both a soluble form or a membrane-bound form of a HDGFX
polypeptide. In the case of cell-free assays comprising the
membrane-bound form of a HDGFX polypeptide, it may be desirable to
utilize a solubilizing agent such that the membrane-bound form of a
HDGFX polypeptide is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as
n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamid- e,
Triton.RTM. X-100, Triton.RTM. X-114, Thesit.RTM.,
Isotridecypoly(ethylene glycol ether).sub.n,
N-dodecyl-N,N-dimethyl-3-amm- onio-1-propane sulfonate,
3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-p- ropane
sulfonate (CHAPSO).
[0242] It may be desirable to immobilize either a HDGFX polypeptide
or its target molecule to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test compound to
a HDGFX polypeptide, or interaction of a HDGFX polypeptide with a
target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtiter plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided that adds a domain that allows one or both
of the proteins to be bound to a matrix. For example, GST-HDGFX
polypeptide fusion proteins or GST-target fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtiter plates, that are
then combined with the test compound or the test compound and
either the non-adsorbed target protein or a HDGFX protein, and the
mixture is incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components, the matrix immobilized in
the case of beads, complex determined either directly or
indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of a
HDGFX binding or activity determined using standard techniques.
[0243] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the HDGFX polypeptide or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated HDGFX protein or target molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques well known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with
HDGFX protein or target molecules, but which do not interfere with
binding of the HDGFX protein to its target molecule, can be
derivatized to the wells of the plate, and unbound target or HDGFX
protein trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the HDGFX protein or target
molecule, as well as enzyme-linked assays that rely on detecting an
enzymatic activity associated with the HDGFX protein or target
molecule.
[0244] In another embodiment, modulators of a HDGFX expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of a HDGFX mRNA or protein in the cell
is determined. The level of expression of a HDGFX mRNA or protein
in the presence of the candidate compound is compared to the level
of expression of a HDGFX mRNA or protein in the absence of the
candidate compound. The candidate compound can then be identified
as a modulator of a HDGFX expression based on this comparison. For
example, when expression of a HDGFX mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of a HDGFX mRNA or protein expression.
Alternatively, when expression of a HDGFX mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of a HDGFX mRNA or protein expression. The level of
a HDGFX mRNA or protein expression in the cells can be determined
by methods described herein for detecting HDGFX mRNA or
protein.
[0245] In yet another aspect of the invention, the HDGFX proteins
can be used as "bait proteins" in a two-hybrid assay or three
hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.
(1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins that bind to or interact with the HDGFX
("HDGFX-binding proteins" or "HDGFX-bp") and modulate HDGFX
activity. Such HDGFX-binding proteins are also likely to be
involved in the propagation of signals by the HDGFX proteins as,
for example, upstream or downstream elements of the HDGFX
pathway.
[0246] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a HDGFX is
fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL-4). In the other construct, a DNA
sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a HDGFX-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) that is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene that encodes the protein which interacts
with the HDGFX.
[0247] Screening can also be performed in vivo. For example, in one
embodiment, the invention includes a method for screening for a
modulator of activity or of latency or predisposition to a
HDGFX-associated disorder by administering a test compound or to a
test animal at increased risk for a HDGFX-associated disorder. In
some embodiments, the test animal recombinantly expresses a HDGFX
polypeptide. Activity of the polypeptide in the test animal after
administering the compound is measured, and the activity of the
protein in the test animal is compared to the activity of the
polypeptide in a control animal not administered said polypeptide.
A change in the activity of said polypeptide in said test animal
relative to the control animal indicates the test compound is a
modulator of latency of or predisposition to a HDGFX-associated
disorder.
[0248] In some embodiments, the test animal is a recombinant test
animal that expresses a test protein transgene or expresses the
transgene under the control of a promoter at an increased level
relative to a wild-type test animal. Preferably, the promoter is
not the native gene promoter of the transgene.
[0249] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0250] Detection Assays
[0251] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample.
[0252] The HDGFX sequences of the present invention can also be
used to identify individuals from minute biological samples. In
this technique, an individual's genomic DNA is digested with one or
more restriction enzymes, and probed on a Southern blot to yield
unique bands for identification. The sequences of the present
invention are useful as additional DNA markers for RFLP
("restriction fragment length polymorphisms," described in U.S.
Pat. No. 5,272,057).
[0253] Furthermore, the sequences of the present invention can be
used to provide an alternative technique that determines the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the HDGFX sequences described herein can be used to
prepare two PCR primers from the 5' and 3' ends of the sequences.
These primers can then be used to amplify an individual's DNA and
subsequently sequence it.
[0254] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The HDGFX sequences of
the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of
these sequences, and to a greater degree in the noncoding regions.
It is estimated that allelic variation between individual humans
occurs with a frequency of about once per each 500 bases. Much of
the allelic variation is due to single nucleotide polymorphisms
(SNPs), which include restriction fragment length polymorphisms
(RFLPs).
[0255] Each of the sequences described herein can, to some degree,
be used as a standard against which DNA from an individual can be
compared for identification purposes. Because greater numbers of
polymorphisms occur in the noncoding regions, fewer sequences are
necessary to differentiate individuals. The noncoding sequences of
SEQ ID NO:1, as described above, can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers that each yield a noncoding amplified sequence of 100
bases. If predicted coding sequences are used, a more appropriate
number of primers for positive individual identification would be
500-2,000.
[0256] Use of Partial HDGFX Sequences in Forensic Biology
[0257] DNA-based identification techniques based on HDGFX nucleic
acid sequences or polypeptide sequences can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0258] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, that can enhance the reliability
of DNA-based forensic identifications by, for example, providing
another "identification marker" (i.e. another DNA sequence that is
unique to a particular individual). As mentioned above, actual base
sequence information can be used for identification as an accurate
alternative to patterns formed by restriction enzyme generated
fragments. Sequences targeted to noncoding regions of SEQ ID NO:1
are particularly appropriate for this use as greater numbers of
polymorphisms occur in the noncoding regions, making it easier to
differentiate individuals using this technique. Examples of
polynucleotide reagents include the HDGFX sequences or portions
thereof, e.g., fragments derived from the noncoding regions of SEQ
ID NO:1, having a length of at least 20 bases, preferably at least
30 bases.
[0259] The HDGFX sequences described herein can further be used to
provide polynucleotide reagents, e.g., labeled or label-able probes
that can be used, for example, in an in situ hybridization
technique, to identify a specific tissue, e.g., brain tissue, etc.
This can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such HDGFX
probes can be used to identify tissue by species and/or by organ
type.
[0260] In a similar fashion, these reagents, e.g., HDGFX primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
[0261] Predictive Medicine
[0262] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining a HDGFX protein and/or
nucleic acid expression as well as HDGFX activity, in the context
of a biological sample (e.g., blood, serum, cells, tissue) to
thereby determine whether an individual is afflicted with a disease
or disorder, or is at risk of developing a disorder, associated
with aberrant HDGFX expression or activity. The invention also
provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder
associated with a HDGFX protein, nucleic acid expression or
activity. For example, mutations in a HDGFX gene can be assayed in
a biological sample. Such assays can be used for prognostic or
predictive purpose to thereby prophylactically treat an individual
prior to the onset of a disorder characterized by or associated
with HDGFX protein, nucleic acid expression or activity.
[0263] Another aspect of the invention provides methods for
determining HDGFX protein, nucleic acid expression or HDGFX
activity in an individual to thereby select appropriate therapeutic
or prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows for the selection of
agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual based on the genotype of the individual (e.g., the
genotype of the individual examined to determine the ability of the
individual to respond to a particular agent.)
[0264] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs, compounds) on the expression
or activity of a HDGFX in clinical trials.
[0265] These and other agents are described in further detail in
the following sections.
[0266] Diagnostic Assays
[0267] Other conditions in which proliferation of cells plays a
role include tumors, restenosis, psoriasis, Dupuytren's
contracture, diabetic complications, Kaposi's sarcoma and
rheumatoid arthritis.
[0268] A HDGFX polypeptide may be used to identify an interacting
polypeptide a sample or tissue. The method comprises contacting the
sample or tissue with the HDGFX, allowing formation of a complex
between the HDGFX polypeptide and the interacting polypeptide, and
detecting the complex, if present.
[0269] The proteins of the invention may be used to stimulate
production of antibodies specifically binding the proteins. Such
antibodies may be used in immunodiagnostic procedures to detect the
occurrence of the protein in a sample. The proteins of the
invention may be used to stimulate cell growth and cell
proliferation in conditions in which such growth would be
favorable. An example would be to counteract toxic side effects of
chemotherapeutic agents on, for example, hematopoiesis and platelet
formation, linings of the gastrointestinal tract, and hair
follicles. They may also be used to stimulate new cell growth in
neurological disorders including, for example, Alzheimer's disease.
