U.S. patent application number 15/169452 was filed with the patent office on 2016-11-24 for fkbp-l and uses thereof.
This patent application is currently assigned to ALMAC DISCOVERY LIMITED. The applicant listed for this patent is ALMAC DISCOVERY LIMITED. Invention is credited to David Hirst, Martin Gerard O'Rourke, Tracy Robson, Andrea Valentine.
Application Number | 20160340401 15/169452 |
Document ID | / |
Family ID | 36745574 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160340401 |
Kind Code |
A1 |
Robson; Tracy ; et
al. |
November 24, 2016 |
FKBP-L AND USES THEREOF
Abstract
Disclosed are methods and compositions that employ FKBP-L
polypeptides for modulating angiogenesis and/or tumor metastasis.
The FKBP-L polypeptides may be used for the treatment of disorders
mediated by angiogenesis such as cancer.
Inventors: |
Robson; Tracy; (Belfast,
GB) ; Valentine; Andrea; (Aghalee, GB) ;
O'Rourke; Martin Gerard; (Aghalee, GB) ; Hirst;
David; (Belfast, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALMAC DISCOVERY LIMITED |
Craigavon |
|
GB |
|
|
Assignee: |
ALMAC DISCOVERY LIMITED
Craigavon
GB
|
Family ID: |
36745574 |
Appl. No.: |
15/169452 |
Filed: |
May 31, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12303343 |
Dec 3, 2008 |
9381228 |
|
|
PCT/GB2007/002107 |
Jun 8, 2007 |
|
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15169452 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 31/4412 20130101; A61P 9/00 20180101; A61P 35/00 20180101;
C12N 9/90 20130101; A61K 45/06 20130101; A61P 35/04 20180101; A61K
31/7105 20130101; A61P 27/02 20180101; A61P 27/00 20180101; A61K
31/00 20130101; C12Y 502/01008 20130101; C07K 14/705 20130101; A61K
38/177 20130101; A61K 39/3955 20130101; A61K 38/1709 20130101; A61P
43/00 20180101; A61K 31/7105 20130101; A61K 38/1709 20130101; A61K
2300/00 20130101; A61P 17/02 20180101; A61K 2300/00 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C12N 9/90 20060101 C12N009/90; A61K 47/48 20060101
A61K047/48; A61K 38/17 20060101 A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2006 |
GB |
0611405.2 |
Claims
1-33. (canceled)
34. An isolated polypeptide selected from the group consisting of
polypeptides whose sequence comprises or consists of: SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28; a sequence that is at least 95% but not
100% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11 SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, or SEQ ID NO:28, wherein the sequence of the polypeptide is
not SEQ ID NO:10 or SEQ ID NO:14; a sequence that is at least 95%
but not 100% identical to a sequence that consists of at least 18
contiguous amino acids of SEQ ID NO:10; and wherein the polypeptide
is not the full length FKBP-L protein.
35. The isolated polypeptide of claim 34, wherein the sequence of
the polypeptide is at least 95% but not 100% identical to a
sequence that consists of at least 18 contiguous amino acids of SEQ
ID NO:10.
36. The isolated polypeptide of claim 34, wherein the sequence of
the polypeptide is at least 95% but not 100% identical to SEQ ID
NO:14.
37. The isolated polypeptide of claim 34, wherein the polypeptide
is linked to a polymer and wherein the polymer is chosen from
dextrans, polyvinyl pyrrolidones, and polyethylene glycol.
38. The isolated polypeptide of claim 37, wherein the polymer is
polyethylene glycol.
39. The isolated polypeptide of claim 34, wherein the polypeptide
is linked to a molecule chosen from carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, glycolipids, heterocyclic
compounds, nucleosides, and nucleotides.
40. The isolated polypeptide of claim 34, wherein the polypeptide
is a modified polypeptide, and wherein the modified polypeptide is
chosen from phosphopeptides, cyclic peptides, peptides containing
D-amino acids, and peptides containing radiolabels.
41. The isolated polypeptide of claim 40, wherein the modified
polypeptide is chosen from peptides containing D-amino acids and
peptides containing radiolabels.
42. The isolated polypeptide of claim 34, wherein the polypeptide
is modified by the addition of biotin.
43. The isolated polypeptide of claim 34, wherein the polypeptide
is modified by the addition of a moiety to facilitate crosslinking,
wherein the moiety is chosen from benzophenone, maleimide, and
activated esters.
44. The isolated polypeptide of claim 34, wherein the polypeptide
is modified by the addition of a moiety to facilitate crosslinking
and wherein the moiety is chosen from heterobifunctional
cross-linking agents containing maleimide and an activated
ester.
45. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is linked to a polymer and
wherein the polymer is chosen from dextrans, polyvinyl
pyrrolidones, and polyethylene glycol.
46. The isolated polypeptide of claim 45, wherein the polymer is
polyethylene glycol.
47. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is linked to a molecule
chosen from carbohydrates, monosaccharides, oligosaccharides,
polysaccharides, glycolipids, heterocyclic compounds, nucleosides,
and nucleotides.
48. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is a modified polypeptide,
and wherein the modified polypeptide is chosen from
phosphopeptides, cyclic peptides, peptides containing D-amino
acids, and peptides containing radiolabels.
49. The isolated polypeptide of claim 48, wherein the modified
polypeptide is chosen from peptides containing D-amino acids and
peptides containing radiolabels.
50. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is modified by the
addition of biotin.
51. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is modified by the
addition of a moiety to facilitate crosslinking, wherein the moiety
is chosen from benzophenone, maleimide, and activated esters.
52. An isolated polypeptide whose sequence consists of SEQ ID NO:10
or SEQ ID NO:14, wherein the polypeptide is modified by the
addition of a moiety to facilitate crosslinking and wherein the
moiety is chosen from heterobifunctional cross-linking agents
containing maleimide and an activated ester.
53. An isolated polypeptide whose sequence consists of SEQ ID NO:6,
SEQ ID NO:20, or SEQ ID NO:23, wherein the polypeptide is linked to
a molecule chosen from carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, glycolipids, heterocyclic
compounds, nucleosides, and nucleotides.
54. An isolated polypeptide whose sequence consists of SEQ ID NO:6,
SEQ ID NO:20, or SEQ ID NO:23, wherein the polypeptide is a
modified polypeptide, and wherein the modified polypeptide is
chosen from phosphopeptides, cyclic peptides, peptides containing
D-amino acids, and peptides containing radiolabels.
55. The isolated polypeptide of claim 54, wherein the modified
polypeptide is chosen from peptides containing D-amino acids and
peptides containing radiolabels.
56. An isolated polypeptide whose sequence consists of SEQ ID NO:6,
SEQ ID NO:20 or SEQ ID NO:23, wherein the polypeptide is modified
by the addition of biotin.
57. An isolated polypeptide whose sequence consists of SEQ ID NO:6,
SEQ ID NO:20, or SEQ ID NO:23 wherein the polypeptide is modified
by the addition of a moiety to facilitate crosslinking, wherein the
moiety is chosen from benzophenone, maleimide, and activated
esters.
58. An isolated polypeptide whose sequence consists of SEQ ID NO:6
SEQ ID NO:20, or SEQ ID NO:23, wherein the polypeptide is modified
by the addition of a moiety to facilitate crosslinking and wherein
the moiety is chosen from heteroblfunctional cross-linking agents
containing maleimide and an activated ester.
Description
PRIORITY CLAIM TO RELATED APPLICATIONS
[0001] This application is a Division of U.S. patent application
Ser. No. 12/303,343, filed Dec. 3, 2008 and issued as U.S. Pat. No.
9,381,228, which is a U.S. National Stage Entry of
PCT/GB2007/002107, filed Jun. 8, 2007, and claims priority to UK
Patent Application No. 0611405.2 to Robson et al., filed Jun. 9,
2006. The entire disclosure of UK Patent Application No. 0611405.2
to Robson et al, is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to FKBP-L polypeptides, FKBP-L
peptides, FKBP-L peptide derivatives, and uses thereof.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis is the formation of new blood vessels from
pre-existing vasculature and may be controlled by intricate
signalling via soluble factors. Pathologies associated with
angiogenesis may include cancer (Folkman J. (1971) N. Engl. J. Med.
285:1182; Folkman J. (1999) Nature Med. 1: 27-31), arteriosclerosis
(Lip, G. Y., Blann, A. D. (2004) Ann Med. 36(2) 119-125), psoriasis
(Powell, J. (1999) Curr. Opin. Pediatr. 11: 457-463), endometriosis
(Olive, D. L., Lindheim, S. R., Pritts, E. A. (2004) Best Pract.
Res. Clinc. Obstet. Gynaecol. 18(2) 319-328) and some ocular
disorders like diabetic retinopathy (Folkman J. (1999) Nature Med.
1: 27-31). Angiogenesis may also be necessary for wound repair
since the new vessels provide nutrients to support the active
cells, promote granulation tissue formation and facilitate the
clearance of debris. Approximately 60% of the granulation tissue
mass may be composed of blood vessels which also supply the
necessary oxygen to stimulate repair and vessel growth. It is well
documented that angiogenic factors are present in wound fluid and
promote repair while antiangiogenic factors inhibit repair. In
tumors, when endothelial cells are exposed to soluble factors which
stimulate angiogenesis, they may undergo several physiological
changes including a massive increase in proliferation, degradation
and invasion through the existing vessel basement membrane, and
migration away from the blood vessel to a new location. At the new
location the endothelial cells may again proliferate and form
capillary tubules before ultimately forming a highly disorganised
tumor vasculature (Garcea G, Lloyd T D, Gescher A, Dennison A R,
Steward W P, Berry D P. (2004) Eur J Cancer. June; 4099):1302-13).
Activated endothelial cells may show a distinct pattern of gene
expression, which leads to modification of the principal cellular
functions involved in angiogenesis. These include the regulation of
proteolytic balance leading to localised pericellular matrix
degradation, synthesis of adhesion molecules involved in
extracellular matrix interaction, and most importantly,
cytoskeletal reorganization involved in cell migration (Garcea G,
Lloyd T D, Gescher A, Dennison A R, Steward W P, Berry D P. (2004)
Eur J Cancer. June; 4099):1302-13).
[0004] Novel anti-angiogenic compounds have been shown to inhibit a
range of endothelial markers, which have been identified as being
up-regulated in activated endothelial cells. These may include
receptors, matrix metalloproteins, and adhesion proteins. The
success rate of these inhibitors has been quite high. Recently the
novel anti-angiogenic compound Avastin, a VEGF antibody, has passed
FDA approval and anti-angiogenesis has now become recognised as the
fourth modality used in the treatment of cancer (Abdollhi A.,
Hlatky L., Huber P. E. (2005) Drug Resistance Updates,
February-April; 8:59-74). These therapies may have the following
advantages over conventional chemotherapeutic treatments. First,
angiogenesis is primarily an onco-foetal mechanism, thus minimal
side effects should be expected when administered, even after
prolonged treatment. Secondly, tumor-associated angiogenesis is a
physiological host mechanism and its pharmacological inhibition
should, consequently, not lead to the development of resistance.
Finally the tumor mass itself is difficult to target, where the
endothelial cells that line the supplying vasculature are
frequently classed as vulnerable.
[0005] Pro-angiogenic compounds may also be therapeutic. For
example, pro-angiogeneic compounds which may promote wound repair
include angiogenic cytokines, such as FGF, VEGF, TGF-beta,
angiopoietin, and mast cell tryptase.
[0006] A novel polypeptide and its gene have been recently
identified and partially characterised. This new polypeptide has
been named FKBP-L, DIR1 or WISP39. This gene has been demonstrated
as having a role in stress responses (Robson, T., Lohrer, H.,
Bailie, J. R., Hirst, D. G., Joiner, M. C., Arrand, J. E. (1997)
Biochemical J. Transactions 25, 335-341). It has also been shown
that repression of the FKBP-L gene can protect against cellular
X-ray and UV-induced oxidative cellular damage (Robson, T., Joiner,
M. C., McCullough, W., Price, M. E., McKeown, S. R., Hirst, D. G.
(1999) Radiation Research 152, 451-461; Robson, T., Price, M. E.,
Moore, M. L., Joiner, M. C., McKelvey-Martin, V. J., McKeown, S.
R., Hirst, D. G., (2000) Int. J. Radiat). FKBP-L may also stabilize
newly synthesised p21 (a cyclin dependent kinase inhibitor and a
critical regulator of cell cycle) by forming a trimeric complex
with p21 and Hsp90 (Jascur, T. et al (2005) Molecular Cell, Vol.
17, 237-249).
[0007] There is a need to provide new therapeutics that can
modulate angiogeneis and cell migration. Such therapeutics may be
important as stand-alone treatments, or to be used in conjunction
with other therapeutic agents.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention relate to the use of
FKBP-L polypeptides to modulate angiogenesis and cell migration.
The present invention may be embodied in a variety of ways.
[0009] For example, in certain embodiments, the FKBP-L and
fragments thereof may be used to modulate angiogenesis. Also, in
some embodiments, the FKBP-L polypeptides may be used to modulate
cell migration and/or metastasis of tumor cells. The action of
FKBP-L may be mediated by CD44. Thus, the FKBP-L polypeptide may,
in certain embodiments, be used to modulate angiogenesis, cell
migration and/or metastasis of cells that express CD44.
[0010] In some embodiments, the present invention comprises methods
of treating a disorder mediated by or associated with at least one
of cell migration, and/or angiogenesis, and/or tumor metastasis.
The method may comprise administering a therapeutically effective
amount of: (i) an active compound comprising an isolated FKBP-L
polypeptide, a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or a fragment thereof, or (ii) a polynucleotide
encoding such a FKBP-L polypeptide to a subject in need
thereof.
[0011] In other embodiments, the present invention comprises the
use of (i) an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or fragment thereof, or (ii) a polynucleotide encoding
such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a medicament for use in treating a disorder mediated
by or associated with at least one of cell migration, angiogenesis,
and/or tumor metastasis.
[0012] There are additional features of the invention which will be
described hereinafter. It is to be understood that the invention is
not limited in its application to the details set forth in the
following claims, description and figures. The invention is capable
of other embodiments and of being practiced or carried out in
various ways.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The present invention may be better understood by reference
to the following non-limiting drawings.
[0014] FIGS. 1A, 1B, and 1C show alternative amino acid sequences
of full-length FKPB-L, fragments of FKBP-L in accordance with
alternative embodiments of the present invention.
[0015] FIGS. 2A, 2B, 2C, 2D, and 2E show polynucleotide sequences
encoding for FKBP-L and some of its deletion mutants and variants
in accordance with alternative embodiments of the present
invention.
[0016] FIG. 3 shows the inhibitory effects of transiently
transfected FKBP-L cDNA (SEQ ID NO: 31) on HMEC-1 wound closure in
accordance with one embodiment of the present invention.