Alternatively, antagonistic treatments may be administered in which
an antibody specifically binding the HDGFX-like proteins of the
invention would abrogate the specific growth-inducing effects of
the proteins. Such antibodies may be useful, for example, in the
treatment of proliferative disorders including various tumors and
benign hyperplasias.
[0270] Polynucleotides or oligonucleotides corresponding to any one
portion of the HDGFX nucleic acids of SEQ ID NO:1 may be used to
detect DNA containing a corresponding ORF gene, or detect the
expression of a corresponding HDGFX gene, or HDGFX-like gene. For
example, a HDGFX nucleic acid expressed in a particular cell or
tissue, as noted in Table 3, can be used to identify the presence
of that particular cell type.
[0271] An exemplary method for detecting the presence or absence of
a HDGFX polypeptide in a biological sample involves obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting a HDGFX
protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes a
HDGFX protein such that the presence of a HDGFX polypeptide is
detected in the biological sample. An agent for detecting a HDGFX
mRNA or genomic DNA is a labeled nucleic acid probe capable of
hybridizing to a HDGFX mRNA or genomic DNA. The nucleic acid probe
can be, for example, a full-length HDGFX nucleic acid, such as the
nucleic acid of SEQ ID NO:1, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to a HDGFX mRNA or genomic DNA, as described above.
Other suitable probes for use in the diagnostic assays of the
invention are described herein.
[0272] An agent for detecting a HDGFX protein is an antibody
capable of binding to a HDGFX protein, preferably an antibody with
a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof
(e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with
regard to the probe or antibody, is intended to encompass direct
labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable substance to the probe or antibody, as well
as indirect labeling of the probe or antibody by reactivity with
another reagent that is directly labeled. Examples of indirect
labeling include detection of a primary antibody using a
fluorescently labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin. The term "biological sample" is intended to
include tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject. That is, the detection method of the invention can be used
to detect a HDGFX mRNA, protein, or genomic DNA in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of a HDGFX mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of a HDGFX protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of a HDGFX
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of a HDGFX protein include introducing
into a subject a labeled anti-HDGFX antibody. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques.
[0273] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0274] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting a
HDGFX protein, mRNA, or genomic DNA, such that the presence of a
HDGFX protein, mRNA or genomic DNA is detected in the biological
sample, and comparing the presence of a HDGFX protein, mRNA or
genomic DNA in the control sample with the presence of a HDGFX
protein, mRNA or genomic DNA in the test sample.
[0275] The invention also encompasses kits for detecting the
presence of a HDGFX polypeptide in a biological sample. For
example, the kit can comprise: a labeled compound or agent capable
of detecting a HDGFX protein or mRNA in a biological sample; means
for determining the amount of a HDGFX polypeptide in the sample;
and means for comparing the amount of a HDGFX polypeptide in the
sample with a standard. The compound or agent can be packaged in a
suitable container. The kit can further comprise instructions for
using the kit to detect a HDGFX protein or nucleic acid.
[0276] Prognostic Assays
[0277] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant HDGFX polypeptide
expression or activity. For example, the assays described herein,
such as the preceding diagnostic assays or the following assays,
can be utilized to identify a subject having or at risk of
developing a disorder associated with a HDGFX protein, nucleic acid
expression or activity in, e.g., proliferative or differentiative
disorders such as hyperplasias, tumors, restenosis, psoriasis,
Alzheimer's disease, etc. Alternatively, the prognostic assays can
be utilized to identify a subject having or at risk for developing
a disease or disorder. Thus, the present invention provides a
method for identifying a disease or disorder associated with
aberrant HDGFX expression or activity in which a test sample is
obtained from a subject and a HDGFX protein or nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of a HDGFX
protein or nucleic acid is diagnostic for a subject having or at
risk of developing a disease or disorder associated with aberrant
HDGFX expression or activity. As used herein, a "test sample"
refers to a biological sample obtained from a subject of interest.
For example, a test sample can be a biological fluid (e.g., serum),
cell sample, or tissue.
[0278] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant HDGFX expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
disorder, such as a proliferative disorder, differentiative
disorder, glia-associated disorders, etc. Thus, the present
invention provides methods for determining whether a subject can be
effectively treated with an agent for a disorder associated with
aberrant HDGFX expression or activity in which a test sample is
obtained and a HDGFX protein or nucleic acid is detected (e.g.,
wherein the presence of a HDGFX protein or nucleic acid is
diagnostic for a subject that can be administered the agent to
treat a disorder associated with aberrant HDGFX expression or
activity.)
[0279] The methods of the invention can also be used to detect
genetic lesions in a HDGFX gene, thereby determining if a subject
with the lesioned gene is at risk for, or suffers from, a
proliferative disorder, differentiative disorder, glia-associated
disorder, etc. In various embodiments, the methods include
detecting, in a sample of cells from the subject, the presence or
absence of a genetic lesion characterized by at least one of an
alteration affecting the integrity of a gene encoding a HDGFX
protein, or the mis-expression of the HDGFX gene. For example, such
genetic lesions can be detected by ascertaining the existence of at
least one of (1) a deletion of one or more nucleotides from a HDGFX
gene; (2) an addition of one or more nucleotides to a HDGFX gene;
(3) a substitution of one or more nucleotides of a HDGFX gene, (4)
a chromosomal rearrangement of a HDGFX gene; (5) an alteration in
the level of a messenger RNA transcript of a HDGFX gene, (6)
aberrant modification of a HDGFX gene, such as of the methylation
pattern of the genomic DNA, (7) the presence of a non-wild type
splicing pattern of a messenger RNA transcript of a HDGFX gene, (8)
a non-wild type level of a protein, (9) allelic loss of a HDGFX
gene, and (10) inappropriate post-translational modification of a
HDGFX protein. As described herein, there are a large number of
assay techniques known in the art which can be used for detecting
lesions in a HDGFX gene. A preferred biological sample is a
peripheral blood leukocyte sample isolated by conventional means
from a subject. However, any biological sample containing nucleated
cells may be used, including, for example, buccal mucosal
cells.
[0280] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al. (1988) Science 241:1077-1080; and
Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be
particularly useful for detecting point mutations in the HDGFX gene
(see Abravaya et al. (1995) Nucl Acids Res 23:675-682). This method
can include the steps of collecting a sample of cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers that specifically hybridize to a HDGFX gene
under conditions such that hybridization and amplification of the
HDGFX gene (if present) occurs, and detecting the presence or
absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
It is anticipated that PCR and/or LCR may be desirable to use as a
preliminary amplification step in conjunction with any of the
techniques used for detecting mutations described herein.
[0281] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al., 1990, Proc Natl Acad Sci USA
87:1874-1878), transcriptional amplification system (Kwoh, et al.,
1989, Proc Natl Acad Sci USA 86:1173-1177), Q-Beta Replicase
(Lizardi et al, 1988, BioTechnology 6:1197), or any other nucleic
acid amplification method, followed by the detection of the
amplified molecules using techniques well known to those of skill
in the art. These detection schemes are especially useful for the
detection of nucleic acid molecules if such molecules are present
in very low numbers.
[0282] In an alternative embodiment, mutations in a HDGFX gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,493,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0283] In other embodiments, genetic mutations in a HDGFX nucleic
acid of the invention can be identified by hybridizing a sample and
control nucleic acids, e.g., DNA or RNA, to high density arrays
containing hundreds or thousands of oligonucleotides probes (Cronin
et al. (1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature
Medicine 2: 753-759). For example, genetic mutations in a HDGFX of
the invention can be identified in two dimensional arrays
containing light-generated DNA probes as described in Cronin et al.
above. Briefly, a first hybridization array of probes can be used
to scan through long stretches of DNA in a sample and control to
identify base changes between the sequences by making linear arrays
of sequential overlapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0284] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
HDGFX gene and detect mutations by comparing the sequence of the
sample HDGFX gene with the corresponding wild-type (control)
sequence. Examples of sequencing reactions include those based on
techniques developed by Maxim and Gilbert (1977) PNAS 74:560 or
Sanger (1977) PNAS 74:5463. It is also contemplated that any of a
variety of automated sequencing procedures can be utilized when
performing the diagnostic assays (Naeve et al., (1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al.
(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl
Biochem Biotechnol 38:147-159).
[0285] Other methods for detecting mutations in the HDGFX gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type HDGFX
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent that
cleaves single-stranded regions of the duplex such as which will
exist due to basepair mismatches between the control and sample
strands. For instance, RNA/DNA duplexes can be treated with RNase
and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al (1988) Proc Natl Acad Sci USA 85:4397; Saleeba et al (1992)
Methods Enzymol 217:286-295. In an embodiment, the control DNA or
RNA can be labeled for detection.
[0286] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in HDGFX
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a HDGFX sequence, e.g., a wild-type
HDGFX sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0287] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in HDGFX genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl Acad Sci
USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; Hayashi
(1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments
of sample and control a HDGFX nucleic acids will be denatured and
allowed to renature. The secondary structure of single-stranded
nucleic acids varies according to sequence, the resulting
alteration in electrophoretic mobility enables the detection of
even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA, rather than DNA, in which the secondary
structure is more sensitive to a change in sequence. In one
embodiment, the subject method utilizes heteroduplex analysis to
separate double stranded heteroduplex molecules on the basis of
changes in electrophoretic mobility. See, e.g., Keen et al. (1991)
Trends Genet 7:5.
[0288] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE). See, e.g., Myers et al (1985) Nature 313:495. When DGGE is
used as the method of analysis, DNA will be modified to insure that
it does not completely denature, for example by adding a GC clamp
of approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA. See, e.g., Rosenbaum and Reissner (1987)
Biophys Chem 265:12753.
[0289] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions that permit hybridization only if a
perfect match is found. See, e.g., Saiki et al. (1986) Nature
324:163); Saiki et al. (1989) Proc Natl Acad. Sci USA 86:6230. Such
allele specific oligonucleotides are hybridized to PCR amplified
target DNA or a number of different mutations when the
oligonucleotides are attached to the hybridizing membrane and
hybridized with labeled target DNA.
[0290] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification may carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res
17:2437-2448) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner (1993) Tibtech 11:238). In addition it may be
desirable to introduce a novel restriction site in the region of
the mutation to create cleavage-based detection. See, e.g.,
Gasparini et al (1992) Mol Cell Probes 6:1. It is anticipated that
in certain embodiments amplification may also be performed using
Taq ligase for amplification. See, e.g., Barany (1991) Proc Natl
Acad Sci USA 88:189. In such cases, ligation will occur only if
there is a perfect match at the 3' end of the 5' sequence, making
it possible to detect the presence of a known mutation at a
specific site by looking for the presence or absence of
amplification.
[0291] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a HDGFX gene.
[0292] Furthermore, any cell type or tissue, preferably peripheral
blood leukocytes, in which a HDGFX of the invention is expressed
may be utilized in the prognostic assays described herein. However,
any biological sample containing nucleated cells may be used,
including, for example, buccal mucosal cells.
[0293] Pharmacogenomics
[0294] Agents, or modulators that have a stimulatory or inhibitory
effect on HDGFX activity (e.g., HDGFX gene expression), as
identified by a screening assay described herein can be
administered to individuals to treat (prophylactically or
therapeutically) disorders (e.g., neurological, cancer-related or
gestational disorders) associated with aberrant HDGFX activity. In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permits the selection of effective agents (e.g., drugs)
for prophylactic or therapeutic treatments based on a consideration
of the individual's genotype. Such pharmacogenomics can further be
used to determine appropriate dosages and therapeutic regimens.
Accordingly, the activity of a HDGFX protein, expression of a HDGFX
nucleic acid, or mutation content of a HDGFX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual.
[0295] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See e.g.,
Eichelbaum, 1996, Clin Exp Pharmacol Physiol, 23:983-985 and
Linder, 1997, Clin Chem, 43:254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0296] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0297] Thus, the activity of a HDGFX protein, expression of a HDGFX
nucleic acid, or mutation content of a HDGFX genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual. In
addition, pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a HDGFX modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0298] Monitoring Clinical Efficacy
[0299] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of a HDGFX (e.g., the ability to
modulate aberrant cell proliferation and/or differentiation) can be
applied in basic drug screening and in clinical trials. For
example, the effectiveness of an agent determined by a screening
assay as described herein to increase HDGFX gene expression,
protein levels, or upregulate HDGFX activity, can be monitored in
clinical trials of subjects exhibiting decreased HDGFX gene
expression, protein levels, or downregulated HDGFX activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease HDGFX gene expression, protein levels,
or downregulate HDGFX activity, can be monitored in clinical trials
of subjects exhibiting increased HDGFX gene expression, protein
levels, or upregulated HDGFX activity. In such clinical trials, the
expression or activity of a HDGFX and, preferably, other genes that
have been implicated in, for example, a proliferative or
neurological disorder, can be used as a "read out" or marker of the
responsiveness of a particular cell. Other HDGFX-associated
disorders include, e.g., cancers, cell proliferation disorders,
neurological disorders; and fertility disorders..
[0300] For example, genes, including genes encoding a HDGFX of the
invention, that are modulated in cells by treatment with an agent
(e.g., compound, drug or small molecule) that modulates a HDGFX
activity (e.g., identified in a screening assay as described
herein) can be identified. Thus, to study the effect of agents on
cellular proliferation disorders, for example, in a clinical trial,
cells can be isolated and RNA prepared and analyzed for the levels
of expression of a HDGFX and other genes implicated in the
disorder. The levels of gene expression (i.e., a gene expression
pattern) can be quantified by Northern blot analysis or RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods as described herein, or by
measuring the levels of activity of a gene or other genes. In this
way, the gene expression pattern can serve as a marker, indicative
of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during, treatment of the individual with the
agent.
[0301] In one embodiment, the invention provides a method for
monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, protein, peptide, nucleic
acid, peptidomimetic, small molecule, or other drug candidate
identified by the screening assays described herein) comprising the
steps of (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of
expression of a HDGFX protein, mRNA, or genomic DNA in the
preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the HDGFX protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the HDGFX protein, mRNA, or
genomic DNA in the pre-administration sample with the HDGFX
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of a
HDGFX to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
a HDGFX to lower levels than detected, i.e., to decrease the
effectiveness of the agent.
[0302] Methods of Treatment
[0303] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant HDGFX expression or activity.
[0304] Diseases and disorders that are characterized by increased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
antagonize (i.e., reduce or inhibit) activity. Therapeutics that
antagonize activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to, (i) a HDGFX polypeptide, or analogs,
derivatives, fragments or homologs thereof, (ii) antibodies to a
HDGFX peptide; (iii) nucleic acids encoding a HDGFX peptide; (iv)
administration of antisense nucleic acid and nucleic acids that are
"dysfunctional" (i.e., due to a heterologous insertion within the
coding sequences of coding sequences to a HDGFX polypeptide) that
are utilized to "knockout" endogenous function of a HDGFX
polypeptide by homologous recombination (see, e.g., Capecchi, 1989,
Science 244: 1288-1292); or (v) modulators (i.e., inhibitors,
agonists and antagonists, including additional peptide mimetic of
the invention or antibodies specific to a peptide of the invention)
that alter the interaction between a HDGFX peptide and its binding
partner.
[0305] Diseases and disorders that are characterized by decreased
(relative to a subject not suffering from the disease or disorder)
levels or biological activity may be treated with Therapeutics that
increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity may be administered in a therapeutic or
prophylactic manner. Therapeutics that may be utilized include, but
are not limited to, a polypeptide, a peptide, or analogs,
derivatives, fragments or homologs thereof; or an agonist that
increases bioavailability.
[0306] Increased or decreased levels can be readily detected by
quantifying peptide and/or RNA, by obtaining a patient tissue
sample (e.g., from biopsy tissue) and assaying it in vitro for RNA
or polypeptide levels, structure and/or activity of the expressed
polypeptides (or mRNAs encoding a HDGFX polypeptide). Methods that
are well-known within the art include, but are not limited to,
immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis, immunocytochemistry, etc.) and/or hybridization
assays to detect expression of mRNAs (e.g., Northern assays, dot
blots, in situ hybridization, etc.).
[0307] In one aspect, the invention provides a method for
preventing, in a subject, a disease or condition associated with
aberrant HDGFX expression or activity, by administering to the
subject an agent that modulates HDGFX expression or at least one
HDGFX activity. Subjects at risk for a disease that is caused or
contributed to by aberrant HDGFX expression or activity can be
identified by, for example, any or a combination of diagnostic or
prognostic assays as described herein. Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the HDGFX aberrancy, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of a HDGFX aberrancy, for
example, a HDGFX agonist or HDGFX antagonist agent can be used for
treating the subject. The appropriate agent can be determined based
on screening assays described herein.
[0308] Another aspect of the invention pertains to methods of
modulating HDGFX expression or activity for therapeutic purposes.