[0017] FIG. 4 illustrates a dose-response graph of the effect of a
full length His-tagged FKBP-L recombinant polypeptide (SEQ ID NO:
1) on HMEC-1 migration in an in vitro wound closure assay in
accordance with one embodiment of the present invention.
[0018] FIG. 5 illustrates that an Haemagglutanin (HA)-tagged full
length FKBP-L polypeptide is actively secreted from HMEC-1 cells in
accordance with one embodiment of the present invention.
[0019] FIG. 6 illustrates the inhibitory effect of full length
FKBP-L recombinant polypeptide on HMEC-1 wound closure over time in
accordance with one embodiment of the present invention.
[0020] FIG. 7 illustrates a dose response graph of the effect of
full length FKBP-L recombinant polypeptide on HMEC-1 tube formation
on Matrigel matrix basement membrane in accordance with one
embodiment of the present invention.
[0021] FIGS. 8A and 8B illustrate the effect of the full-length
recombinant protein FKBP-L on angiogenesis in vivo using the mouse
sponge assay, in accordance with an embodiment of the present
invention where FIG. 8A shows treatment of cells with bovine
fibroblast growth factor (bFGF) alone, and FIG. 8B shows treatment
of cells with bFGF with full length FKBP-L polypeptide.
[0022] FIG. 9 shows a reduction in numbers of vessels seen upon
treatment with bFGF and full length recombinant FKBP-L polypeptide
(SEQ ID NO: 1) as compared to bFGF alone in accordance with
alternate embodiments of the present invention.
[0023] FIG. 10 illustrates a dose response of the effect of full
length FKBP-L recombinant polypeptide on the ex vivo rat aortic
ring explant model of angiogenesis in accordance with alternate
embodiments of the present invention.
[0024] FIGS. 11A and 11B show the effect of full length recombinant
FKBP-L polypeptide at a range of concentrations on the viability or
proliferation of HMEC-1 in the MTT assay after 24 hours (FIG. 11A)
and 48 hours (FIG. 11B) in accordance with alternate embodiments of
the present invention.
[0025] FIG. 12 shows changes in cytoskeletal morphology of
migrating endothelial cells on exposure to full length FKBP-L
recombinant polypeptide in accordance with an embodiment of the
present invention, where HMEC-1 microtubules were stained using
anti-tubulin.
[0026] FIG. 13 shows changes in cytoskeletal morphology of
migrating endothelial cells on exposure to full length FKBP-L
recombinant polypeptide in accordance with an embodiment of the
present invention, where HMEC-1 intermediate filaments were stained
using anti-vimentin.
[0027] FIGS. 14A, 14B, and 14C illustrate the effect of full length
recombinant polypeptide FKBP-L on PC3 (FIG. 14A), MDA (FIG. 14B)
and HT29 (FIG. 14C) tumor cell migration in accordance with
alternate embodiments of the present invention.
[0028] FIG. 15 illustrates the effect of direct intratumoral
injection of a FKBP-L cDNA construct on DU145 human prostate tumor
xenograft cell growth in vivo in accordance with an embodiment of
the present invention.
[0029] FIG. 16 shows that inhibition of cell migration is
correlated to expression of CD44 in HMEC-1 and the five tumor cell
lines DU145, PC3, HT29, MCF-7, MDA-231 in accordance with an
embodiment of the present invention.
[0030] FIGS. 17A, 17B, 17C, 17D, and 17E show the effect of full
length recombinant FKBP-L on DU145 (CD44 -ve) (FIG. 17A), HT29
(CD44 +ve) (FIG. 17B), PC3 (CD44 +ve) (FIG. 17C), MDA (CD44 +ve)
(FIG. 17D), and MCF-7(CD44 -ve), (FIG. 17E) tumor cell migration in
accordance with an embodiment of the present invention.
[0031] FIG. 18 shows that knock-down of CD44 in PC3 cells via an
siRNA targeted approach inhibits the FKBP-L-mediated inhibition of
PC3 migration in accordance with an embodiment of the present
invention.
[0032] FIG. 19 shows that FKBP-L can interact with endogenous CD44
in wounded HMEC-1 monolayers in accordance with an embodiment of
the present invention.
[0033] FIGS. 20A, 20B, and 20C illustrate FKBP-L deletion mutants,
where FIG. 20A and FIG. 20B illustrate the sequencing results of
several of the FKBP-L deletion mutants, and FIG. 20C illustrates
the inhibitory effects of transiently transfected FKBP-L deletion
mutants on wound closure in accordance with alternate embodiments
of the present invention.
[0034] FIG. 21 shows an evaluation of full-length recombinant
FKBP-L (SEQ ID NO: 1), candidate peptides FKBP-L 1-57 (1-57) (SEQ
ID NO: 6) and the FKBP-L 24mer (24mer) (SEQ ID NO: 10) spanning the
active domain of FKBP-L, using the wound scrape assay in accordance
with alternate embodiments of the present invention.
[0035] FIG. 22 shows an evaluation of full-length recombinant
FKBP-L (SEQ ID NO: 1), candidate peptides FKBP-L 1-57 (1-57) (SEQ
ID NO: 6), and the FKBP-L 24mer (24mer) (SEQ ID NO: 10) spanning
the active domain of FKBP-L, on the formation of endothelial
cell-to-cell contacts using the synthetic basement membrane
Matrigel in the tube formation assay in accordance with alternate
embodiments of the present invention.
[0036] FIGS. 23A and 23B show the effect of the FKBP-L 24mer
peptide (SEQ ID NO: 10) (FIG. 23A) and the FKBP-L 57mer (SEQ ID NO:
6) (FIG. 23B) peptides spanning the active domain of FKBP-L on
angiogenic sprouting using the rat aortic ring assay in accordance
with alternate embodiments of the present invention.
[0037] FIGS. 24A and 24B show the effect of candidate peptides
spanning the active domain of FKBP-L (i.e, FKBP-L 24mer peptide,
SEQ ID NO: 10; the FKBP-L 57mer, SEQ ID NO: 6; and full length
recombinant His-tagged FKBP-L, SEQ ID NO: 1) on the mean length,
maximum length (max length), and number of vessels (no. of vessels)
for angiogenic sprouting using the rat aortic ring assay in
accordance with alternate embodiments of the present invention.
[0038] FIG. 25 shows the effect of the FKBP-L 24mer (SEQ ID NO: 10)
on endothelial (HMEC-1) and tumor cell invasion (MDA231 and PC3) in
a modified Boyden chamber system in accordance with alternate
embodiments of the present invention.
[0039] FIG. 26 shows the effect of the FKBP-L 24mer (SEQ ID NO: 10)
on endothelial (HMEC-1) cell adhesion in accordance with alternate
embodiments of the present invention.
[0040] FIGS. 27A and 27B show the effect of the FKBP-L 24mer (SEQ
ID NO: 10) on MDA-231 (FIG. 27A) and PC3 (FIG. 27B) tumor cell
migration, in accordance with alternate embodiments of the present
invention.
[0041] FIGS. 28A and 28B show that the FKBP-L 24 mer is an
angiostatic inhibitor, where FIG. 28A shows the effect of addition
of the FKBP-L 24mer at day 7, and FIG. 28B shows an experiment
where aortic rings were initially exposed to FKBP-L 24mer and then
the 24mer removed, in accordance with alternate embodiments of the
present invention.
[0042] FIG. 29 illustrates that the FKBP-L 24mer inhibits
angiogenesis in vivo using the mouse sponge assay; shown are
numbers of vessels seen upon treatment with bFGF alone as compared
to bFGF and full length recombinant FKBP-L polypeptide (rFKBP-L)
(SEQ ID NO: 1), or bFGF in combination with the FKBP-L 24mer
(24mer) (SEQ ID NO: 10) polypeptide, in accordance with alternate
embodiments of the present invention.
[0043] FIG. 30 illustrates inhibition of mouse endothelial cell
(2H-11) migration by the FKBPL 24mer peptide (SEQ ID NO: 10) in
accordance with an embodiment of the present invention.
[0044] FIGS. 31A, 31B, 31C, 31D, and 31E show that the FKBP-L 24mer
peptide (SEQ ID NO: 10) inhibits DU145 tumor growth in vivo after
daily IP injection (FIG. 31A); increases survival (FIG. 31B, FIG.
31C, and FIG. 31D); and is not toxic (FIG. 31E), in accordance with
alternate embodiments of the present invention.
[0045] FIG. 32 shows the effect of the FKBP-L 24mer peptide (SEQ ID
NO: 10) on the viability or proliferation of HMEC-1 cells using the
MTT assay in accordance with alternate embodiments of the present
invention.
[0046] FIGS. 33A and 33B show the effect of candidate peptides
spanning active domain of FKBP-L on the viability or proliferation
of HMEC-1 cells upon administration for 24 hours (FIG. 33A) or 48
hours (FIG. 33B) using the MTT assay in accordance with alternate
embodiments of the present invention.
[0047] FIGS. 34A, 34B, 34C, 34D, 34E, 34F, 34G, 34H, 34I, 34J, 34K,
and 34L show the response of various modified/truncated versions of
the FKBP-L 24 mer: a PEG-modified FKBP-L 24mer (FIG. 34A), a FKBP-L
24mer with an N-terminal pyroglutamic acid (FIG. 34B), and
truncated forms of 24mer FKBP-L peptide (FIG. 34C, FIG. 34D, FIG.
34E, FIG. 34F, FIG. 34G, FIG. 34H, FIG. 34I, FIG. 34J, FIG. 34K,
and FIG. 34L). All are compared to the 24mer peptide in the in
vitro HMEC-1 wound scrape assay in accordance with alternate
embodiments of the present invention.
[0048] FIGS. 35A, 35B, and 35C show purification of recombinant
FKBP-L in accordance with alternate embodiments of the present
invention, where FIG. 35A shows an SDS PAGE gel run under reducing
conditions showing purified recombinant FKBP-L protein before and
after dialysis (lanes 1 & 2 respectively); and FIG. 35B shows
an SDS PAGE comparison of dialysed recombinant FKBP-L before and
after treatment with DTT (lanes 3 & 4). Lane 3 is non-reduced
sample, lane 4 is sample reduced with 50 mM DTT. FIG. 35C shows
further purification of recombinant FKBP-L by gel filtration.
Inserts show native PAGE analysis of both peaks from gel filtration
purification along with dialysed protein, with (+) and without (-)
100 mM DTT.
[0049] FIG. 36 shows gel permeation chromatographic analysis of
recombinant FKBP-L in accordance with alternate embodiments of the
present invention.
[0050] FIG. 37 shows glutaraldehyde cross-linking of recombinant
FKBP-L in the presence (+) and absence (-) of 100 mM DTT in
accordance with alternate embodiments of the present invention.
Lane c is the control (no DTT).
DETAILED DESCRIPTION
Definitions
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Practitioners are particularly directed
to Current Protocols in Molecular Biology (Ausubel) for definitions
and terms of the art. Abbreviations for amino acid residues are the
standard 3-letter and/or 1-letter codes used in the art to refer to
one of the 20 common L-amino acids.
[0052] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges subsumed therein. For example, a
stated range of "1 to 10" should be considered to include any and
all subranges between (and inclusive of) the minimum value of 1 and
the maximum value of 10; that is, all subranges beginning with a
minimum value of 1 or more, e.g. 1 to 6.1, and ending with a
maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any
reference referred to as being "incorporated herein" is to be
understood as being incorporated in its entirety.
[0053] It is further noted that, as used in this specification, the
singular forms "a," "an," and "the" include plural referents unless
expressly and unequivocally limited to one referent. The term "or"
is used interchangeably with the term "and/or" unless the context
clearly indicates otherwise.
[0054] Also, the terms "portion" and "fragment" are used
interchangeably to refer to parts of a polypeptide, nucleic acid,
or other molecular construct.
[0055] As used herein, the term "biologically active FKBP-L
polypeptide" (e.g., fragment and/or modified polypeptides) is used
to refer to a polypeptide that displays the same or similar amount
and type of activity as the full-length FKBP-L polypeptide. In this
context "biological activity" of an FKBP-L polypeptide, fragment or
derivative includes any one of anti-angiogenic activity, inhibition
of tumour cell growth and/or proliferation, inhibition of tumour
cell migration and/or metastasis. Biological activity of FKBP-L
fragments or derivatives may be tested in comparison to full length
FKBP-L using any of the in vitro or in vivo assays described in the
accompanying examples, such as for example wound closure or wound
scrape assay, in vitro cell migration assay, Matrigel.TM. assay for
cell-cell adhesion, mouse sponge assay, aortic ring explant assay,
MTT proliferation assay, HMEC-1 tube formation assay in vivo tumour
cell growth assay. In this regard, deliberate amino acid
substitutions may be made in the polypeptide on the basis of
similarity in polarity, charge, solubility, hydrophobicity, or
hydrophilicity of the residues, as long as the specificity of
activity (i.e., function) is retained.
[0056] As used herein a "subject" may be an animal. For example,
the subject may be a mammal. Also, the subject may be a human. In
alternate embodiments, the subject may be either a male or a
female. In certain embodiments, the subject may be a patient, where
a patient is an individual who is under medical care and/or
actively seeking medical care for a disorder or disease.
[0057] "Polypeptide" and "protein" are used interchangeably herein
to describe protein molecules that may comprise either partial or
full-length proteins. The term "peptide" is used to denote a less
than full-length protein or a very short protein unless the context
indicates otherwise.
[0058] As is known in the art, "proteins", "peptides,"
"polypeptides" and "oligopeptides" are chains of amino acids
(typically L-amino acids) whose alpha carbons are linked through
peptide bonds formed by a condensation reaction between the
carboxyl group of the alpha carbon of one amino acid and the amino
group of the alpha carbon of another amino acid. Typically, the
amino acids making up a protein are numbered in order, starting at
the amino terminal residue and increasing in the direction toward
the carboxy terminal residue of the protein.
[0059] As used herein, the term "upstream" refers to a residue that
is N-terminal to a second residue where the molecule is a protein,
or 5' to a second residue where the molecule is a nucleic acid.
Also as used herein, the term "downstream" refers to a residue that
is C-terminal to a second residue where the molecule is a protein,
or 3' to a second residue where the molecule is a nucleic acid.
[0060] A "nucleic acid" is a polynucleotide such as
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term is
used to include single-stranded nucleic acids, double-stranded
nucleic acids, and RNA and DNA made from nucleotide or nucleoside
analogues.
[0061] The term "vector" refers to a nucleic acid molecule that may
be used to transport a second nucleic acid molecule into a cell. In
one embodiment, the vector allows for replication of DNA sequences
inserted into the vector. The vector may comprise a promoter to
enhance expression of the nucleic acid molecule in at least some
host cells. Vectors may replicate autonomously (extrachromasomal)
or may be integrated into a host cell chromosome. In one
embodiment, the vector may comprise an expression vector capable of
producing a protein derived from at least part of a nucleic acid
sequence inserted into the vector.