The modulatory method of the invention involves contacting a cell
with an agent that modulates one or more of the activities of a
HDGFX protein activity associated with the cell. An agent that
modulates a HDGFX protein activity can be an agent as described
herein, such as a nucleic acid or a protein, a naturally-occurring
cognate ligand of a HDGFX protein, a peptide, a HDGFX
peptidomimetic, or other small molecule. In one embodiment, the
agent stimulates one or more a HDGFX protein activity. Examples of
such stimulatory agents include active a HDGFX protein and a
nucleic acid molecule encoding a HDGFX polypeptide that has been
introduced into the cell. In another embodiment, the agent inhibits
one or more a HDGFX protein activity. Examples of such inhibitory
agents include antisense a HDGFX nucleic acid molecules and
anti-HDGFX antibodies. These modulatory methods can be performed in
vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant expression or activity of a HDGFX protein
or nucleic acid molecule. In one embodiment, the method involves
administering an agent (e.g., an agent identified by a screening
assay described herein), or combination of agents that modulates
(e.g., upregulates or downregulates) HDGFX expression or activity.
In another embodiment, the method involves administering a HDGFX
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant HDGFX expression or activity.
[0309] The invention will be further illustrated in the following
examples, which do not limit the scope of the claims.
EXAMPLES
Example 1
[0310] Molecular Cloning of HDGFX
[0311] The predicted open reading frame codes for a 251 amino acid
long protein with an overall 59% identity, on the amino acid level,
to the bovine hepatoma derived growth factor related protein 3
(HRP-3) (TREMBLNEW-ACC:CAB40348). The predicted full length ORF has
been cloned and verified.
[0312] Cloning the Full Length HDGFX
[0313] Oligonucleotide primers were designed to PCR amplify a DNA
segment coding for the full length HDGFX gene product. The forward
primer includes an in-frame BamHI restriction site and a consensus
Kozak sequence. The reverse primer contains an in-frame XhoI
restriction site. The sequences of the PCR primers are the
following:
5 HDGFX Forw: GGATCCACCATGTCGGCCTACGGCATGCCCATGTAC, and (SEQ ID
NO:11) HDGFX Rev: CTCGAGCAGGCTGTCGCGATCTCCGCCGCC. (SEQ ID
NO:12)
[0314] PCR reactions were set up using a total of 5 ng mixture of
cDNA template containing equal amounts of cDNAs derived from human
fetal brain, human testis, human mammary and human skeletal muscle
tissues, 1 microM of each of the HDGFX Forw and HDGFX Rev primers,
5 micromoles dNTP (Clontech Laboratories, Palo Alto Calif.) and 1
microliter of 50.times. Advantage-HF 2 polymerase (Clontech
Laboratories, Palo Alto Calif.) in a 50 microliter volume. The
following PCR reaction conditions were used:
6 a) 96.degree. C. 3 minutes b) 96.degree. C. 30 seconds
denaturation c) 70.degree. C. 30 seconds, primer annealing. This
temperature was gradually decreased by 1.degree. C./cycle d)
72.degree. C. 1 minute extension. Repeat steps (b)-(d) 10 times e)
96.degree. C. 30 seconds denaturation f) 60.degree. C. 30 seconds
annealing g) 72.degree. C. 1 minute extension Repeat steps (e)-(g)
25 times h) 72.degree. C. 10 minutes final extension
[0315] A PCR product having an approximate size of 750 bp was
isolated after electrophoresis in agarose gel and ligated to the
pCR2.1 vector (Invitrogen, Carlsbad, Calif.). The cloned insert was
sequenced using vector specific M13 Forward(-40) and M13 Reverse
primers, as well as the following gene specific primers:
7 HDGFX S1: GACAAGCCGACCCACGCTGG and (SEQ ID NO:13) HDGFX S2:
CCAGCGTGGGTCGGCTTGTC. (SEQ ID NO:14)
[0316] The sequence was verified as an open reading frame coding
for the predicted HDGFX full length protein. The cloned sequence is
100% identical to the predicted translation product of SEQ ID NO:1.
The clone is called pCR2.1-AL033539-S321-4C.
Example 2
[0317] Construction of the Mammalian Expression Vector
pCEP4/V5His.
[0318] The oligonucleotide primers,
8 pSec-V5-His Forward: CTCGTCCTCGAGGGTAAGCCTATCCCTAAC and (SEQ ID
NO:15) pSec-V5-His Reverse: CTCGTCGGGCCCCTGATCAGCGGGTTTA- AAC (SEQ
ID NO:16)
[0319] were designed to amplify a fragment from the expression
vector pcDNA3.1-V5His (Invitrogen, Carlsbad, Calif.). The PCR
product was digested with XhoI and ApaI and ligated into the
XhoI/ApaI digested pSecTag2B vector (Invitrogen, Carlsbad Calif.).
The correct structure of the resulting vector, pSecV5His, was
verified by DNA sequence analysis. The vector pSecV5His was
digested with PmeI and NheI, and the PmeI-NheI fragment was ligated
into the BamHI/Klenow and NheI treated vector pCEP4 (Invitrogen,
Carlsbad, Calif.). The resulting vector was named pCEP4/Sec.
Subsequently, a KpnI and XhoI fragment from pcDNA3.1A (Invitrogen,
Carlsbad, Calif.) was isolated and ligated into pCEP4/Sec that had
been treated KpnI and XhoI. The resulting vector, named pCEP4/V5His
lacks the IgKappa secretion signal originally present in
pSecTag2B.
Example 3
[0320] Expression of HDGFX in Human Embryonic Kidney 293 Cells.
[0321] A 0.8 kb BamHI-XhoI fragment containing the HDGFX sequence
was isolated from pCR2.1-AL033539-S321-4C (Example 1) and subcloned
into BamHI-XhoI digested pCEP4/V5His (Example 2) to generate
expression vector pCEP4/V5His-AL033539. The pCEP4/V5His-AL033539
vector was transfected into 293 cells using the LipofectaminePlus
reagent following the manufacturer's instructions (Gibco/BRL,
Rockville, Md.). The cell pellet and supernatant were harvested 72
hours after transfection and examined for HDGFX expression by
Western blotting run under reducing conditions with an anti-V5
antibody. FIG. 1 shows that the expressed HDGFX polypeptide has a
molecular weight of approximately 50 kDa, which is secreted by 293
cells, as indicated by using SeeBlue molecular weight markers
(Invitrogen, Carlsbad, Calif.).
Example 4
[0322] Construction of Recombinant E. coli Expression Vector
pET(C).
[0323] The vector pBADgIII (Invitrogen Inc., Carlsbad, Calif.) was
digested with NcoI and PmeI restriction enzymes to release a
fragment of 150 bp containing BglII and SalI cloning sites and
C-terminal 6xHis tag. The plasmid pET28a (Novagen, Madison, Wis.)
was linearized by digestion with the restriction enzyme XhoI and
filled in with the Klenow fragment of the E. coli DNA polymerase.
It was then digested with the restriction enzyme NcoI.
Subsequently, the NcoI-PmeI fragment from pBADgIII was ligated into
the linearized pET28a and the resulting E. coli expression vector
was named as pET(C).
Example 5
[0324] Expression of HDGFX in Recombinant E. coli Using the
Expression Vector pET(C).
[0325] The BamHI-XhoI fragment containing the HDGFX gene (Examples
1 and 3) was ligated into the pET(C) vector (Example 4) that had
been digested with BamHI and XhoI restriction enzymes. The
resulting expression vector is named pET(C)-AL033539. In this
vector, HDGFX was fused to the 6xHis tag at its C-terminus. The
plasmid pET(C)-AL033539 was then transformed into the E. coli
expression host BL21(DE3, pLys) (Novagen, Madison, Wis.).
Expression of the HDGFX polypeptide was induced according to the
manufacturer's instructions. After induction, total cells were
harvested, and proteins were analyzed by Western blotting using
anti-His antibody (Invitrogen, Carlsbad, Calif.). FIG. 2 shows that
a HDGFX polypeptide was expressed as a polypeptide whose molecular
weight is approximately 45 kDa in E. coli cells, as indicated by
using SeeBlue molecular weight markers (Invitrogen, Carlsbad,
Calif.).
Example 6
[0326] Cytokine Production in Monocytes in Response to Treatment
with HDGFX.
[0327] Monocytes were isolated from fresh human peripheral blood
mononuclear cells using CD14 microbeads (Miltenyi Biotec) according
to the manufacture's procedure. Monocytes were plated in a 96-well
flat bottom tissue culture treated plate at 1.times.10.sup.5 cells
per well in a volume of 100 .mu.l per well in DMEM medium (Gibco,
Rockville Md.) containing 10% fetal bovine serum (HyClone) and
supplemented with L-glutamine, sodium pyruvate, non-essential amino
acids, HEPES, and .beta.-mercaptoethanol (supplements from
Gibco/BRL, Rockville, Md.). HDGFX was purified from the secreted
protein in the conditioned medium described in Example 3. HDGFX or
vehicle control (20 mM Tris-HCl pH 7.4; 50 mM NaCl) was added to
the wells and then the samples were incubated at 37.degree. C. in a
tissue culture incubator with 10% CO.sub.2 for 24 hr. Tumor
necrosis factor alpha (TNF .alpha.) or interleukin-6 (IL-6)
production was measured by assaying the cell supernatant by ELISA
(Pharmingen).