[0062] As is known in the art, conditions for hybridizing nucleic
acid sequences to each other can be described as ranging from low
to high stringency. Generally, highly stringent hybridization
conditions refer to washing hybrids in low salt buffer at high
temperatures. Hybridization may be to filter bound DNA using
hybridization solutions standard in the art such as 0.5M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), at 65.degree. C., and
washing in 0.25 M NaHPO.sub.4, 3.5% SDS followed by washing
0.1.times.SSC/0.1% SDS at a temperature ranging from room
temperature to 68.degree. C. depending on the length of the probe
(see e.g. Ausubel, F. M. et al., Short Protocols in Molecular
Biology, 4.sup.th Ed., Chapter 2, John Wiley & Sons, N.Y). For
example, a high stringency wash comprises washing in
6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C. for a 14
base oligonucleotide probe, or at 48.degree. C. for a 17 base
oligonucleotide probe, or at 55.degree. C. for a 20 base
oligonucleotide probe, or at 60.degree. C. for a 25 base
oligonucleotide probe, or at 65.degree. C. for a nucleotide probe
about 250 nucleotides in length. Nucleic acid probes may be labeled
with radionucleotides by end-labeling with, for example,
[.gamma.-.sup.32P]ATP, or incorporation of radiolabeled nucleotides
such as [.alpha.-.sup.32P]dCTP by random primer labeling.
Alternatively, probes may be labeled by incorporation of
biotinylated or fluorescein labeled nucleotides, and the probe
detected using Streptavidin or anti-fluorescein antibodies.
[0063] The terms "identity" or "percent identical" refers to
sequence identity between two amino acid sequences or between two
nucleic acid sequences. Percent identity can be determined by
aligning two sequences and refers to the number of identical
residues (i.e., amino acid or nucleotide) at positions shared by
the compared sequences. Sequence alignment and comparison may be
conducted using the algorithms standard in the art (e.g. Smith and
Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970,
J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad.
Sci., USA, 85:2444) or by computerized versions of these algorithms
(Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Drive, Madison, Wis.) publicly available as
BLAST and FASTA. Also, ENTREZ, available through the National
Institutes of Health, Bethesda Md., may be used for sequence
comparison. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., BLASTN;
available at the Internet site for the National Center for
Biotechnology Information) may be used. In one embodiment, the
percent identity of two sequences may be determined using GCG with
a gap weight of 1, such that each amino acid gap is weighted as if
it were a single amino acid mismatch between the two sequences. Or,
the ALIGN program (version 2.0), which is part of the GCG
(Accelrys, San Diego, Calif.) sequence alignment software package
may be used.
[0064] The binding properties of a protein comprising either a
receptor or a ligand can be expressed in terms of binding
specificity, which may be determined as a comparative measure
relative to other known substances that bind to the receptor.
Standard assays for quantifying binding and determining binding
affinity are known in the art and include, e.g., equilibrium
dialysis, equilibrium binding, gel filtration, surface plasmon
resonance, the use of a labeled binding partners, ELISAs and
indirect binding assays (e.g., competitive inhibition assays). For
example, as is well known in the art, the dissociation constant of
a protein can be determined by contacting the protein with a
binding partner and measuring the concentration of bound and free
protein as a function of its concentration.
[0065] As used herein, the term "conserved residues" refers to
amino acids that are the same among a plurality of proteins having
the same structure and/or function. A region of conserved residues
may be important for protein structure or function. Thus,
contiguous conserved residues as identified in a three-dimensional
protein may be important for protein structure or function. To find
conserved residues, or conserved regions of 3-D structure, a
comparison of sequences for the same or similar proteins from
different species, or of individuals of the same species, may be
made.
[0066] As used herein, the term "similar" or "homologue" when
referring to amino acid or nucleotide sequences means a polypeptide
having a degree of homology or identity with the wild-type amino
acid sequence. Homology comparisons can be conducted by eye, or
more usually, with the aid of readily available sequence comparison
programs. These commercially available computer programs can
calculate percent homology between two or more sequences (e.g.
Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA,
80:726-730). For example, homologous sequences may be taken to
include an amino acid sequences which in alternate embodiments are
at least 70% identical, 75% identical, 80% identical, 85%
identical, 90% identical, 95% identical, 96% identical, 97%
identical, or 98% identical to each other.
[0067] As used herein, the term at least 90% identical thereto
includes sequences that range from 90 to 99.99% identity to the
indicated sequences and includes all ranges in between. Thus, the
term at least 90% identical thereto includes sequences that are 91,
91.5, 92, 92.5, 93, 93.5. 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5,
98, 98.5, 99, 99.5 percent identical to the indicated sequence.
Similarly the term "at least 70% identical includes sequences that
range from 70 to 99.99% identical, with all ranges in between. The
determination of percent identity is determined using the
algorithms described herein.
[0068] As used herein, a polypeptide or protein "domain" comprises
a region along a polypeptide or protein that comprises an
independent unit. Domains may be defined in terms of structure,
sequence and/or biological activity. In one embodiment, a
polypeptide domain may comprise a region of a protein that folds in
a manner that is substantially independent from the rest of the
protein. Domains may be identified using domain databases such as,
but not limited to PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE,
ISREC PROFILES, SAMRT, and PROCLASS.
[0069] As used herein, the term "linked" identifies a covalent
linkage between two different groups (e.g., nucleic acid sequences,
polypeptides, polypeptide domains) that may have an intervening
atom or atoms between the two groups that are being linked. As used
herein, "directly linked" identifies a covalent linkage between two
different groups (e.g., nucleic acid sequences, polypeptides,
polypeptide domains) that does not have any intervening atoms
between the two groups that are being linked.
[0070] As used herein, "ligand binding domain" refers to a domain
of a protein responsible for binding a ligand. The term ligand
binding domain includes homologues of a ligand binding domain or
portions thereof. In this regard, deliberate amino acid
substitutions may be made in the ligand binding site on the basis
of similarity in polarity, charge, solubility, hydrophobicity, or
hydrophilicity of the residues, as long as the binding specificity
of the ligand binding domain is retained.
[0071] As used herein, a "ligand binding site" comprises residues
in a protein that directly interact with a ligand, or residues
involved in positioning the ligand in close proximity to those
residues that directly interact with the ligand. The interaction of
residues in the ligand binding site may be defined by the spatial
proximity of the residues to a ligand in the model or structure.
The term ligand binding site includes homologues of a ligand
binding site, or portions thereof. In this regard, deliberate amino
acid substitutions may be made in the ligand binding site on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, or hydrophilicity of the residues, as long as the
binding specificity of the ligand binding site is retained. A
ligand binding site may exist in one or more ligand binding domains
of a protein or polypeptide.
[0072] As used herein, the term "interact" refers to a condition of
proximity between two molecules or portions of a single molecule
(e.g., different domains in a peptide). The interaction may be
non-covalent, for example, as a result of hydrogen-bonding, van der
Waals interactions, or electrostatic or hydrophobic interactions,
or it may be covalent.
[0073] As used herein, a "ligand" refers to a molecule or compound
or entity that interacts with a ligand binding site, including
substrates or analogues or parts thereof. As described herein, the
term "ligand" may refer to compounds that bind to the protein of
interest. A ligand may be an agonist, an antagonist, or a
modulator. Or, a ligand may not have a biological effect. Or, a
ligand may block the binding of other ligands thereby inhibiting a
biological effect. Ligands may include, but are not limited to,
small molecule inhibitors. These small molecules may include
peptides, peptidomimetics, organic compounds and the like. Ligands
may also include polypeptides and/or proteins.
[0074] As used herein, "modulate" refers to changing or altering
the biological activity of a molecule of interest. A "modulator"
compound may increase or decrease activity, or change the physical
or chemical characteristics, or functional or immunological
properties, of the molecule of interest. A modulator compound of
the present invention may include natural and/or chemically
synthesized or artificial FKBP-L peptides, peptide mimetics,
modified peptides (e.g., phosphopeptides, cyclic peptides, peptides
containing D- and unnatural amino-acids, stapled peptides, peptides
containing radiolabels), or peptides linked to antibodies,
carbohydrates, monosaccharides, oligosaccharides, polysaccharides,
glycolipids, heterocyclic compounds, nucleosides or nucleotides or
parts thereof, and/or small organic or inorganic molecules (e.g.,
peptides modified with PEG or other stabilizing groups). Thus, the
FKBP-L polypeptides of the invention also includes a chemically
modified peptides or isomers and racemic forms.
[0075] An "agonist" comprises a compound that binds to a receptor
to form a complex that elicits a pharmacological response specific
to the receptor involved.
[0076] An "antagonist" comprises a compound that binds to an
agonist or to a receptor to form a complex that does not give rise
to a substantial pharmacological response and can inhibit the
biological response induced by an agonist.
[0077] The term "peptide mimetics" refers to structures that serve
as substitutes for peptides in interactions between molecules
(Morgan et al., 1989, Ann. Reports Med. Chem., 24:243-252). Peptide
mimetics may include synthetic structures that may or may not
contain amino acids and/or peptide bonds but that retain the
structural and functional features of a peptide, or agonist, or
antagonist. Peptide mimetics also include peptoids, oligopeptoids
(Simon et al., 1972, Proc. Natl. Acad, Sci., USA, 89:9367); and
peptide libraries containing peptides of a designed length
representing all possible sequences of amino acids corresponding to
a peptide, or agonist or antagonist of the invention.
[0078] As used herein, the term "EC50" is defined as the
concentration of an agent that results in 50% of a measured
biological effect. For example, the EC50 of a therapeutic agent
having a measurable biological effect may comprise the value at
which the agent displays 50% of the biological effect.
[0079] As used herein, the term "IC50" is defined as the
concentration of an agent that results in 50% inhibition of a
measured effect. For example, the IC50 of an antagonist of binding
may comprise the value at which the antagonist reduces ligand
binding to a ligand binding site by 50%.
[0080] As used herein, an "effective amount" means the amount of an
agent that is effective for producing a desired effect in a
subject. The term "therapeutically effective amount" denotes that
amount of a drug or pharmaceutical agent that will elicit
therapeutic response of an animal or human that is being sought.
The actual dose which comprises the effective amount may depend
upon the route of administration, the size and health of the
subject, the disorder being treated, and the like.
[0081] The term "pharmaceutically acceptable carrier" as used
herein may refer to compounds and compositions that are suitable
for use in human or animal subjects, as for example, for
therapeutic compositions administered for the treatment of a
disorder or disease of interest.
[0082] The term "pharmaceutical composition" is used herein to
denote a composition that may be administered to a mammalian host,
e.g., orally, parenterally, topically, by inhalation spray,
intranasally, or rectally, in unit dosage formulations containing
conventional non-toxic carriers, diluents, adjuvants, vehicles and
the like.
[0083] The term "parenteral" as used herein, includes subcutaneous
injections, intravenous, intramuscular, intracisternal injection,
or infusion techniques.
[0084] A "stable" formulation is one in which the polypeptide or
protein therein essentially retains its physical and chemical
stability and biological activity upon storage. Various analytical
techniques for measuring protein stability are available in the art
and are reviewed in Peptide and Protein Drug Delivery, 247-301,
Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)
and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability
can be measured at a selected temperature for a selected time
period. For rapid screening, the formulation of interest may be
kept at 40.degree. C. for 1 week to 1 month, at which time
stability is measured. The extent of aggregation following
lyophilization and storage can be used as an indicator of peptide
and/or protein stability. For example, a "stable" formulation is
one wherein less than about 10% and preferably less than about 5%
of the polypeptide or protein is present as an aggregate in the
formulation. An increase in aggregate formation following
lyophilization and storage of the lyophilized formulation can be
determined. For example, a "stable" lyophilized formulation may be
one wherein the increase in aggregate in the lyophilized
formulation is less than about 5% or less than about 3%, when the
lyophilized formulation is incubated at 40.degree. C. for at least
one week. Stability of the fusion protein formulation may be
measured using a biological activity assay such as a binding assay
as described herein.
[0085] FKBP-L Polypeptides as Modulators of Cell Migration,
Angiogenesis, and Tumor Metastasis
[0086] The present invention recognizes that FKBP-L, fragments of
FKBP-L and modified FKBP-L and fragments thereof, can inhibit cell
migration and may possess potent angiogenesis modulating
properties. Embodiments of the present invention relate to FKBP-L
derived peptides and their use. The present invention may be
embodied in a variety of ways.
[0087] Thus, in certain embodiments, the FKBP-L polypeptides of the
present invention may show anti-angiogenic properties. Also, in
some embodiments, the FKBP-L polypeptides of the present invention
may be used to modulate cell migration and/or metastasis of tumor
cells. The action of the FKBP-L polypeptides of the present
invention may, in certain embodiments, be mediated by CD44. Thus,
in some embodiments of the present invention, FKBP-L polypeptides
may be used to modulate angiogenesis, cell migration, and/or
metastasis of cells that express CD44.
[0088] In certain embodiments, the invention may be used to treat
disorders mediated by or associated with cell migration. For
example, FKBP-L peptides can be used to inhibit or combat tumor
invasion and metastasis. Or, in some embodiments, FKBP-L peptides
may be used to inhibit the migration of cells involved in wound
healing. In yet other embodiments, FKBP-L peptides may be used to
inhibit angiogenesis to thereby treat disorders mediated by
angiogenesis.
[0089] Thus, in some embodiments, the present invention comprises a
method of treating a disorder mediated by or associated with at
least one of cell migration, angiogenesis, or tumor metastasis,
where the method comprises administering a therapeutically
effective amount of: (i) an active compound comprising an isolated
FKBP-L polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or a fragment thereof, or (ii) a polynucleotide
encoding such a FKBP-L polypeptide, fragment, or derivative to a
patient in need thereof.
[0090] For example, in some embodiments, the present invention
comprises a method of modulating angiogenesis or tumor metastasis,
the method comprising administering a therapeutically effective
amount of an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or a fragment thereof, or a polynucleotide encoding
such a FKBP-L polypeptide, fragment or derivative to a subject in
need thereof.
[0091] In other embodiments, the present invention comprises the
use of: (i) an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or a fragment thereof, or (ii) a polynucleotide
encoding such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a composition or medicament for the treatment of a
disorder mediated by or associated with at least one of cell
migration and/or angiogenesis. For example, in one embodiment, the
present invention comprises the use of (i) an active compound
comprising an isolated FKBP-L polypeptide or a biologically active
fragment of a FKBP-L polypeptide, or a biologically active
derivative of a FKBP-L polypeptide or fragment thereof or (ii) a
polynucleotide encoding such a FKBP-L polypeptide, fragment or
derivative in the manufacture of a medicament for use as an
inhibitor of angiogenesis.
[0092] A variety of disorders that are mediated by or associated
with angiogenesis and/or cell migration may be treated with the
compositions and/or medicaments of the present invention. Thus, in
alternate embodiments, the medicament may be used in the treatment
of at least one of angiogenesis-associated inflammation, ocular
disorders mediated by angiogenesis, wound healing, or cancer.
[0093] Thus, in one embodiment, the present invention comprises the
use of (i) an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or fragment thereof or (ii) a polynucleotide encoding
such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a medicament for use in the treatment of
angiogenesis-associated inflammation.
[0094] In other embodiments, the disorder associated with
angiogenesis is an ocular disorder, for example, macular
degeneration and other ocular disorders described herein.
Alternatively the disorder associated with angiogenesis is
arteriosclerosis, arthritis, psoriasis or endometriosis. Thus, in
alternate embodiments, the invention provides a method of treatment
of at least one of an ocular disorder, arteriosclerosis, arthritis,
psoriasis or endometriosis, the method comprising administering a
therapeutically effective amount of an active compound comprising
an isolated FKBP-L polypeptide, a biologically active fragment of a
FKBP-L polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or a fragment thereof, or a polynucleotide encoding
such a FKBP-L polypeptide, fragment or a derivative thereof, to a
subject in need thereof. Or, the present invention may comprise the
use of (i) an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or fragment thereof or (ii) a polynucleotide encoding
such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a medicament for use in the treatment of ocular
disorders mediated by angiogenesis. For example, in alternate
embodiments, the FKBP-L peptide or polynucleotide may be used for
the manufacture of a medicament for the treatment of macular
degenerative disease or diabetic retinopathy. Or, the present
invention may comprise the use of (i) an active compound comprising
an isolated FKBP-L polypeptide or a biologically active fragment of
a FKBP-L polypeptide, or a biologically active derivative of a
FKBP-L polypeptide or fragment thereof or (ii) a polynucleotide
encoding such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a medicament for use in the treatment of at least
one of arteriosclerosis, psoriasis, arthritis, or
endometriosis.
[0095] In certain embodiments, the invention provides methods of
treatment of cancer. For example, in some embodiments the present
invention provides a method of treating cancer comprising
administering a therapeutically effective amount of an active
compound comprising an isolated FKBP-L polypeptide, a biologically
active fragment of a FKBP-L polypeptide, or a biologically active
derivative of a FKBP-L polypeptide or a fragment thereof, or a
polynucleotide encoding such a FKBP-L polypeptide, fragment or
derivative thereof, for at least one of treating cancer, inhibiting
tumor cell migration and/or metastasis, or inhibiting tumor cell
growth and/or proliferation. In an embodiment, the inhibition of
tumor cell migration and metastasis is by inhibition of
angiogenesis. For example, the present invention may comprise the
use of (i) an active compound comprising an isolated FKBP-L
polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of a FKBP-L
polypeptide or fragment thereof or (ii) a polynucleotide encoding
such a FKBP-L polypeptide, fragment or derivative in the
manufacture of a medicament for use in the treatment of cancer. In
certain embodiments, the compounds and compositions of the present
invention may prevent tumor cell growth and/or metastasis. In an
embodiment, the inhibition of tumor cell migration and metastasis
is by inhibition of angiogenesis. Thus, in one embodiment, the
present invention may comprise the use of (i) an active compound
comprising an isolated FKBP-L polypeptide or a biologically active
fragment of a FKBP-L polypeptide, or a biologically active
derivative of a FKBP-L polypeptide or fragment thereof or (ii) a
polynucleotide encoding such a FKBP-L polypeptide, fragment or
derivative in the manufacture of a medicament for use as an
inhibitor of tumor cell migration and/or metastasis. In yet other
embodiments, the present invention may comprise the use of (i) an
active compound comprising an isolated FKBP-L polypeptide or a
biologically active fragment of a FKBP-L polypeptide, or a
biologically active derivative of a FKBP-L polypeptide or fragment
thereof or (ii) a polynucleotide encoding such a FKBP-L
polypeptide, fragment or derivative in the manufacture of a
medicament for use as an inhibitor of tumor cell growth and/or
proliferation.
[0096] The expression FKBP-L polypeptides is used in the
specification according to its broadest meaning. It designates the
naturally occurring proteins as shown in SEQ ID NOS: 1, 2 and 29
together with homologues due to polymorphisms, other variants,
mutants and portions of said polypeptide which retain their
angiogenesis modulating activities. For example, in certain
embodiments, the FKBP-L polypeptide comprises SEQ ID NO: 1 with an
N-terminal sequence (see amino acid residues in bold font in SEQ ID
NO: 1 as shown in FIG. 1) that includes a poly-histidine tag of six
histidine residues attached to the N-terminus of the protein, or
SEQ ID NO: 2 with a Threonine at position 181 and a Glycine at
position 186 of the wild-type sequence. Or, a polypeptide of SEQ ID
NO: 29 (GENBank Accession No. NP 071393; NM 022110; [gi:34304364])
may be used. Example constructs of other FKBP-L polypeptides (e.g.,
fragments and other modifications) of the present invention are
shown in FIG. 1. Also, example constructs of polynucleotide
constructs encoding for FKBP-L polypeptide constructs are provided
in FIG. 2.
[0097] Embodiments of the present invention comprise an isolated
FKBP-L polypeptide or a biologically active fragment of a FKBP-L
polypeptide, or a biologically active derivative of such a FKBP-L
polypeptide or fragment for use as a medicament. Thus, alternate
embodiments of the present invention comprise use of a FKBP-L
peptide or nucleotide that encodes a FKBP-L peptide as described
herein wherein the FKBP-L polypeptide comprises the amino acid
sequence shown in SEQ ID NO: 10, or the amino acid sequence shown
in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 29, or the amino acid
sequence shown in any one of SEQ ID NOs: 3 to 7, or 11 to 28, or an
amino acid sequence at least 90% identical to the amino acid
sequence shown in any one of SEQ ID NOs: 1 to 29. Or, a sequence
that comprises at least 18 contiguous amino acids of SEQ ID NO: 10
(e.g., SEQ ID NOs: 11, 16, 23) may be used. References herein to
peptides (and to uses thereof) which are shown as modified, such as
SEQ ID NOs: 12, 13 and 28, should be interpreted as encompassing
peptides of identical amino acid sequence without the listed
modification (and uses thereof) unless otherwise stated.
[0098] As described herein, the methods and compositions of the
present invention may utilize a full-length FKBP-L polypeptide, or
fragments of the polypeptide. Thus, certain embodiments of the
present invention comprise a FKBP-L derivative which comprises or
consists of an effective portion of the N-terminal amino acid
sequence of naturally occurring FKBP-L. This sequence may comprise
or consist of an active N-terminal portion of the FKBP-L
polypeptide. In alternate embodiments, the polypeptide may comprise
or consist of amino acids 1 to 57 of SEQ ID NO: 2 (i.e., SEQ ID NO:
6), or amino acids 34-57 of SEQ ID NO:2 (i.e., SEQ ID NO: 10). Or,
the peptide may comprise or consist of a sequence that comprises at
least 18 contiguous amino acids of SEQ ID NO: 10 (e.g., SEQ ID NOs:
11, 16, or 23). In alternate embodiment, the polypeptide used in
the methods and compositions of the present invention may comprise
or consist of one of the amino acid sequences shown in any one of
SEQ ID NOs: 1-7, 10-29. In certain embodiments, the present
invention comprises a biologically active fragment of FKBP-L,
wherein said polypeptide includes no more than 200 consecutive
amino acids of the amino acid sequence shown in SEQ ID NO:1, SEQ ID
NO:2, or SEQ ID NO: 29.
[0099] As described herein, the peptides may be modified (e.g., to
contain PEG and/or His tags or other modifications). Or, the
present invention may comprise isolated polypeptides having a
sequence at least 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or
96%, or 97%, or 98%, or 99% identical to the amino acid sequences
as set forth in any one of SEQ ID NOS: 1-29. Or, the isolated
peptide or the peptide used for preparation of a medicament may
comprise or consist of a sequence having at least 70%, or 75%, or
80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%
identity to at least 18 contiguous amino acids of SEQ ID NO: 10
(e.g., SEQ ID NOs: 11, 16, 23).
[0100] The FKBP-L derivative of the invention may be of variable
length as long as it retains its antiangiogenic/proangiogenic
activity and can be used according to the various aspects of the
invention described above. Functional equivalents of FKBP-L are
also encompassed by the present invention. For example, in certain
embodiments, a functional equivalent may comprise or consist of a
small molecule which can bind CD44 and/or prevent binding of a
ligand (e.g. MIF) to a complex containing CD44 and CD74. Or, a
functional equivalent may comprise or consist of a small molecule
that will act in a similar manner as FKBP-L and its peptide
derivatives to inhibit at least of cell migration, angiogenesis
and/or metastasis.
[0101] The dose of the FKBP-L polypeptide administered may vary
depending upon the disorder being treated. In alternate
embodiments, a dosage to be achieved in vivo would be equivalent to
an in vitro level of greater than 10.sup.-12 M, or 10.sup.-11 M, or
10.sup.-10 M, or 10.sup.-9 M, or 10.sup.-8 M, or 10.sup.-7 M, or
10.sup.-6 M, or 10.sup.-5 M. Thus, a dosage to be achieved in vivo
may be equivalent to an in vitro level of 10.sup.-12 M to 10.sup.-5
M, or 10.sup.-11 M to 10.sup.-6 M, or 10.sup.-10 M to 10.sup.-7 M,
or 10.sup.-9 M to 10.sup.-7 M or ranges therein. In alternate
embodiments, the dosage used may be equivalent to an in vitro level
of about 1-10000 ngml.sup.-1, or about 10-5000 ngml.sup.-1, or
about 100-1000 ngml.sup.-1. Or, in certain embodiments, the dosage
may comprise from about 0.00001 to 500 mg/kg/day, or from about
0.0001 to 300 mg/kg/day, or from about 0.003 to 100 mg/kg/day, or
from about 0.03 to 30 mg/kg/day, or from about 0.1 mg/kg/day to 10
mg/kg/day, or from about 0.3 mg/kg/day to 3 mg/kg/day.
[0102] In an embodiment, the FKBP-L polypeptide is administered to
a subject in need thereof. As used herein, a subject in need
thereof is a subject who may be benefited by the administration of
FKBP-L.
[0103] In yet other embodiments, the present invention comprises an
isolated nucleic acid molecule which encodes a protein or
polypeptide comprising the amino acid sequence as set forth in any
one of SEQ ID NOs: 1-29, or a biologically active fragment thereof,
and the use of such molecules for the preparation of medicaments
and/or as therapeutic agents. In an embodiment, a biologically
active fragment comprises or consists of at least 18 contiguous
amino acids of SEQ ID NO: 10 (e.g., SEQ ID NOS:11, 16, 23).
[0104] For example, embodiments of the present invention comprise
the use of a polynucleotide that encodes a FKBP-L peptide, a
biologically active fragment of a FKBP-L peptide, or biologically
active derivative thereof, wherein the polynucleotide encoding the
FKBP-L polypeptide, fragment or derivative comprises the nucleotide
sequence shown in any one of SEQ ID NOs: 30-39.
[0105] Also, the present invention comprises isolated nucleic acids
that encode for FKBP-L peptides. The nucleic acid molecule may
comprise a nucleic acid molecule having the sequence as set forth
in SEQ ID NOs: 30-39, or a fragment thereof, wherein the nucleic
acid molecule encodes for a polypeptide having the sequence of SEQ
ID NOs: 1-28, or a fragment of these polypeptides. In an
embodiment, a fragment comprises or consists of at least 18
contiguous amino acids of SEQ ID NO: 10 (e.g., SEQ ID NOS: 11, 16,
23). In certain embodiments, degenerate nucleic acid molecules,
comprising a degenerate variation in the third position of the
amino acid codon such that the same amino acid is encoded by the
degenerate sequence, may be used to encode the FKBP-L polypeptides,
fragments and/or derivatives of the present invention. Thus, in
certain embodiments, the present invention may comprise isolated
nucleic acid molecules having a sequence at least 70%, or 75%, or
80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%
identical to SEQ ID NOS: 30-39 or fragments thereof.
[0106] The present invention also include primers that may be used
to produce polynucleotide fragments of SEQ ID NO: 31, where such
fragments encode the FKBP-L peptides shown in FIG. 1. Thus, in
alternate embodiments, the present invention include
oligonucleotide primers comprising the sequences as set forth in
SEQ ID NOS: 45-58 or a sequence at least 70%, or at least 80%, or
at least 85%, or at least 90%, or at least 95%, or at least 96%, or
at least 97%, or at least 98%, or at least 99% identical
thereto.
[0107] In yet other embodiments, the present invention comprises
vectors containing the isolated nucleic acid molecules of the
present invention. In certain embodiments, the present invention
also comprise cells transfected with such vectors, such that a
FKBP-L polypeptide is expressed. Such embodiments are described in
more detail herein.
[0108] In yet other embodiments, the present invention comprises an
isolated nucleic acid molecule which is antisense to the coding
strand of the FKBP-L gene or portion thereof and the use of such
molecules for the preparation of medicaments and/or as therapeutic
agents. Thus, in yet another embodiment, the present invention
comprises a polynucleotide that is at least 70%, or 75%, or 80%, or
85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% identical to a
nucleic acid sequence that is antisense to the coding strand of an
mRNA encoding a FKBP-L polypeptide of the invention.
[0109] In certain embodiments, the anti-sense molecules can be used
to advantageously promote angiogenesis and/or cell migration and in
the treatment of disorders mediated by or associated with at least
one of angiogenesis or cell migration. For example, in one
embodiment, the present invention comprises the use of an antisense
oligonucleotide or siRNA capable of specifically down-regulating
expression of FKBP-L in the manufacture of a medicament for use as
a modulator to promote angiogenesis. Also, in certain embodiments,
the present invention comprises the use of an antisense
oligonucleotide or siRNA capable of specifically down-regulating
expression of FKBP-L in the manufacture of a medicament for use as
a modulator to promote at least one of hematopoiesis or
vasculogenesis. In one embodiment, the present invention comprises
the use of an antisense oligonucleotide or siRNA capable of
specifically down-regulating expression of FKBP-L in the
manufacture of a medicament for use to promote wound healing. Also,
the present invention may comprise the use of an antisense
oligonucleotide or siRNA capable of specifically down-regulating
expression of FKBP-L in the manufacture of a medicament for use in
the treatment of at least one of peptic ulcer, a bone fracture, or
keloids.
[0110] In other embodiments, the present invention may comprise the
use of an antisense oligonucleotide or siRNA capable of
specifically down-regulating expression of FKBP-L in the
manufacture of a medicament for use in the treatment of pardentitis
or pardontopathy mediated by angiogenesis. In other embodiments,
the present invention may comprise the use of an antisense
oligonucleotide or siRNA capable of specifically down-regulating
expression of FKBP-L in the manufacture of a medicament for use in
the treatment or regulation of the reproductive system, such as
ovulation, mestruation and placentation. In yet other embodiments,
the present invention may comprise the use of an antisense
oligonucleotide or siRNA capable of specifically down-regulating
expression of FKBP-L in the manufacture of a medicament for use in
the treatment or regulation of the dysfunction in the brain and
nervous system, such as may be caused by stroke. Use of an
antisense oligonucleotide or siRNA capable of specifically
down-regulating expression of FKBP-L may therefore be useful in the
treatment of certain types of dementia and/or mental
retardation.