[0328] The results are shown in FIGS. 3 and 4. Monocytes treated
with 100 ng/ml HDGFX produced 23.8 ng/ml IL-6 and 0.66 ng/ml TNF
.alpha.. Monocytes treated with 10 ng/ml HDGFX produced 8.3 ng/ml
IL-6, while TNF .alpha. production was not detected with treatment
of 10 ng/ml HDGFX. Monocytes treated with the vehicle control did
not produce detectable levels of IL-6 or TNF .alpha.. These results
are representative of those obtained in a second, separate,
experiment.
Example 7
[0329] In vivo Effects of Administration of HDGFX in Mice.
[0330] HDGFX was purified from the secreted protein in the
conditioned medium described in Example 3. Normal female BALB/c
mice from Harlan Labs were given single daily ip injections of
HDGFX (5 mg/kg) or control vehicle (10 ml/kg) for 7 days. On the
eighth day, animals were injected with HDGFX (5 mg/kg) and BrdU
(100 mg/kg) and 1 hr later were anesthetized with Isoflurane and
bled for determination of complete blood count (CBC) and clinical
chemistry alterations. Tissues and organs were removed and weighed
and collected into formalin for histopathologic evaluation which
included BrdU immunohistochemistry for detection of proliferative
changes. The results are shown in Table 5.
[0331] Relative spleen weights were increased 68% in mice treated
with HDGFX compared to mice treated with vehicle control
(0.683.+-.0.044 grams (gr)/100 gr body wt vs. 0.407.+-.0.08 gr/100
gr body wt). Also, relative liver weights were increased 11% in
mice treated with HDGFX compared to mice treated with vehicle
control (4.73.+-.0.96 gr/100 gr body wt vs. 4.279.+-.0.117 gr/100
gr body wt). Mice treated with HDGFX showed marked increase in
granulocytopoiesis in the bone marrow compared to mice treated with
vehicle control (myeloid:erythroid ratio 3:1 vs. 1:1). Treatment
with HDGFX resulted in marked increases in splenic extramedullary
hematopoiesis and lymphoid hyperplasia. Histopathology showed
subacute inflammation affecting the pancreatic ducts with
neutrophil and mononuclear cell infiltratation and BrdU labeling
was evident in the parenchyma in mice treated with HDGFX.
9TABLE 5 3/1/00 3/8/00 Body 3/3/00 3/5/00 Final Liver Rel. Spleen
Rel. Comments/ Animal WT Body Body WT Weight Liv. Wt. Weight Spl.
Wt. Gross # BP # grams WT WT grams grams gr/100 gr grams gr/100 gr
observations Vehicle (Tris/PBS 10 ml/kg) 1 548 19.03 19.4 19.3 19.5
0.81 4.154 0.077 0.395 * 2 549 19.02 19.5 19.3 19.8 0.864 4.364
0.086 0.434 NSL 3 550 19.6 19.7 19.4 19.6 0.786 4.010 0.076 0.388
NSL 4 551 19.4 20 19.7 19.6 0.819 4.179 0.081 0.413 NSL 5 552 19.2
19.9 19.6 19.6 0.919 4.689 0.079 0.403 NSL Mean 19.250 19.700
19.460 19.620 0.840 4.279 0.080 0.407 SE 0.112 0.114 0.081 0.049
0.024 0.117 0.002 0.008 HDGFX (5 mg/kg) 1 558 18.85 18.4 18.5 18.7
0.84 4.492 0.111 0.594 NSL 2 559 20.5 19.3 19.6 19.8 0.934 4.717
0.145 0.732 NSL 3 560 20.1 19.2 19.7 19.1 0.969 5.073 0.149 0.780
NSL 4 561 21 19.9 20.6 20.7 0.957 4.623 0.116 0.560 NSL 5 562 18.1
18.1 18.3 19.8 0.939 4.742 0.148 0.747 NSL Mean 19.710 18.980
19.340 19.620 0.928 4.730 0.134 0.683 SE 0.537 0.325 0.423 0.343
0.023 0.096 0.008 0.044 p<0.05 p<0.05 p<0.01 p<0.01
*Diffuse mineralization of the heart NSL No significant lesions
Example 8
[0332] Proliferative Activity of HDGFX.
[0333] The results in Example 7 suggest that HDGFX affects the
growth of pancreatic duct epithelium. Therefore the proliferative
effect of HDGFX was assessed on cell cultures of these cells.
[0334] A. Demonstration of Cell Proliferation
[0335] The proliferative effect of HDGFX on H6c7 human pancreatic
duct epithelial (HPDE) cells, obtained from fresh surgical samples
of pancreas, was examined. Secreted HDGFX was purified from the
conditioned medium described in Example 3.
[0336] Day 1: HPDE cells were plated at .about.10,000/well in
6-well Nunc Tissue Culture plates in KSF medium (a keratinocyte
growth supporting medium) supplemented with bovine pituitary
extract and epidermal growth factor (EGF) (5 ng/ml).
[0337] Day 2: The medium was changed to KSF without supplement
[0338] Day 3: The medium was changed to KSF medium supplemented
with HDGF (EXTRACT; 0.5-5 .mu.l/ml medium), MOCK (1 and 5 .mu.l/ml)
or purified HDGF (ng/ml). Cell number at this time is
.about.1.07.times.10.sup.4/well- . HDGF EXTRACT is a designation
for minimally purified conditioned medium containing HDGFX, and
MOCK relates to a control conditioned medium.
[0339] Day 7: The number of cells was counted using a Coulter ZM
cell counter.
[0340] The results are shown in FIG. 5, where HDGFX is designated
HDGF. The extract effects in FIG. 5 appear to originate with the
conditioned medium, and not HDGFX, since mock doses provide the
same effect. HDGFX purified using the fused purification tags
(Example 3) provides a significant proliferative effect (FIG.
5).
[0341] B. Suppression of Proliferation at High Concentrations of
HDGF.
[0342] The experiment in section A was extended by using higher
concentrations of HDGF. All groups are in triplicates.
[0343] Day 1: H6c7 cells (P28) were plated in 6 well plates at
.about.10,000 cells/well in KSF medium without growth factor
supplements.
[0344] Day 2: The medium was refreshed with the same
supplement-free KSF medium.
[0345] Day 3: The medium was exchanged to medium containing various
concentrations of purified HDGF. No HDGF was added to the control
group.
[0346] Day 8: The cells were counted as above. The results are
presented in FIG. 6.
[0347] It is seen that the lowest concentration, which is the same
as the highest concentration used in part A (FIG. 5) also provides
a moderate degree of proliferation. Progressively higher
concentrations of HDGF appear to suppress this proliferation.
[0348] This experiment was repeated twice. In the first experiment
the proliferation over control levels and suppression at high
concentrations of HDGF was not found. It is believed this was due
to loss of response by the cells upon repeated cell passages. In
the second experiment, a proliferation to the extent of
approximately 10% was observed at HDGF concentrations of 56 and 112
ng/ml, with 28 and 224 ng/ml concentrations providing a cell count
indistinguishable from control (.about.180,000/well), and 14 ng/ml
having a cell count slightly below control. The cell count in the
presence of bovine pituitary extract and EGF was about
270,000/well.
Example 9
[0349] Northern Blot Analysis of Early Gene Expression by
HDGFX.
[0350] Northern blot analysis of c-myc and c-fos expression was
conducted on samples of HPDE cells after treating them with HDGF
(350 ng/ml) for various time periods. The results are shown in FIG.
7.
[0351] There appears to be upregulation of c-myc and c-fos mRNA
expression following treatment with HDGF, compared to the
constitutive expression of GADPH at all time points. This is
consistent with activation of transcription early genes,
characteristic of c-myc and c-fos, by HDGF.
Example 10
[0352] Quantitative Expression Analysis of HDGFX Nucleic Acids
[0353] The quantitative expression of various clones was assessed
in 40 normal and 54 tumor samples by real time quantitative PCR
(TAQMAN.RTM.) performed on a Perkin-Elmer Biosystems ABI PRISM.RTM.
7700 Sequence Detection System. Cell lines are shown in TABLE
10TABLE 4 TISSUES ANALYZED FOR HDGFX EXPRESSION well # TISSUE 1
Endothelial cells 2 Endothelial cells (treated) 3 Pancreas 4
Pancreatic cancer CAPAN 2 5 Adipose 6 Adrenal gland 7 Thyroid 8
Salivary gland 9 Pituitary gland 10 Brain (fetal) 11 Brain (whole)
12 Brain (amygdala) 13 Brain (cerebellum) 14 Brain (hippocampus) 15
Brain (hypothalamus) 16 Brain (substantia nigra) 17 Brain
(thalamus) 18 Spinal cord 19 CNS cancer (glio/astro) U87-MG 20 CNS
cancer (glio/astro) U-118-MG 21 CNS cancer (astro) SW1783 22 CNS
cancer* (neuro; met) SK-N-AS 23 CNS cancer (astro) SF-539 24 CNS
cancer (astro) SNB-75 25 CNS cancer (glio) SNB-19 26 CNS cancer
(glio) U251 27 CNS cancer (glio) SF-295 28 Heart 29 Skeletal muscle
30 Bone marrow 31 Thymus 32 Spleen 33 Lymph node 34 Colon
(ascending) 35 Stomach 36 Small intestine 37 Colon cancer SW480 38
Colon cancer* (SW480 met)SW620 39 Colon cancer HT29 40 Colon cancer
HCT-116 41 Colon cancer CaCo-2 42 Colon cancer HCT-15 43 Colon
cancer HCC-2998 44 Gastric cancer* (liver met) NCI-N87 45 Bladder
46 Trachea 47 Kidney 48 Kidney (fetal) 49 Renal cancer 786-0 50
Renal cancer A498 51 Renal cancer RXF 393 52 Renal cancer ACHN 53
Renal cancer UO-31 54 Renal cancer TK-10 55 Liver 56 Liver (fetal)
57 Liver cancer (hepatoblast) HepG2 58 Lung 59 Lung (fetal) 60 Lung
cancer (small cell) LX-1 61 Lung cancer (small cell) NCI-H69 62
Lung cancer (small cell variant) SHP-77 63 Lung cancer (large cell)
NCI-H460 64 Lung cancer (non-small cell) A549 65 Lung cancer
(non-small cell) NCI-H23 66 Lung cancer (non-small cell) HOP-62 67
Lung cancer (non-small cell) NCI-H522 68 Lung cancer (squamous
cell) SW 900 69 Lung cancer (squamous cell) NCI-H596 70 Mammary
gland 71 Breast cancer* (plural. effusion) MCF-7 72 Breast cancer*
(plural effusion) MDA-MB-231 73 Breast cancer* (plural effusion)
T47D 74 Breast cancer BT-549 75 Breast cancer MDA-N 76 Ovary 77
Ovarian cancer OVCAR-3 78 Ovarian cancer OVCAR-4 79 Ovarian cancer
OVCAR-5 80 Ovarian cancer OVCAR-8 81 Ovarian cancer IGROV-1 82
Ovarian cancer* (ascites) SK-OV-3 83 Myometrium 84 Uterus 85
Placenta 86 Prostate 87 Prostate cancer* (bone met)PC-3 88 Testis
89 Melanoma Hs688(A).T 90 Melanoma* (met) Hs688(B).T 91 Melanoma
UACC-62 92 Melanoma M14 93 Melanoma LOX IMVI 94 Melanoma* (met)
SK-MEL-5 95 Melanoma SK-MEL-28 96 Melanoma UACC-257 KEY: glio. =
gliocyte; astro. = astrocyte; neuro. = neurocyte; met. =
metastatic; CNS = central nervous system
[0354] First, 96 RNA samples were normalized to .beta.-actin and
GAPDH. RNA (.about.50 ng total or .about.1 ng polyA+) was converted
to cDNA using the TAQMAN.RTM. Reverse Transcription Reagents Kit
(PE Biosystems, Foster City, Calif.; cat #N808-0234) and random
hexamers according to the manufacturer's protocol. Reactions were
performed in 20 .mu.l and incubated for 30 min. at 48.degree. C.
cDNA (5 .mu.l) was then transferred to a separate plate for the
TAQMAN.RTM. reaction using .beta.-actin and GAPDH TAQMAN.RTM. Assay
Reagents (PE Biosystems; cat. #'s 4310881E and 4310884E,
respectively) and TAQMAN.RTM. universal PCR Master Mix (PE
Biosystems; cat #4304447) according to the manufacturer's protocol.
Reactions were performed in 25 .mu.l using the following
parameters: 2 min. at 50.degree. C.; 10 min. at 95.degree. C.; 15
sec. at 95.degree. C./1 min. at 60.degree. C. (40 cycles). Results
were recorded as CT values (cycle at which a given sample crosses a
threshold level of fluorescence) using a log scale, with the
difference in RNA concentration between a given sample and the
sample with the lowest CT value being represented as 2 to the power
of delta CT. The percent relative expression is then obtained by
taking the reciprocal of this RNA difference and multiplying by
100. The average CT values obtained for .beta.-actin and GAPDH were
used to normalize RNA samples. The RNA sample generating the
highest CT value required no further diluting, while all other
samples were diluted relative to this sample according to their
.beta.-actin/GAPDH average CT values.
[0355] Normalized RNA (5 .mu.l) was converted to cDNA and analyzed
via TAQMAN.RTM. using One Step RT-PCR Master Mix Reagents (PE
Biosystems; cat. #4309169) and gene-specific primers according to
the manufacturer's instructions. Probes and primers were designed
for each assay according to Perkin Elmer Biosystem's Primer Express
Software package (version I for Apple Computer's Macintosh Power
PC) using the sequence of clone HDGFX_A as input. Default settings
were used for reaction conditions and the following parameters were
set before selecting primers: primer concentration=250 nM, primer
melting temperature (T.sub.m) range=58.degree.-60.degree. C.,
primer optimal T.sub.m=59.degree. C., maximum primer
difference=2.degree. C., probe does not have 5' G, probe T.sub.m
must be 10.degree. C. greater than primer T.sub.m, amplicon size 75
bp to 100 bp. The probes and primers selected (see below) were
synthesized by Synthegen (Houston, Tex., USA). Probes were double
purified by HPLC to remove uncoupled dye and evaluated by mass
spectroscopy to verify coupling of reporter and quencher dyes to
the 5' and 3' ends of the probe, respectively. Their final
concentrations were: forward and reverse primers, 900 nM each, and
probe, 200 nM.
[0356] PCR conditions: Normalized RNA from each tissue and each
cell line was spotted in each well of a 96 well PCR plate (Perkin
Elmer Biosystems). PCR cocktails including two probes
(HDGFX_A-specific and another gene-specific probe multiplexed with
the HDGFX_A probe) were set up using 1.times. TaqMan.TM. PCR Master
Mix for the PE Biosystems 7700, with 5 mM MgCl2, dNTPs (dA, G, C, U
at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq Gold.TM. (PE Biosystems),
and 0.4 U/.mu.l RNase inhibitor, and 0.25 U/.mu.l reverse
transcriptase. Reverse transcription was performed at 48.degree. C.
for 30 minutes followed by amplification/PCR cycles as follows:
95.degree. C. 10 min, then 40 cycles of 95.degree. C. for 15
seconds, 60.degree. C. for 1 minute.
11 A. Probe set Ag082b Start SEQ ID Primers Sequences nt Position
NO: Forward 5'-ACCAGGTGTTTTTCTTCGGGA-3' 21 191 17 Probe
FAM-5'-CCACGAGACGGCCTTCCTGAGTCC-3'-TAMRA 24 213 18 Reverse
5'-TTGTACGGGAACAGGCGTTT-3' 20 238 19
[0357] Expression of clone HDGFX_A is detected by primer-probe set
Ag082b only in normal testis (100.0% relative expression) and
pancreas (9.0% relative expression), of all the normal and cancer
tissues assayed.
12 B. Probe set Ag082c SEQ Primers Sequences ID NO: Forward
5'-ACCAGGTGTTTTTCTTCGGGA-3' 20 Probe
FAM-CCACGAGACGGCCTTCCTGAGTCC-TAMRA 21 Reverse
5'-TTGTACGGGAACAGGCGTTT-3' 22
[0358] Expression of clone HDGFX_A is detected by primer-probe set
Ag082c only in normal testis (100.0% relative expression) and
pancreas (9.7% relative expression), of all the normal and cancer
tissues assayed.