[0111] Additional aspects of certain embodiments of the present
invention are discussed in more detail below.
FKBP-L Modulates Cell Migration, Angiogenesis and Metastasis
[0112] In certain embodiments, FKBP-L and fragments thereof may be
used to modulate angiogenesis. In one embodiment, FKBP-L or
fragments thereof may be used to inhibit angiogenesis. For example,
transfection of cells with FKBP-L may inhibit endothelial cell
migration and angiogenesis (FIG. 3) indicating that FKBP-L protein
is a potential anti-migratory protein. The dose-dependent nature
effect of FKBP-L on cell migration is shown in FIG. 4. Thus, it can
be seen that a dose of 10.sup.-6 M full length His-tagged FKBP-L is
effective to prevent cell migration.
[0113] In certain embodiments, FKBP-L may be secreted from certain
types of cells such as endothelial cells (FIG. 5), and tumor cells.
Thus, in an embodiment, the anti-angiogenic action of FKBP-L may be
via receptor activation. The secretion of FKBP-L from endothelial
cells indicates that application of FKBP-L protein or
over-expression of FKBP-L using a cDNA construct may both be able
to exert anti-angiogenic effects observed both in vitro and in
vivo.
[0114] In certain embodiments, FKBP-L exhibits an effect on cell
migration over a physiologically relevant time period. For example,
HMEC-1 cells treated with full length His-tagged recombinant FKBP-L
polypeptide (SEQ ID NO: 1) may exhibit decreased wound closure for
up to 2 to 3 days (FIG. 6). Thus, application of FKBP-L protein may
be for hours, days or weeks as required to inhibit cell migration
and/or angiogenesis.
[0115] The effect of FKBP-L on cell migration and/or angiogenesis
may, in certain embodiments, be effective for any cells that are
influenced by cell migration and/or angiogensis. Thus, as described
in detail in the Examples herein, full length recombinant FKBP-L
(e.g., SEQ ID NO: 1) exhibits anti-migratory action over a broad
dose range in a variety of models for angiogenesis, including
HMEC-1 wound closure (FIGS. 3, 4, and 6), and HMEC-1 tube formation
(FIG. 7), the mouse sponge assay (FIGS. 8, 9A and 9B), and the
aortic ring explant model (FIG. 10).
[0116] Also, in an embodiment, the effect of FKBP-L on cell
mobility and/or angiogenesis is not due to toxicity of the
compound. Thus, where cells are exposed to recombinant full-length
FKBP-L for up to 48 hours, the may be no indication of toxicity
(FIGS. 11A and 11B).
[0117] There may be a variety of mechanisms by which FKBP-L acts on
the cell. In an embodiment, the mechanism of FKBP-L mediated
inhibition of migration may be directed at the cytoskeleton (FIGS.
12 and 13). For example, in certain embodiments, FKBP-L may lead to
disruption or other changes in the cytoskeletal filaments.
[0118] The anti-angiogenic effects of FKBP-L indicate that FKBP-L
may have antitumorigenic and/or antimetastatic activity. For
example, as shown in FIG. 14, panels A, B, and C, full length
recombinant FKBP-L polypeptide may inhibit tumor cell migration in
a dose-dependent manner, indicating that FKBP-L may be useful as a
therapeutic agent to reduce tumor cell invasion and metastasis of
tumor cells that depend on migration to metastasize. In certain
embodiments, treatment of tumors in vivo with an expression
construct that encodes a full length FKBP-L polypeptide by gene
therapy (FIG. 15) leads to a reduction in tumor growth.
FKBP-L Interaction with Genes Involved in Angiogenesis
[0119] A variety of biochemical pathways may be modulated by
FKBP-L. In certain embodiments, FKPB-L may lead to an increase in
the expression of certain genes associated with angiogenesis and/or
cell migration. For example, in certain embodiments, transfection
with an anti-sense FKPB-L nucleic acid may lead to an increase in
the expression of PI3K, Rho GTPase activating protein-oligophrenia
1, ROCK, Microtubule associated protein 1B, MMP-like 1 protein,
and/or TNF ligand superfamily member 1 protein (see Example 12
herein). Elevated RhoA, RhoC, ROCK I, and ROCK II expression is
known to be associated with tumor progression and it has been
suggested that Rho and ROCK signalling contribute to the
morphologic changes and metastatic behaviour of some tumor cells.
Thus, in certain embodiments, overexpression of FKBP-L may inhibit
angiogenesis, and FKBP-L repression using antisense
oligonucleotides may promote angiogenesis by activation of genes
associated with angiogenesis, such as Rho and ROCK.
FKBP-L Interaction with CD44
[0120] CD74 is expressed in antigen presenting cells. A primary
function of CD74 is the intracellular sorting of MHC class II
molecules. CD74 is expressed on carcinomas of renal, lung, gastric
and thymic origin and by certain sarcomas. Additionally, CD74 may
be expressed in response to certain tumor genes. For example,
INF-.gamma.-induced CD74 surface expression in breast carcinoma
lines may be enhanced by retinoblastoma protein. Thus, the
restricted expression of CD74 by normal tissues, and its rapid
internalization may make CD74 an attractive therapeutic agent for
both cancer and immunologic disease.
[0121] Macrophage Inhibitory Factor (MIF) may also be involved in
tumorigenesis. High levels of MIF are seen in human tumors and
correlate with grading and prognosis. Moreover MIF may be involved
in angiogenesis, tumor growth and metastasis via a Rho-dependent
pathway (Amin et al., 2006, Blood, 107:2252-2261; Ren et al., 2006,
Oncogene, 25:3501-3508; Sun et al., 2005, Clin. Cancer Res.,
11:1050-1058; Sun et al., 2003, Int. J. Mol. Med., 12:633-641). MIF
signal transduction can be initiated by binding to CD74 (Leng et
al., 2003, J. Exp. Med., 197:1467-1476). It is also thought that
activation of CD74 requires interaction with CD44 (Naujokas et al.,
1993, Cell, 74:257-268; and Naujokas et al., 1995, Immunity,
3:359-372). MIF has been shown to interact in a complex with both
CD74 and CD44 and inhibition of this complex results in decreased
proliferation in bladder cancer cells (Meyer-Siegler et al., 2004,
BMC Cancer, July 12; 4:34; see also Leng et al., 2006, Cell Res.,
16:162-168).
[0122] The formation of a complex between MIF, CD44 and CD74 may be
important for MIF-mediated biological signalling (Shi et al.,
Immunity, 2006, 25(4):595-606).
[0123] In certain embodiments, FKBP-L may act by interacting with
CD44. In an embodiment, FKBP-L may bind to CD44 and prevent CD44
from interacting with CD74. If FKBP-L, or a portion thereof, is
able to displace CD74 from CD44, the FKBP-L polypeptide may prevent
the formation of the complex of CD44-CD74-MIF that is required for
MIF-induced signal transduction. Or, in other embodiments, FKBP-L
may act by alternative mechanisms.
[0124] CD44 is believed to be expressed by most epithelial cells
and has been implicated in angiogenesis (Cao et al., 2006, Am. J.
Pathol., 169:325-336). Thus, in one embodiment, CD44 may be
required for FKBP-L inhibition of endothelial cell migration and/or
angiogenesis. Also, in an embodiment, CD44 can be required for
FKBP-L inhibition of tumor cell migration. Thus, as shown in FIGS.
16 and 17A-17E, full length recombinant FKBP-L, in certain
embodiments, can inhibit tumor cell migration in tumor cell lines
that express CD44 (i.e., CD44 positive or CD44 +ve), but not in
CD44 negative (CD44 -ve) tumor cell lines, suggesting that FKBP-L
may inhibit tumor metastases in a subset of CD44 positive tumor
cell lines. HMEC-1 cells are also positive for CD44 (not shown). In
an embodiment, inactivation of CD44 (e.g., using an siRNA specific
to CD44) results in preventing FKBP-L mediated inhibition of tumor
cell migration (e.g., FIG. 18), demonstrating that CD44 may be
involved in FKBP-L inhibition of tumor cell migration and/or
metastasis.
[0125] In an embodiment, FKBP-L may interact directly with CD44.
For example, exogenously overexpressed FKBP-L (e.g., SEQ ID NO: 1
generated from SEQ ID NO: 31) may interact with endogenous CD44 in
wounded monolayers (FIG. 19; Example 16). In an embodiment, there
is no significant interaction between endogenous FKBP-L and CD44 in
non-wounded monolayers, suggesting that a critical level of FKBP-L
needs to be expressed before the interaction with CD44 can be
detected. Furthermore, this interaction may only occur in
endothelial cells that are primed for migration (i.e. in wounded
monolayers).
[0126] Thus, in embodiments, full length FKBP-L is active against
CD44 positive microvascular endothelial cells (FIG. 16) and
therefore can target these cells within solid tumors to prevent
further microvessel outgrowth to support tumour growth. As such,
FKBP-L may target the vasculature rather than a specific tumor
type, and may be active against a majority, if not all, solid
tumours and micrometastases. Also, as discussed in more detail
below, FKBP-L peptides display similar activity. For example, amino
acids 34-57 of FKBP-L (i.e., the FKBP-L "24mer"), amino acids 1-57
of FKBP-L (i.e., the FKBP-L "1-57mer") and other FKBP-L peptides
from the N-terminus of FKBP-L protein may inhibit migration of
tumor cells that express CD44. Thus, FKBPL polypeptide and its
derivatives can inhibit endothelial cell migration and/or tumor
cell migration with implications for angiogenesis and invasion in a
manner that is consistant with FKBP-L interacting with CD44.
Fragments of FKBP-L
[0127] Embodiments of the present invention recognize that certain
regions of the N-terminus of the FKBP-L protein may display
biological activity. Thus, in certain embodiments, expression
constructs that express full length wild-type (WT) FKBP-L, or, in
alternate embodiments, truncated mutants, such as but not limited
to .DELTA.48, .DELTA.58, .DELTA.86, .DELTA.151, .DELTA.200 may
inhibit wound closure (FIG. 20). The amino acid sequence of each of
these constructs is shown in FIG. 1. For example, in certain
embodiments, WT-FKBP-L and .DELTA.58 inhibited wound closure by
36.2% and 48.8% respectively. There may be a minimum amount of
sequence that is required for activity. For example, in certain
embodiments, truncated FKBP-L .DELTA.34 may fail to significantly
inhibit wound closure, suggesting that the active domain is deleted
in this mutant. These experiments may therefore indicate that the
active domain resides between amino acids 34 and 57 of full-length
(e.g., SEQ ID NO: 2) FKBP-L.
[0128] Thus, as shown in FIG. 20A-20C, in certain embodiments, the
domain important for its anti-angiogenic activity may be located
between amino acids 34 to 57 (i.e. in the N-terminus) of
FKBP-L.
[0129] In certain embodiments, the portion of FKBP-L between amino
acids 34 and 57 exhibits the same biological activity as
full-length FKBP-L. In some embodiments, the FKBP-L 24mer may
display increased potency as compared to the full-length FKBP-L.
For example, the FKBP-L 24mer peptide (SEQ ID NO: 10) may exhibit
similar or more potent biological activity as compared to
full-length recombinant FKBP-L (e.g., SEQ ID NO: 1) with respect to
inhibition of endothelial cell migration/wound closure (FIG. 21),
the inhibition of the formation of endothelial cell-to-cell
contacts in the Matrigel tube formation assay (FIG. 22), angiogenic
sprouting (FIGS. 23A, 23B, 24A and 24B), the ability of cells to
invade (FIG. 25), and/or the ability of cells to adhere (FIG. 26).
In certain embodiments, however, the FKBP-L 24mer and the FKBP-L
1-57mer display increased potency as compared to full length FKBP-L
(see e.g., FIGS. 21, 22 and 24).
[0130] In certain embodiments, the biological activity of FKBP-L
can require CD44. For example, the FKBP-L 24mer peptide (SEQ ID NO:
10) may act in a similar manner to full-length recombinant FKBP-L
(rFKBP-L), and inhibit MDA-231 and PC3 tumor cell migration. These
tumor cells are both CD44 positive (CD44 +ve) (i.e., express CD44
protein) (FIGS. 27A and 27B) indicating that the FKBP-L 24mer may
be able to inhibit tumor metastases in a subset of CD44 +ve tumor
cell lines. In an embodiment, FKBP-L and its derivatives can
inhibit tumor cell migration and invasion and endothelial cell
migration in a manner that is consistant with FKBP-L interacting
with CD44.
[0131] Also in certain embodiments, the FKBP-L 24mer peptide (SEQ
ID NO: 10) is an angiostatic inhibitor (FIGS. 28A and 28B). Thus,
the FKPB-L 24mer may inhibit vessel development when the vessels
are either mature or freshly embedded. However, in an embodiment,
the FKBP-L polypeptide may act by a static mechanism in that it
stops vessel development when added, but has little to no residual
effect when removed.
[0132] Also, in certain embodiments, the FKBP-L 24mer inhibits
angiogenesis in vivo using the mouse sponge assay (FIG. 29) and
also inhibits mouse endothelial cell migration in vitro (FIG. 30)
over a broad dose range, demonstrating that this human peptide is
also active in mouse. This is supported by the data provided in
FIGS. 29 and 31.
[0133] Similar to the full length FKBP-L protein, the FKBP-L 24mer
peptide (SEQ ID NO: 10) may, in certain embodiments, inhibit tumor
cell growth in vivo (FIG. 31A). Also, mice treated with the FKBP-L
24mer showed significantly increased survival (FIG. 31 B-D). Thus,
as shown in FIG. 31A, treatment by i.p. injection with the 24mer
FKBP-L peptide at doses of either 0.3 mg/kg/day or
3.times.10.sup.-3 mg/kg/day significantly slowed the growth of
DU145 tumors in SCID mice compared to vehicle only treated tumors.
In an embodiment, tumors treated with these doses of 24mer FKBP-L
peptide show evidence of a necrotic center as is typical of the
effects seen with anti-angiogenics.
[0134] In an embodiment, the activity of the FKBP-L 24mer peptide,
like the full-length FKBP-L, is not due to toxicity of the peptide
(FIGS. 31E, 32 and 33).
[0135] In certain embodiments, portions or fragments of the FKBP-L
24mer peptide (SEQ ID NO: 10) may be used as therapeutic agents.
Example 29 (FIG. 34) provides examples of peptide fragments of the
FKBP-L 24mer that may have similar activity and potency as the
FKBP-L 24, FKBP-L 1-57, and full length FKBP-L.