EQUIVALENTS
[0359] From the foregoing detailed description of the specific
embodiments of the invention, it should be apparent that particular
novel compositions and methods involving nucleic acids,
polypeptides, antibodies, detection and treatment have been
described. Although these particular embodiments have been
disclosed herein in detail, this has been done by way of example
for purposes of illustration only, and is not intended to be
limiting with respect to the scope of the appended claims that
follow. In particular, it is contemplated by the inventors that
various substitutions, alterations, and modifications may be made
as a matter of routine for a person of ordinary skill in the art to
the invention without departing from the spirit and scope of the
invention as defined by the claims. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
Sequence CWU 1
1
22 1 880 DNA Homo sapiens CDS (79)..(831) 1 gcagccgccc ttactgcgcg
cgcgcagact tcggcgtcta cttccggtgt ggcccaggcg 60 gggtccgcag aaccagct
atg tcg gcc tac ggc atg ccc atg tac aag agc 111 Met Ser Ala Tyr Gly
Met Pro Met Tyr Lys Ser 1 5 10 ggg gac ctg gtg ttt gcc aag tta aag
ggc tat gcc cac tgg ccg gcg 159 Gly Asp Leu Val Phe Ala Lys Leu Lys
Gly Tyr Ala His Trp Pro Ala 15 20 25 agg ata gag cac atg acc cag
ccc aac cgc tac cag gtg ttt ttc ttc 207 Arg Ile Glu His Met Thr Gln
Pro Asn Arg Tyr Gln Val Phe Phe Phe 30 35 40 ggg acc cac gag acg
gcc ttc ctg agt ccc aaa cgc ctg ttc ccg tac 255 Gly Thr His Glu Thr
Ala Phe Leu Ser Pro Lys Arg Leu Phe Pro Tyr 45 50 55 aag gag tgc
aag gag aag ttc ggc aag ccc aac aag agg cgc ggc ttc 303 Lys Glu Cys
Lys Glu Lys Phe Gly Lys Pro Asn Lys Arg Arg Gly Phe 60 65 70 75 agc
gcg ggg ctg tgg gaa atc gag aac aac ccc acg gtc cag gcc tcc 351 Ser
Ala Gly Leu Trp Glu Ile Glu Asn Asn Pro Thr Val Gln Ala Ser 80 85
90 gac tgc cca tta gcc tca gag aag ggc agc gga gac ggg cct tgg ccg
399 Asp Cys Pro Leu Ala Ser Glu Lys Gly Ser Gly Asp Gly Pro Trp Pro
95 100 105 gag ccc gag gcc gca gag ggc gac gag gac aag ccg acc cac
gct ggt 447 Glu Pro Glu Ala Ala Glu Gly Asp Glu Asp Lys Pro Thr His
Ala Gly 110 115 120 ggc ggc ggc gac gaa ttg ggg aag ccg gac gac gac
aag ccc act gag 495 Gly Gly Gly Asp Glu Leu Gly Lys Pro Asp Asp Asp
Lys Pro Thr Glu 125 130 135 gag gag aag ggg ccg ctg aag agg agc gcg
ggg gac ccg ccg gag gac 543 Glu Glu Lys Gly Pro Leu Lys Arg Ser Ala
Gly Asp Pro Pro Glu Asp 140 145 150 155 gcc ccc aaa cga ccc aag gag
gca gcc ccc gac caa gag gag gag gcg 591 Ala Pro Lys Arg Pro Lys Glu
Ala Ala Pro Asp Gln Glu Glu Glu Ala 160 165 170 gag gcg gag agg gcg
gcg gaa gcg gag agg gcg gcg gcg gcg gcg gcg 639 Glu Ala Glu Arg Ala
Ala Glu Ala Glu Arg Ala Ala Ala Ala Ala Ala 175 180 185 gcg acg gcc
gtc gac gag gag agt ccg ttc ctc gtg gcg gtg gag aac 687 Ala Thr Ala
Val Asp Glu Glu Ser Pro Phe Leu Val Ala Val Glu Asn 190 195 200 ggc
agc gcc cct agc gag ccg ggc ctg gtc tgc gag ccg cct cag cca 735 Gly
Ser Ala Pro Ser Glu Pro Gly Leu Val Cys Glu Pro Pro Gln Pro 205 210
215 gag gag gag gag ctc cgg gag gaa gaa gtc gcg gac gag gag gcc tcc
783 Glu Glu Glu Glu Leu Arg Glu Glu Glu Val Ala Asp Glu Glu Ala Ser
220 225 230 235 cag gag tgg cat gcc gag gca ccg ggc ggc gga gat cgc
gac agc ctg 831 Gln Glu Trp His Ala Glu Ala Pro Gly Gly Gly Asp Arg
Asp Ser Leu 240 245 250 tagttaccag cgtttccaga agagcccctg ccccgttcct
gctgcggcc 880 2 251 PRT Homo sapiens 2 Met Ser Ala Tyr Gly Met Pro
Met Tyr Lys Ser Gly Asp Leu Val Phe 1 5 10 15 Ala Lys Leu Lys Gly
Tyr Ala His Trp Pro Ala Arg Ile Glu His Met 20 25 30 Thr Gln Pro
Asn Arg Tyr Gln Val Phe Phe Phe Gly Thr His Glu Thr 35 40 45 Ala
Phe Leu Ser Pro Lys Arg Leu Phe Pro Tyr Lys Glu Cys Lys Glu 50 55
60 Lys Phe Gly Lys Pro Asn Lys Arg Arg Gly Phe Ser Ala Gly Leu Trp
65 70 75 80 Glu Ile Glu Asn Asn Pro Thr Val Gln Ala Ser Asp Cys Pro
Leu Ala 85 90 95 Ser Glu Lys Gly Ser Gly Asp Gly Pro Trp Pro Glu
Pro Glu Ala Ala 100 105 110 Glu Gly Asp Glu Asp Lys Pro Thr His Ala
Gly Gly Gly Gly Asp Glu 115 120 125 Leu Gly Lys Pro Asp Asp Asp Lys
Pro Thr Glu Glu Glu Lys Gly Pro 130 135 140 Leu Lys Arg Ser Ala Gly
Asp Pro Pro Glu Asp Ala Pro Lys Arg Pro 145 150 155 160 Lys Glu Ala
Ala Pro Asp Gln Glu Glu Glu Ala Glu Ala Glu Arg Ala 165 170 175 Ala
Glu Ala Glu Arg Ala Ala Ala Ala Ala Ala Ala Thr Ala Val Asp 180 185
190 Glu Glu Ser Pro Phe Leu Val Ala Val Glu Asn Gly Ser Ala Pro Ser
195 200 205 Glu Pro Gly Leu Val Cys Glu Pro Pro Gln Pro Glu Glu Glu
Glu Leu 210 215 220 Arg Glu Glu Glu Val Ala Asp Glu Glu Ala Ser Gln
Glu Trp His Ala 225 230 235 240 Glu Ala Pro Gly Gly Gly Asp Arg Asp
Ser Leu 245 250 3 223 PRT Bos taurus 3 Met Ser Arg Phe Tyr Arg Arg
Lys Tyr Lys Cys Gly Asp Leu Val Phe 1 5 10 15 Ala Lys Leu Lys Gly
Tyr Ala His Trp Pro Ala Arg Ile Glu Gln Thr 20 25 30 Ala Glu Ala
Asn Arg Tyr Gln Val Phe Phe Phe Gly Thr His Glu Thr 35 40 45 Ala
Phe Leu Gly Pro Arg His Leu Phe Pro Tyr Glu Glu Ser Lys Glu 50 55
60 Lys Phe Gly Lys Pro Asn Lys Arg Arg Gly Phe Ser Glu Gly Leu Trp
65 70 75 80 Glu Ile Glu Asn Asn Pro Thr Val Gln Ala Ser Asp Tyr Gln
Cys Ala 85 90 95 Leu Glu Lys Ser Cys Pro Glu Glu Pro Glu Pro Glu
Val Ala Glu Gly 100 105 110 Gly Glu Asp Pro Lys Ser His Thr Asn Gly
Gly Asp Asp Asp Asp Gln 115 120 125 Gly Lys Leu Gly Val Asp Leu Pro
Ala Glu Glu Glu Asn Lys Lys Glu 130 135 140 Thr Leu Lys Arg Thr Ala
Glu Asp Pro Pro Glu Asp Ile Pro Lys Arg 145 150 155 160 Pro Lys Glu
Ala Asp Pro Glu Glu Gly Glu Glu Arg Lys Glu Ala Ala 165 170 175 Ala
Val Ala Glu Glu Ala Glu Asp Ala Arg Pro Leu Leu Val Glu Val 180 185
190 Glu Asn Asp Pro Ala Ala Ser Val Leu Gly Leu Ala Trp Gly Leu Pro
195 200 205 Val Met Glu Gln Glu Pro Glu Glu Glu Ser Ala Glu Arg Glu
Ala 210 215 220 4 221 PRT Homo sapiens 4 Tyr Lys Cys Gly Asp Leu
Val Phe Ala Lys Met Lys Gly Tyr Pro His 1 5 10 15 Trp Pro Ala Arg
Ile Asp Glu Met Pro Glu Ala Ala Val Lys Ser Thr 20 25 30 Ala Asn
Lys Tyr Gln Val Phe Phe Phe Gly Thr His Glu Thr Ala Phe 35 40 45
Leu Gly Pro Lys Asp Leu Phe Pro Tyr Glu Glu Ser Lys Glu Lys Phe 50
55 60 Gly Lys Pro Asn Lys Arg Lys Gly Phe Ser Glu Gly Leu Trp Glu
Ile 65 70 75 80 Glu Asn Asn Pro Thr Val Lys Ala Ser Gly Tyr Gln Ser
Ser Gln Lys 85 90 95 Lys Ser Cys Val Glu Glu Pro Glu Pro Glu Pro
Glu Ala Ala Glu Gly 100 105 110 Asp Gly Asp Lys Lys Gly Asn Ala Glu
Gly Ser Ser Asp Glu Glu Gly 115 120 125 Lys Leu Val Ile Asp Glu Pro
Ala Lys Glu Lys Asn Glu Lys Gly Ala 130 135 140 Leu Lys Arg Arg Ala
Gly Asp Leu Leu Glu Asp Ser Pro Lys Arg Pro 145 150 155 160 Lys Glu
Ala Glu Asn Pro Glu Gly Glu Glu Lys Glu Ala Ala Thr Leu 165 170 175
Glu Val Glu Arg Pro Leu Pro Met Glu Val Glu Lys Asn Ser Thr Pro 180
185 190 Ser Glu Pro Gly Ser Gly Arg Gly Pro Pro Gln Glu Glu Glu Glu
Glu 195 200 205 Glu Asp Glu Glu Glu Glu Ala Thr Lys Glu Asp Ala Glu
210 215 220 5 145 PRT Homo sapiens 5 Lys Ala Ser Gly Tyr Gln Ser
Ser Gln Lys Lys Ser Cys Val Glu Glu 1 5 10 15 Pro Glu Pro Glu Pro
Glu Ala Ala Glu Gly Asp Gly Asp Lys Lys Gly 20 25 30 Asn Ala Glu
Gly Ser Ser Asp Glu Glu Gly Lys Leu Val Ile Asp Glu 35 40 45 Pro
Ala Lys Glu Lys Asn Glu Lys Gly Ala Leu Lys Arg Arg Ala Gly 50 55
60 Asp Leu Leu Glu Asp Ser Pro Lys Arg Pro Lys Glu Ala Glu Asn Pro
65 70 75 80 Glu Gly Glu Glu Lys Glu Ala Ala Thr Leu Glu Val Glu Arg
Pro Leu 85 90 95 Pro Met Glu Val Glu Lys Asn Ser Thr Pro Ser Glu
Pro Gly Ser Gly 100 105 110 Arg Gly Pro Pro Gln Glu Glu Glu Glu Glu
Glu Asp Glu Glu Glu Glu 115 120 125 Ala Thr Lys Glu Asp Ala Glu Ala
Pro Gly Ile Arg Asp His Glu Ser 130 135 140 Leu 145 6 108 PRT Mus
sp. 6 Tyr Lys Cys Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr Pro
His 1 5 10 15 Trp Pro Ala Arg Ile Asp Glu Met Pro Glu Ala Ala Val
Lys Ser Thr 20 25 30 Ala Asn Lys Tyr Gln Val Phe Phe Phe Gly Thr
His Glu Thr Ala Phe 35 40 45 Leu Gly Pro Lys Asp Leu Phe Pro Tyr
Glu Glu Ser Lys Glu Lys Phe 50 55 60 Gly Lys Pro Asn Lys Arg Lys
Gly Phe Ser Glu Gly Leu Trp Glu Ile 65 70 75 80 Glu Asn Asn Pro Thr
Val Lys Ala Ser Gly Tyr Gln Ser Ser Gln Lys 85 90 95 Lys Ser Cys
Ala Ala Glu Pro Glu Val Glu Pro Glu 100 105 7 123 PRT Mus sp. 7 Met
Pro His Ala Phe Lys Pro Gly Asp Leu Val Phe Ala Lys Met Lys 1 5 10
15 Gly Tyr Pro His Trp Pro Ala Arg Ile Asp Asp Ile Ala Asp Gly Ala
20 25 30 Val Lys Pro Pro Pro Asn Lys Tyr Pro Ile Phe Phe Phe Gly
Thr His 35 40 45 Glu Thr Ala Phe Leu Gly Pro Lys Asp Leu Phe Pro
Tyr Asp Lys Cys 50 55 60 Lys Asp Lys Tyr Gly Lys Pro Asn Lys Arg
Lys Gly Phe Asn Glu Gly 65 70 75 80 Leu Trp Glu Ile Gln Asn Asn Pro
His Ala Ser Tyr Ser Ala Pro Pro 85 90 95 Pro Val Ser Ser Ser Asp
Ser Glu Ala Pro Glu Ala Asp Leu Gly Cys 100 105 110 Gly Ser Asp Val
Asp Lys Asp Lys Glu Ser Arg 115 120 8 127 PRT Homo sapiens 8 Met
Thr Arg Asp Phe Lys Pro Gly Asp Leu Ile Phe Ala Lys Met Lys 1 5 10
15 Gly Tyr Pro His Trp Pro Ala Arg Val Asp Glu Val Pro Asp Gly Ala
20 25 30 Val Lys Pro Pro Thr Asn Lys Leu Pro Ile Phe Phe Phe Gly
Thr His 35 40 45 Glu Thr Ala Phe Leu Gly Pro Lys Asp Ile Phe Pro
Tyr Ser Glu Asn 50 55 60 Lys Glu Lys Tyr Gly Lys Pro Asn Lys Arg
Lys Gly Phe Asn Glu Gly 65 70 75 80 Leu Trp Glu Ile Asp Asn Asn Pro
Lys Val Lys Phe Ser Ser Gln Gln 85 90 95 Ala Ala Thr Lys Gln Ser
Asn Ala Ser Ser Asp Val Glu Val Glu Glu 100 105 110 Lys Glu Thr Ser
Val Ser Lys Glu Asp Thr Asp His Glu Glu Lys 115 120 125 9 127 PRT
Homo sapiens 9 Met Thr Arg Asp Phe Lys Pro Gly Asp Leu Ile Phe Ala
Lys Met Lys 1 5 10 15 Gly Tyr Pro His Trp Pro Ala Arg Val Asp Glu
Val Pro Asp Gly Ala 20 25 30 Val Lys Pro Pro Thr Asn Lys Leu Pro
Ile Phe Phe Phe Gly Thr His 35 40 45 Glu Thr Ala Phe Leu Gly Pro
Lys Asp Ile Phe Pro Tyr Ser Glu Asn 50 55 60 Lys Glu Lys Tyr Gly
Lys Pro Asn Lys Arg Lys Gly Phe Asn Glu Gly 65 70 75 80 Leu Trp Glu
Ile Asp Asn Asn Pro Lys Val Lys Phe Ser Ser Gln Gln 85 90 95 Ala
Ala Thr Lys Gln Ser Asn Ala Ser Ser Asp Val Glu Val Glu Glu 100 105
110 Lys Glu Thr Ser Val Ser Lys Glu Asp Thr Asp His Glu Glu Lys 115
120 125 10 109 PRT Mus sp. 10 Tyr Lys Thr Gly Asp Leu Val Phe Ala
Lys Leu Lys Gly Tyr Ala His 1 5 10 15 Trp Pro Ala Arg Ile Glu His
Val Ala Glu Ala Asn Arg Tyr Gln Val 20 25 30 Phe Phe Phe Gly Thr
His Glu Thr Ala Leu Leu Gly Pro Arg His Leu 35 40 45 Phe Pro Tyr
Glu Glu Ser Lys Glu Lys Phe Gly Lys Pro Asn Lys Arg 50 55 60 Arg
Gly Phe Ser Glu Gly Leu Trp Glu Ile Glu His Asp Pro Met Val 65 70
75 80 Glu Ala Ser Ser Ser Leu Cys Ser Glu Glu Asp Gln Ser Tyr Thr
Glu 85 90 95 Asp Pro Gly Leu Ala Glu Glu Pro Glu Leu Gly Gln Glu
100 105 11 36 DNA Artificial Sequence Description of Artificial
Sequence oligonucleotide 11 ggatccacca tgtcggccta cggcatgccc atgtac
36 12 30 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide 12 ctcgagcagg ctgtcgcgat ctccgccgcc 30 13 20 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide 13 gacaagccga cccacgctgg 20 14 20 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide 14
ccagcgtggg tcggcttgtc 20 15 30 DNA Artificial Sequence Description
of Artificial Sequence oligonucleotide 15 ctcgtcctcg agggtaagcc
tatccctaac 30 16 31 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide 16 ctcgtcgggc ccctgatcag
cgggtttaaa c 31 17 21 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide 17 accaggtgtt tttcttcggg a 21
18 24 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide 18 ccacgagacg gccttcctga gtcc 24 19 20 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide 19 ttgtacggga acaggcgttt 20 20 21 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide 20
accaggtgtt tttcttcggg a 21 21 24 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide 21 ccacgagacg
gccttcctga gtcc 24 22 20 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide 22 ttgtacggga acaggcgttt 20
* * * * *