FKBP-L Derivatives
[0136] As described above, a FKBP-L derivative for use in the
invention means a polypeptide modified by varying the amino acid
sequence of FKBP-L, e.g. SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID
NO:29, or a fragment thereof, or a polypeptide at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or
such peptides that have be modified by the addition of a functional
group (e.g., PEG). Generation of such peptides may be performed by
manipulation of the nucleic acid encoding the polypeptide or by
altering the protein itself.
[0137] In SEQ ID NO: 2, the FKBP-L insert(originally cloned into
PUC18 by Cambridge Bioscience and now cloned into pcDNA3.1); had
two inserted point mutations compared to the sequence that is
deposited on the PUBMED database (SEQ ID NO: 29). There is a point
mutation at 540 bp (from start codon): TCT to ACT which therefore
converts a serine (S) to a Threonine (T) (amino acid: 181). There
is also a point mutation at 555 bp (from start codon): AGG to GGG
which therefore converts an Arginine (R) to a Glycine (G) (amino
acid: 186). Both FKBP-L polypeptides (SEQ ID NO: 2 and SEQ ID NO:
29) display biological activity.
[0138] FKBP-L derivatives include analogues of the natural FKBP-L
amino acid sequence and may involve insertion, addition, deletion
and/or substitution of one or more amino acids, while providing a
polypeptide capable of effecting similar angiogenic effects to the
portions corresponding to the truncated mutants, .DELTA.48 (SEQ ID
NO:7), .DELTA.58 (SEQ ID NO:6), .DELTA.86 (SEQ ID NO: 5),
.DELTA.151 (SEQ ID NO:4), or .DELTA.200 (SEQ ID NO:3) (FIG. 1).
Also included in the FKBP-L derivatives of the present invention
are polypeptides derived from .DELTA.58 (SEQ ID NO:6), including
the FKBP-L 24 mer (SEQ ID NO 10) and peptides 1-17 (SEQ ID NOs:
12-28) shown in FIG. 1.
[0139] Thus, in certain embodiments, the N-terminal domain (amino
acids 34-57) of FKBP-L is important for the anti-angiogenic
properties. FIG. 20C and Example 17 shows a study in which various
FKBP-L fragments where compared for effectiveness in inhibiting
migration of cells as compared to time-matched negative controls.
In an embodiment, the .DELTA.58 fragment displays maximum
inhibitory activity of the tested fragments.
[0140] The portion of the FKBP-L polpeptide providing inhibition of
angiogenesis may be found in the polypeptide comprising the portion
of FKBP-L in common to active peptides .DELTA.48 (SEQ ID NO:7) and
.DELTA.58 (SEQ ID NO:6). This polypeptide may comprise SEQ ID NO:
10 (FIG. 1).
[0141] Thus, FKBP-L derivatives used in the methods and
compositions of the present invention also include fragments,
portions or mutants of the naturally occurring FKBP-L. In certain
embodiments, the fragments are selected from the N-terminal domain
of FKBP-L. In certain embodiment, the fragments are selected from
amino acids 1 to 85 of full-length FKBP-L (e.g., SEQ ID NOs: 2 or
29). Preferably such analogues involve the insertion, addition,
deletion and/or substitution of 5 or fewer amino acids, more
preferably of 4 or fewer, even more preferably of 3 or fewer, most
preferably of 1 or 2 amino acids only.
[0142] FKBP-L derivatives according to the invention also include
multimeric peptides including such FKBP-L polypeptide, analogue or
fragment sequences e.g. SEQ ID NOs: 1-7, SEQ ID NO: 10-28, and
prodrugs including such sequences. For example, in certain
embodiments FKBP-L or fragments of FKBP-L may form multimers by the
formation of disulfide bonds between monomers.
[0143] Derivatives of the FKBP-L polypeptide of the invention may
include the polypeptide linked to a coupling partner, e.g., an
effector molecule, a label, a drug, a toxin and/or a carrier or
transport molecule. Techniques for coupling the polypeptides of the
invention to both peptidyl and non-peptidyl coupling partners are
well known in the art.
[0144] A "fragment" of a FKBP-L polypeptide means a stretch of
amino acid residues of at least 6 amino acids.
[0145] FKBP-L derivatives of the invention include fusion peptides.
For example, derivatives may comprise polypeptide peptides of the
invention linked, for example, to antibodies that target the
peptides to diseased tissue, for example, tumor tissue or the
retina.
[0146] The FKBP-L polypeptide or their analogues may be fused with
the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or
portions thereof (CH1, CH2, CH3, or any combination thereof),
resulting in chimeric polypeptides. These fusion polypeptides or
proteins can facilitate purification and may show an increased
half-life in vivo. Such fusion proteins may be more efficient in
binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995).
[0147] Fusion proteins of the invention also include FKBP-L
polypeptides fused with albumin, for example recombinant human
serum albumin or fragments or variants thereof (see, e.g., U.S.
Pat. No. 5,876,969, EP Patent 0413622 and U.S. Pat. No.
5,766,883).
[0148] The use of polynucleotides encoding such fusion proteins
described herein is also encompassed by the invention. The use of a
polynucleotide fused to a cytotoxic agent is also encompassed by
the invention. In this instance the FKBP-L polypeptide may bind to
a receptor and the cytotoxic drug could be internalised.
[0149] For example, in alternate embodiments, derivatives may
include: site-specific PEGylation (or the like) of peptide to
increase half life; or incorporation unnatural amino acids and back
bone modifications to stabilize against proteolysism; or cyclic
derivatives (to provide proteolytic resistance); or to block the N-
and C-termini to prevent or reduce exopeptidase and/or proteinase
activity; or to join together multiple copies of peptides either in
a contiguous chain via linkers chain or in a dendrimer type of
approach to increase `avidity` with cell surface CD44. For example,
trimeric covalently linked derivatives of 24mer may be used as
derivatives of FKBP-L. Or, the FKBP-L 24mer may be attached to a
domain which homotrimerises to form non-covalent trimers. Or,
biotin derivatives of peptides which will form tetrameric complexes
with streptavidin may be used as derivatives of FKBP-L. Or, FKBP-L
or fragments of FKBP-L may form multimers by the formation of
disulfde bonds between monomers. In addition, FKBP-L may form
oligomers through non-covalent associations, possibly through the
predicted tetratricopeptide repeat domains within the protein
sequence.
Reverse Peptide Analogues
[0150] Analogues for use in the present invention further include
reverse- or retro-analogues of natural FKBP-L proteins, portion
thereof or their synthetic derivatives. See, for example, EP 0497
366, U.S. Pat. No. 5,519,115, and Merrifield et al., 1995, PNAS,
92:3449-53, the disclosures of which are herein incorporated by
reference. As described in EP 0497 366, reverse peptides are
produced by reversing the amino acid sequence of a naturally
occurring or synthetic peptide. Such reverse-peptides may retain
the same general three-dimensional structure (e. g., alpha-helix)
as the parent peptide except for the conformation around internal
protease-sensitive sites and the characteristics of the N- and
C-termini. Reverse peptides are purported not only to retain the
biological activity of the non-reversed "normal" peptide but may
possess enhanced properties, including increased biological
activity. (See Iwahori et al., 1997, Biol. Pharm. Bull. 20:
267-70). Derivatives for use in the present invention may therefore
comprise reverse peptides of natural and synthetic FKBP-L
proteins.
[0151] Peptides (including reverse peptides and fragments of
either) for use in the invention may be generated wholly or partly
by chemical synthesis or by expression from nucleic acid. The
peptides for use in the present invention can be readily prepared
according to well-established, standard liquid or, preferably,
solid-phase peptide synthesis methods known in the art (see, for
example, J. M. Stewart and J. D. Young, Solid Phase Peptide
Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill.
(1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide
Synthesis, Springer Verlag, New York (1984).
Multimeric Peptides
[0152] As described above, the peptides may be in the form of
multimers. Thus multimers of 2, 3 or more individual FKBP-L
polypeptide monomeric units, or two or more fragments of FKBP-L,
are within the scope of the invention.
[0153] In one embodiment, such multimers may be used to prepare a
monomeric peptide by preparing a multimeric peptide that includes
the monomeric unit, and a cleavable site (i.e., an enzymatically
cleavable site), and then cleaving the multimer to yield a desired
monomer.
[0154] In one embodiment, the use of multimers can increase the
binding affinity for a receptor.
[0155] The multimers can be homomers or heteromers. As used herein,
the term homomer, refers to a multimer containing only polypeptides
corresponding to a specific amino acid sequence (e.g., SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 29), or variants,
splice variants, fusion proteins, or other FKBP-L analogues or
derivatives described herein. These homomers may contain FKBP-L
peptides having identical or different amino acid sequences. For
example, the multimers can include only FKBP-L peptides having an
identical amino acid sequence, or can include different amino acid
sequences. The multimer can be a homodimer (e.g., containing only
FKBP-L peptides, these in turn may have identical or different
amino acid sequences), homotrimer or homotetramer.
[0156] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e., non-FKBP-L
peptide or polypeptides) in addition to the FKBP-L (poly)peptides
described herein.
[0157] The multimers may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked,
by for example, liposome formation. Thus, in one embodiment,
multimers are formed when the FKBP-L peptides described herein
contact one another in solution. In another embodiment,
heteromultimers are formed when FKBP-L and non-FKBP-L
(poly)peptides contact antibodies to the (poly)peptides described
herein (including antibodies to the heterologous (poly)peptide
sequence in a fusion protein described herein) in solution. In
other embodiments, multimers described herein may be formed by
covalent associations with and/or between the FKBP-L peptides (and
optionally non-FKBP-L peptides) described herein.
[0158] Such covalent associations can involve one or more amino
acid residues contained in the FKBP-L sequence (e.g., that recited
in SEQ ID NOs: 1-28. In one embodiment, the covalent associations
are the consequence of chemical or recombinant manipulation.
Alternatively, such covalent associations can involve one or more
amino acid residues contained in the heterologous polypeptide
sequence in a FKBP-L fusion protein. In one example, covalent
associations are between the heterologous sequence contained in a
fusion protein described herein (see, e.g., U.S. Pat. No.
5,478,925). In another specific example, covalent associations of
fusion proteins described herein are using heterologous
polypeptides sequence from another protein that is capable of
forming covalently associated multimers, for example,
oesteoprotegerin (see, e.g., International Publication NO: WO
98/49305). In another embodiment, two or more polypeptides
described herein are joined through peptide linkers. Examples
include those peptide linkers described in U.S. Pat. No. 5,073,627.
Proteins comprising multiple FKBP-L peptides separated by peptide
linkers can be produced using conventional recombinant DNA
technology.
[0159] Multimers may also be prepared by fusing the FKBP-L
(poly)peptides to a leucine zipper or isoleucine zipper polypeptide
sequence. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
multimeric proteins described herein are those described in PCT
application WO 94/10308. Recombinant fusion proteins comprising a
polypeptide described herein fused to a polypeptide sequence that
dimerizes or trimerizes in solution can be expressed in suitable
host cells, and the resulting soluble multimeric fusion protein can
be recovered from the culture supernatant using techniques known in
the art.
[0160] The multimers may also be generated using chemical
techniques known in the art. For example, polypeptides to be
contained in the multimers described herein may be chemically
cross-linked using linker molecules and linker molecule length
optimisation techniques known in the art (see, e.g., U.S. Pat. No.
5,478,925). Additionally, the multimers can be generated using
techniques known in the art to form one or more inter-molecule
cross-links between the cysteine residues located within the
sequence of the polypeptides desired to be contained in the
multimer (see, e.g., U.S. Pat. No. 5,478,925). Further,
polypeptides described herein may be routinely modified by the
addition of cysteine or biotin to the C-terminus or N-terminus of
the polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925). Additionally,
techniques known in the art can be used to prepare liposomes
containing two or more C-12-C peptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925).
[0161] Alternatively, those multimers including only
naturally-occurring amino acids can be formed using genetic
engineering techniques known in the art. Alternatively, those that
include post-translational or other modifications can be prepared
by a combination of recombinant techniques and chemical
modifications. In one embodiment, the FKBP-L peptides are produced
recombinantly using fusion protein technology described herein or
otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety). For
example, polynucleotides coding for a homodimer described herein
can be generated by ligating a polynucleotide sequence encoding a
FKBP-L peptide described herein to sequence encoding a linker
polypeptide and then further to a synthetic polynucleotide encoding
the translated product of the polypeptide in the reverse
orientation from the original C-terminus to the N-terminus (lacking
the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925). The
recombinant techniques described herein or otherwise known in the
art can be applied to generate recombinant FKBP-L (poly)peptides
that contain a transmembrane domain (or hydrophobic or signal
peptide) and that can be incorporated by membrane reconstitution
techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925).
Pro-Drugs
[0162] The polypeptides described herein are intended, at least in
some embodiments, to be administered to a human or other mammal to
treat or prevent a disorder associated with angiogenesis. Peptides
are typically administered parenterally, e.g., by intravenous,
subcutaneous, or intramuscular injection, or via the intranasal
cavity, and may be readily metabolized by plasma proteases. In some
cases the FKBP-L peptide may be delivered in microcapsules of
poly(DL-lactide-co-glycolide)--controlled release over 30 days.
[0163] Various prodrugs have been developed that enable parenteral
and oral administration of therapeutic peptides. Peptides or
polypeptides can be conjugated to various moieties, such as
polymeric moieties, to modify the physiochemical properties of the
peptide drugs, for example, to increase resistance to acidic and
enzymatic degradation and to enhance penetration of such drugs
across mucosal membranes. For example, Abuchowski and Davis have
described various methods for derivatizating enzymes to provide
water-soluble, non-immunogenic, in vivo stabilized products
("Soluble polymers-Enzyme adducts," Enzymes as Drugs, Eds.
Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y.
(1981)).
[0164] Thus, in certain embodiments, the FKBP-L peptides may be
conjugated to polymers, such as dextrans, polyvinyl pyrrolidones,
glycopeptides, polyethylene glycol and polyamino acids. The
resulting conjugated polypeptides retain their biological
activities and solubility in water for parenteral applications. In
an embodiment, the FKBP-L peptides may be coupled to polyethylene
glycol or polypropropylene glycol having a molecular weight of 500
to 20,000 Daltons to provide a physiologically active
non-immunogenic water soluble polypeptide composition (see e.g.,
U.S. Pat. No. 4,179,337 and Garman, A. J., and Kalindjian, S. B.,
FEBS Lett., 1987, 223, 361-365). The polyethylene glycol or
polypropylene glycol may protect the polypeptide from loss of
activity and the composition can be injected into the mammalian
circulatory system with substantially no immunogenic response. In
other embodiments, the FKBP-L is coupled to an oligomer that
includes lipophilic and hydrophilic moieties (see e.g., U.S. Pat.
Nos. 5,681,811, 5,438,040 and 5,359,030).
[0165] Prodrugs can be prepared for example, by first preparing a
maleic anhydride reagent from polydispersed MPEG5000 and then
conjugating this reagent to the polypeptides disclosed herein. The
reaction of amino acids with maleic anhydrides is well known. The
hydrolysis of the maleyl-amide bond to reform the amine-containing
drug is aided by the presence of the neighbouring free carboxyl
group and the geometry of attack set up by the double bond. The
peptides can be released (by hydrolysis of the prodrugs) under
physiological conditions.
[0166] The polypeptides can also be coupled to polymers, such as
polydispersed PEG, via a degradable linkage, for example, the
degradable linkage shown (with respect to pegylated interferon
.alpha.-2b) in Roberts, M. J., et al., Adv. Drug Delivery Rev.,
2002, 54, 459-476.
[0167] The polypeptides can also be linked to polymers such as PEG
using 1,6 or 1,4 benzyl elimination (BE) strategies (see, for
example, Lee, S., et al., Bioconjugate Chem., (2001), 12, 163-169;
Greenwald, R. B., et al., U.S. Pat. No. 6,180,095, 2001; Greenwald,
R. B., et al., J. Med. Chem., 1999, 42, 3657-3667.); the use of
trimethyl lock lactonization (TML) (Greenwald, R. B., et al., J.
Med. Chem., 2000, 43, 475-487); the coupling of PEG carboxylic acid
to a hydroxy-terminated carboxylic acid linker (Roberts, M. J., J.
Pharm. Sci., 1998, 87(11), 1440-1445), and PEG prodrugs involving
families of MPEG phenyl ethers and MPEG benzamides linked to an
amine-containing drug via an aryl carbamate (Roberts, M. J., et
al., Adv. Drug Delivery Rev., 2002, 54, 459-476), including a
prodrug structure involving a meta relationship between the
carbamate and the PEG amide or ether (U.S. Pat. No. 6,413,507 to
Bently, et al.); and prodrugs involving a reduction mechanism as
opposed to a hydrolysis mechanism (Zalipsky, S., et al.,
Bioconjugate Chem., 1999, 10(5), 703-707).
[0168] The FKBP-L polypeptides of the present invention have free
amino, amido, hydroxy and/or carboxylic groups, and these
functional groups can be used to convert the peptides into
prodrugs. Prodrugs include compounds wherein an amino acid residue,
or a polypeptide chain of two or more (e.g., two, three or four)
amino acid residues which are covalently joined through peptide
bonds to free amino, hydroxy or carboxylic acid groups of various
polymers, for example, polyalkylene glycols such as polyethylene
glycol.
[0169] Prodrugs also include compounds wherein PEG, carbonates,
carbamates, amides and alkyl esters which are covalently bonded to
the above peptides through the C-terminal carboxylic acids. For
example, Peptide 1 as used herein is FKBP-L peptide having
C-terminal PEG groups. Thus, embodiments of the present invention
comprise site-specific PEG addition.
[0170] In certain embodiments, enzyme inhibitors may be used to
slow the rate of degradation of proteins and peptides in the
gastrointestinal tract. Or, the pH in the digestive tract may be
manipulated to inactivate local digestive enzymes. Or, permeation
enhancers may be used to improve the absorption of peptides by
increasing their paracellular and transcellular transports. Or,
nanoparticles may be used as particulate carriers to facilitate
intact absorption by the intestinal epithelium, especially, Peyer's
patches, and to increase resistance to enzyme degradation. In other
embodiments, liquid emulsions may be used to protect the drug from
chemical and enzymatic breakdown in the intestinal lumen, or
micelle formulations may be used for poorly water-solubilised
drugs.
[0171] Thus, in alternate embodiments, the polypeptides can be
provided in a suitable capsule or tablet with an enteric coating,
so that the peptide is not released in the stomach. Alternatively,
or additionally, the polypeptide can be provided as a prodrug, such
as the prodrugs described above. In one embodiment, the
polypeptides are present in these drug delivery devices as
prodrugs.
[0172] Prodrugs comprising the polypeptides of the invention or
pro-drugs from which peptides of the invention (including analogues
and fragments) are released or are releaseable are considered to be
analogues of the invention.
[0173] Use of isotopically-labelled peptides or peptide prodrugs
are also encompassed by the invention. Such peptides or peptide
prodrugs are identical to the peptides or peptide prodrugs of the
invention, but for the fact that one or more atoms are replaced by
an atom having an atomic mass or mass number different from the
atomic mass or mass number usually found in nature. Examples of
isotopes that can be incorporated into compounds of the invention
include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorous, sulfur, fluorine, iodine and chlorine, such as
.sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
.sup.125I, and .sup.35S, respectively. Polypeptides of the present
invention, prodrugs thereof, and/or the prodrugs which contain the
aforementioned isotopes and/or other isotopes of other atoms are
within the scope of this invention. Certain isotopically-labelled
compounds of the present invention, for example those into which
radioactive isotopes such as .sup.3H and .sup.14C are incorporated,
are useful in drug and/or substrate tissue distribution assays.
Tritiated, i.e., .sup.3H, and carbon-14, i.e., .sup.14C, isotopes
are particularly preferred for their ease of preparation and
detectability. Further, substitution with heavier isotopes such as
deuterium, i.e., .sup.2H, can afford certain therapeutic advantages
resulting from greater metabolic stability, for example increased
in vivo half-life or reduced dosage requirements and, hence, may be
preferred in some circumstances. Isotopically-labelled peptides and
prodrugs thereof can generally be prepared by carrying out readily
known procedures, including substituting a readily available
isotopically-labelled reagent for a non-isotopically-labelled
reagent, e.g., a labelled amino acid.
Nucleic Acids
[0174] Peptides for use in the present invention may be produced by
use of nucleic acid in an expression system. For example, in one
aspect, nucleic acids which may be used in the invention include
any isolated polynucleotide encoding the polypeptides of the
invention. In a preferred embodiment, the polynucleotide comprises
any one of the nucleic acid sequences as shown in SEQ ID NOs: 30-39
(FIG. 2). Sequences that encode for additional fragments of FKBP-L,
e.g., SEQ ID NOs: 10-28, may be derived from the full-length
nucleic acid sequence, and include degenerate nucleic acid
sequences, as is known in the art. Examples 1, 2, and 17 provide
descriptions of vectors that may be used to express FKBP-L
polypeptides of the present invention.
[0175] Nucleic acid molecules that encode the FKBP-L polypeptides
for use in the present invention may comprise DNA or RNA. The
nucleic acid constructs may be produced recombinantly,
synthetically, or by any means available to those in the art,
including cloning using standard techniques.
[0176] The nucleic acid molecule may be inserted into any
appropriate vector. A vector comprising a nucleic acid of the
invention forms a further aspect of the present invention. In one
embodiment the vector is an expression vector and the nucleic acid
is operably linked to a control sequence which is capable of
providing expression of the nucleic acid in a host cell. A variety
of vectors may be used. For example, suitable vectors may include
viruses (e. g. vaccinia virus, adenovirus, etc.), baculovirus);
yeast vectors, phage, chromosomes, artificial chromosomes,
plasmids, cosmid DNA and lipososmes, polyplexes, or cells (e.g.
mesenchymal stem cells, macrophages).
[0177] The vectors may be used to introduce the nucleic acids of
the invention into a host cell. A wide variety of host cells may be
used for expression of the nucleic acid of the invention. Suitable
host cells for use in the invention may be prokaryotic or
eukaryotic. They include bacteria, e.g. E. coli, yeast, insect
cells and mammalian cells. Mammalian cell lines which may be used
include but are not limited to, Chinese hamster ovary (CHO) cells,
baby hamster kidney cells, NSO mouse melanoma cells, monkey and
human cell lines and derivatives thereof and many others.
[0178] A host cell strain that modulates the expression of,
modifies, and/or specifically processes the gene product may be
used. Such processing may involve glycosylation, ubiquination,
disulfide bond formation and general post-translational
modification.
[0179] For further details relating to known techniques and
protocols for manipulation of nucleic acid, for example, in
preparation of nucleic acid constructs, mutagenesis, sequencing,
introduction of DNA into cells and gene expression, and analysis of
proteins, see, for example, Current Protocols in Molecular Biology,
2nd ed., Ausubel et al. eds., John Wiley & Sons, 1992 and,
Molecular Cloning: a Laboratory Manual: 3.sup.rd edition Sambrook
et al., Cold Spring Harbor Laboratory Press, 2000.
Pharmaceutical Compositions
[0180] The invention further provides pharmaceutical compositions
comprising a FKBP-L polypeptide (or nucleic acid encoding a FKBP-L
polypeptide). Pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material will depend on the route of administration, which may be,
for example, oral, intravenous, or topical.
[0181] The formulation may be a liquid, for example, a physiologic
salt solution containing non-phosphate buffer at pH 6.8-7.6, or a
lyophilised powder.
Dose
[0182] The compositions are preferably administered to an
individual in a "therapeutically effective amount", this being
sufficient to show benefit to the individual. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is
ultimately within the responsibility and at the discretion of
general practitioners and other medical doctors, and typically
takes account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners.
[0183] In alternate embodiments, a dose range of the FKBPL 24 mer
would be from 30 mg/kg/day to 0.00003 mg/kg/day, or 3 mg/kg/day to
0.0003 mg/kg/day, to 0.3 mg/kg/day to 0.03 mg/kg/day. These doses
are equivalent to 10.sup.-5M to 10.sup.-12M, or 10.sup.-6M to
10-11M, or 10.sup.-7M-10.sup.-1.degree. M in vitro,
respectively.
Administration
[0184] A. FKBP-L Peptides
[0185] Polypeptides of and for use in the present invention may be
administered alone but will preferably be administered as a
pharmaceutical composition, which will generally comprise a
suitable pharmaceutical excipient, diluent or carrier selected
dependent on the intended route of administration.
[0186] The polypeptides may be administered to a patient in need of
treatment via any suitable route. The precise dose will depend upon
a number of factors, including the precise nature of the
peptide.
[0187] Some suitable routes of administration include (but are not
limited to) oral, rectal, nasal, topical (including buccal and
sublingual), subcutaneous, vaginal or parenteral (including
subcutaneous, intramuscular, intravenous, intradermal, intrathecal
and epidural) administration.
[0188] For intravenous, injection, or injection at the site of
affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability. Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilisers, buffers, antioxidants and/or other additives may be
included, as required.
[0189] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may comprise a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally comprise a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0190] The composition may also be administered via microspheres,
liposomes, other microparticulate delivery systems or sustained
release formulations placed in certain tissues including blood.
Suitable examples of sustained release carriers include
semipermeable polymer matrices in the form of shared articles, e.g.
suppositories or microcapsules. Implantable or microcapsular
sustained release matrices include polylactides (U.S. Pat. No.
3,773,919; EP-A-0058481) copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985),
poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate
(Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981, and
Langer, Chem. Tech. 12:98-105, 1982). Liposomes containing the
polypeptides are prepared by well-known methods: DE 3,218, 121A;
Epstein et al, PNAS USA, 82: 3688-3692, 1985; Hwang et al, PNAS
USA, 77: 4030-4034, 1980; EP-A-0052522; E-A-0036676; EP-A-0088046;
EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045
and 4,544,545. Ordinarily, the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is
greater than about 30 mol. percent cholesterol, the selected
proportion being adjusted for the optimal rate of the polypeptide
leakage.
[0191] Examples of the techniques and protocols mentioned above and
other techniques and protocols which may be used in accordance with
the invention can be found in Remington's Pharmaceutical Sciences,
16th edition, Oslo, A. (ed), 1980.
[0192] Also, targeting therapies may be used to deliver the active
agent e.g. polypeptide more specifically, e.g. to neoplastic tissue
or retinal tissue, by the use of targeting systems such as antibody
or cell specific ligands.
[0193] In other embodiments, purified recombinant or synthetic
peptides can be treated with agents to attach mioties to the
protein that can facilitate crosslinking. These moieties can be
photoactivatable crosslinkers such as benzophenone or chemical
crosslinkers such as maleimide or activated esters. Thus for
example, it is possible to react cysteine residues in FKBPL with
maleimide derivatives of benzophenone, or maleimide derivatives of
phenyl azide for photoactivatable crosslinking or with
heterobifunctional cross-linking agents containing maleimide and an
activated ester for example. As is known in the art, there are a
variety of hetero and homo bifunctional crosslinkers that could be
attached to FKBPL and then used to crosslink to other biomolecules
through amide, thioether, hydrazone, oxime etc forming reactions.
In an embodiment, it is possible to introduce these cross-linking
agents into synthetic peptides in a site-specific manner using
total chemical synthesis procedures. Alternatively, photactivatable
groups may be introduced specifically at the C-terminus, or
crosslinking agents may be introduced into recombinant FKBPL in a
specific fashion using protein ligation approaches.
[0194] The FKBP-L peptide may also be administered with additional
therapeutic agents as described in more detail herein.
[0195] B. Nucleic Acids Encoding FKBP-L or Anti-Sense/siRNA
FKBP-L
[0196] In an embodiment, the coding sequence of a FKBP-L
polypeptide or an nucleic acid is inserted into an expression
vector. A regulatory sequence comprising a promoter that is
operable in the host cell of interest may then be linked to cDNA
sequence using molecular techniques. Other regulatory sequences can
also be used, such as one or more of an enhancer sequence, an
intron with functional splice donor and acceptance sites, a signal
sequence for directing secretion of the recombinant polypeptide, a
polyadenylation sequence, other transcription terminator sequences,
and a sequence homologous to the host cell genome. Other sequences,
such as an origin of replication, can be added to the vector as
well to optimize expression of the desired product. Also, a
selectable marker may be included in the vector for selection of
the presence thereof in the transformed host cells.
[0197] The regulatory sequences may be derived from various
sources. For example, one or more of them can be normally
associated with the coding sequence, or may be derived from, or
homologous with, regulator systems present in the host cell of
interest. The various components of the expression vector can be
linked together directly or via linkers that constitute sites of
recognition by restriction enzymes as is known in the art.
[0198] Any promoter that would allow expression of the nucleic acid
that encodes for FKBP-L polypeptide can be used in the present
invention. For example, mammalian promoter sequences that can be
used are those from mammalian viruses that are highly expressed and
that have a broad host range.
[0199] The promoter may be a promoter that is expressed
constitutively in most mammalian cells. Examples of suitable
elements which make possible constitutive expression in eukaryotes
are promoters which are recognized by the RNA polymerase III or
viral promoters, CMV enhancer, CMV promoter, SV40 promoter or LTR
promoters, e.g. from MMTV (mouse mammary tumor virus (e.g., Lee et
al., 1981, Nature, 214, 228-232) and other viral promoter and
activator sequences, derived from, for example, HBV, HCV, HSV, HPV,
EBV, HTLV or HIV. Other examples of elements which make possible
regulated expression in eukaryotes are the tetracycline operator in
combination with a corresponding repressor (Gossen M., et al.,
1994, Curr. Opin. Biotechnol., 5, 516-20). In an embodiment, the
expression of the FKBP-L sequence may takes place under the control
of tissue-specific promoters.
[0200] Alternatively, the promoter may be a promoter that is turned
on at a particular time in the cell cycle or developmental phase.
For example, the constructs may comprise regulatable elements which
make possible tissue-specific expression in eukaryotes, such as
promoters or activator sequences from promoters or enhancers of
those genes which code for proteins which are only expressed in
certain cell types. Examples of regulatable elements which make
possible cell cycle-specific expression in eukaryotes are promoters
of the following genes: cdc25A, cdc25B, cdc25C, cyclin A, cyclin E,
cdc2, E2F-1 to E2F-5, B-myb or DHFR (see e.g., U.S. Pat. No.
6,856,185; U.S. Pat. No. 6,903,078; and Zwicker J. and Muller R.,
1997, Trends Genet., 13, 3-6). The use of cell cycle regulated
promoters may be used where expression of the polypeptides or
nucleic acids used according to the invention is to be restricted
to proliferating cells. Other examples include promoters controlled
by hypoxia, radiation, heat, or the like.
[0201] In another embodiment, an enhancer element can be combined
with a promoter sequence. Such enhancers may not only amplify, but
also can regulate expression of the gene of interest. Suitable
enhancer elements for use in mammalian expression systems are, for
example, those derived from viruses that have a broad host range,
such as the SV40 early gene enhancer, the enhancer/promoters
derived from the LTR of the Rous Sarcoma Virus, and from human
cytomegalovirus. Additionally, other suitable enhancers include
those that can be incorporated into promoter sequences that will
become active only in the presence of an inducer, such as a
hormone, a metal ion, or an enzyme substrate, as is known in the
art.
[0202] In another embodiment of the present invention, a
transcription termination sequence may be placed 3' to the
translation stop codon of the coding sequence for the gene of
interest. Thus, the terminator sequence, together with the
promoter, would flank the coding sequence.
[0203] The expression vector may also contain an origin of
replication such that the vector can be maintained as a replicon,
capable of autonomous replication and stable maintenance in a host.
Such an origin of replication includes those that enable an
expression vector to be reproduced at a high copy number in the
presence of the appropriate proteins within the cell, for example,
the 2p and autonomously replicating sequences that are effective in
yeast, and the origin of replication of the SV40 vital T-antigen,
that is effective in COS-7 cells. Mammalian replication systems may
include those derived from animal viruses that require trans-acting
factors to replicate. For example, the replication system of
papovaviruses, such as SV40, the polyomavirus that replicate to
extremely high copy number in the presence of the appropriate vital
T antigen may be used, or those derived from bovine papillomavirus
and Epstein-Barr virus may be used.
[0204] In some cases, the expression vector can have more than one
replication system, thus, allowing it to be maintained, for
example, in mammalian cells for expression and in a procaryotic
host for cloning and amplification (see e.g., U.S. Pat. No.
5,677,278).
[0205] In one embodiment, the expression vector can be made to
integrate into the host cell genome as an integrating vector. The
integrating vector herein may contain at least one polynucleotide
sequence that is homologous to the host cell genome that allows the
vector to integrate. For example, in one embodiment, bacteriophage
or transposon insertion sequences may be used.
[0206] In certain embodiments of the present invention, one or more
selectable markers can be included in the expression vector to
allow for the selection of the host cells that have been
transformed. Selectable markers that can be expressed in a host
cell include genes that can render the host cell resistant to drugs
such as tunicamycin, G418, ampicillin, chloramphenicol,
erythromycin, kanamycin (neomycin), and tetracycline. Selectable
markers also include biosynthetic genes, such as those in the
histidine, tryptophan, and leucine biosynthetic pathways, such as
ade2, his4, leu2, trp1, or that provide the host cells with the
ability to grow in the presence of toxic compounds, such as a
metal, may be used.
[0207] A variety of methods may be used to transfer a
polynucleotide encoding for FKBP-L polypeptide and/or a nucleic
acid encoding FKBP-L anti-sense DNA or FKBP-L siRNA into host
cells. Thus, the formulations of the present invention may comprise
specific components that facilitate transfer of nucleic acids into
cells.
[0208] For example, to allow for the introduction of nucleic acids
in a eukaryotic and/or prokaryotic cell by transfection,
transformation or infection, the nucleic acid can be present as a
plasmid, as part of a viral or non-viral vector. Suitable viral
vectors may include baculoviruses, vaccinia viruses, lentiviruses
(see e.g., Siprashvili and Khavari, Mol. Ther., 2004, 9, 93-100),
adenoviruses, adeno-associated viruses and herpesviruses. Examples
of vectors having gene therapy activity are virus vectors, for
example adenovirus vectors or retroviral vectors (Lindemann et al.,
1997, Mol. Med., 3, 466-76; Springer et al., 1998, Mol. Cell., 2,
549-58). Also, eukaryotic expression vectors are suitable in
isolated form for gene therapy use as naked DNA can penetrate
certain cells(Hengge et al., 1996, J. Clin. Invest., 97, 2911-6; Yu
et al., 1999, J. Invest. Dermatol., 112, 370-5). Another form of
gene therapy vectors can be obtained by applying the above
described nucleic acid to gold particles and shooting these into
tissue, preferably into the skin, or cells with the aid of the
so-called gene gun (Wang et al., 1999, J. Invest. Dermatol., 112,
775-81, Tuting et al., 1998, J. Invest. Dermatol., 111, 183-8).
[0209] In alternate embodiments, liposomes may be used to
facilitate transfer of a polynucleotide encoding FKBP-L into cells.
Liposomes are artificially-made small vesicles with a lipid bilayer
membrane comprised of phospholipids (Jeschke, M. G. et al., Gene
Ther., 12, 1718-24 (2005); U.S. Pat. No. 6,576,618). Nucleic acids,
proteins, and other biological materials can be enclosed in
liposomes for delivery to mammalian cells through fusion with the
cell's plasma membrane. Liposomes may be an attractive delivery
system because they are non-viral, stable and can interact with the
cell membrane.
[0210] Liposomes can be comprised of cationic, anionic, or neutral
lipids, and mixtures thereof (Luo, D. & Saltzman, W. M., Nat.
Biotech., 18, 33-37 (1999)). For DNA transfer, the lipids can also
be modified chemically to incorporate chemical groups to facilitate
DNA condensation or release. Cationic lipids, such as quaternary
ammonium detergents, cationic derivatives of cholesterol and
diacylglycerol, and lipid derivatives of polyamines, may be favored
for cell transfection because they decrease the net negative charge
of the DNA and facilitate its interaction with cell membranes
(Nishikawa, M. & Huang, L., Hum. Gene Ther., 12, 861-70
(2001)). Neutral lipids, such as dioleoylphosphitylethanolamine
(DOPE), glycerol dilaurate, polyoxyethylene-10-stearyl ether
(POE-10), and cholesterol, may be added as `helper lipids` in
cationic-lipid DNA complexes to facilitate the release of the DNA
from the endosome after endocytic uptake of the complex.
Auxiliaries that increase DNA transfer, such as polymers or
proteins that are bound to the DNA or synthetic peptide-DNA
molecules that make it possible to transport DNA into the nucleus
of the cell more efficiently can also be used (see e.g., Niidome,
T. & Huang, L., Gene Ther., 9, 1647-52 (2002)). Thus, cationic
polymers, such as polylysine or protamine, can be used in lipid-DNA
complexes as they cause tight condensation of DNA, which prevents
complex aggregation and nuclease degradation. For example, mixing
1,2-dioleoyl-3-(trimethylammonium)propane) (DOTAP) liposomes with
protamine sulfate prior to mixing with plasmid DNA produced small
135 nm particles that were stable and resulted in a high level of
gene expression in a variety of tissues (e.g., lung., liver, heart)
(Li, S. et al., Gene Ther., 5, 930-37 (1998)). Inclusion of
cholesterol as a helper lipid may increase the transfection
efficiency of liposome-peptide-DNA complexes. Also, luciferase or
.beta.-galactosidase gene DNA may be precompacted with short
peptides derived from human histone or protamine before addition of
a cationic lipid (Lipofectamine RPR 115335 or RPR 120535) or
polymer (polyethylenimine) to achieve enhanced transfection
efficiency, even in the presence of serum (see e.g., Schwartz, B.
et al., Gene Ther., 6, 282-92 (1999)).
[0211] As is known in the art, liposomes may be made by heating
lipids to form a lipid phase (Wu, H. et al., Int. J. Pharmaceut.,
221, 23-24 (2001)). An aqueous phase containing water, salts or
buffer may then be mixed with the lipid phase by passing the
mixture back and forth between syringes under cooling conditions,
followed by sonication until a final liposome size of 100 to 140 nm
is reached. The DNA or protein to be included in the liposome is
then added (as a solution) by inversion mixing. The choice of
lipids used, their ratio, the concentration of DNA used in creating
the liposomes and the amount of liposomes added will generally
require empirical determination for optimization. Auxiliaries to
facilitate DNA transfer, such as peptides, can be mixed with the
DNA prior to adding to the liposome mixture but the DNA-auxiliary
must maintain sufficiently high aqueous solubility to be properly
encapsulated within the external lipid phase of the liposome.
[0212] Alternatively, small unilamellar vesicles can be prepared by
ultrasonic treatment of a liposome suspension comprised of cationic
lipids, such as Cytofectin GS 2888, mixed with
1,2-dioleyloxypropyl-3-trimethylammonium bromide (DOTMA) or
dioleoylphosphati-dylethanolamine bromide (DPOE). After inversion
mixing, the DNA or protein may be bound ionically to the surface of
the liposomes, in a ratio that maintains a positive net charge on
the complex while having DNA complexed to 100% of the liposomes.
Also, dimerizable cationic thiol detergents may be used to prepare
liposomes for delivery of DNA (see e.g., Dauty, E. et al., J. Am.
Chem. Soc., 123, 9227-34 (2001)). Upon oxidation, the thiol groups
in the lipid can convert to disulfides and cause the DNA-lipid
complex to form a stable nanometric particle that can bind
electrostatically to cell surface anionic heparin sulfate
proteoglycans for cellular uptake. Once inside the cell, the
reductive environment provided by intracellular glutathione reduces
the disulfides back to thiols and releases the DNA.
Therapeutic Antibodies
[0213] In another embodiment the invention relates to therapeutic
use of an antibody having immunological specificity for FKBP-L (or
fragments or functional equivalents thereof, as discussed below) to
specifically down-regulate the activity of FKBP-L in vivo. Such
antibodies are useful in the treatment of disease conditions which
benefit from specific down-regulation of FKBP-L activity, in
particular diseases/conditions which benefit from
stimulation/up-regulation of angiogenesis. In specific embodiments
the invention encompasses use of an antibody having immunological
specificity for FKBP-L (or a fragment or functional equivalent
thereof) to promote angiogenesis. An embodiment relates to use of
an antibody having immunological specificity for FKBP-L (or a
fragment or functional equivalent thereof) to promote wound
healing.
[0214] The term "antibody" as used herein encompasses purified or
isolated naturally occurring antibodies of any isotype having the
required immunological specificity, as well as synthetically
produced antibodies or structural analogs thereof. Preparations of
antibody can be polyclonal or monoclonal. Reference to such an
"antibody" as described above includes not only complete antibody
molecules, but also fragments thereof which retain substantial
antigen (i.e. FKBP-L) binding capability. It is not necessary for
any effector functions to be retained in such fragments, although
they may be included. Suitable antibody fragments which may be used
include, inter alia, F(ab').sub.2 fragments, scAbs, Fv, scFv
fragments and nanoantibodies etc. Antibody fragments which contain
the idiotype of the molecule can be generated by known techniques,
for example, such fragments include but are not limited to the
F(ab')2 fragment which can be produced by pepsin digestion of the
antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab')2 fragments and the Fab
fragments which can be generated by treating the antibody molecule
with papain and a reducing agent. Other antibody fragments with the
required antigen binding activity can be prepared by recombinant
expression techniques generally known in the art.
[0215] Chimeric humanized and fully humanized monoclonal antibodies
can be made by recombinant engineering. By addition of the human
constant chain to F(ab')2 fragments it is possible to create a
humanized monoclonal antibody which is useful in immunotherapy
applications where patients making antibodies against the mouse Ig
would otherwise be at a disadvantage. Breedveld F. C. Therapeutic
Monoclonal Antibodies. Lancet 2000 Feb. 26; 335, P735-40.
Recombinant therapeutic monoclonal antibodies may be advantageously
prepared by recombinant expression in mammalian host cells (e.g.
CHO cells).
[0216] Monoclonal antibodies with immunological specificity for
FKBP-L can be prepared by immunisation of a suitable host animal
(e.g. mouse or rabbit) with a suitable challenging antigen (e.g.
full length FKBP-L or an epitope thereof).
Therapeutic Uses
[0217] The polypeptides and nucleic acids of and for use in the
invention may be used in the control and/or treatment of a wide
variety of clinical conditions in mammals, including humans. The
polypeptides and methods of the invention may be used in the
treatment of a condition or disorder for which anti-angiogenic or
pro-angiogenic agents may be therapeutically useful.
[0218] As used herein, "treatment" or "therapy" includes any regime
that can benefit a human or non-human animal. The treatment may be
in respect of an existing condition or may be prophylactic
(preventative treatment). Treatment may include curative,
alleviation or prophylactic effects.
[0219] Cell migration, angiogenesis and related indications (e.g.,
tumor growth and/or metastasis) can be inhibited by administering
an effective amount of a FKBP-L polypeptide or a nucleic acid
encoding said peptide to a patient in need of such treatment. The
methods can be used to treat tumors, various autoimmune disorders,
hereditary disorders, ocular disorders and other
angiogenesis-mediated or angiogenesis-associated disorders.
[0220] Alternatively, angiogenesis may be promoted by administering
an antisense FKBP-L nucleic acid (e.g., siRNA) or antibodies to
FKBP-L to a patient in need of such treatment. The methods could be
used to treat wound healing, including that of most tissues such as
skin and bone and the treatment of chronic ulcers (diabetic or
otherwise).
[0221] The therapeutic and diagnostic methods described herein
typically involve administering an effective amount of the
peptides, nucleic acids or compositions including the polypeptide
or nucleic acid of the invention to a patient. The exact dose to be
administered will vary according to the use of the compositions and
on the age, sex and condition of the patient, and can readily be
determined by the treating physician. The compositions may be
administered as a single dose or in a continuous manner over a
period of time. Doses may be repeated as appropriate.
[0222] The compositions and methods can be used to treat
angiogenesis-mediated disorders including haemangioma, solid
tumors, leukemia, lymphoma metastasis, telangiectasia, psoriasis,
endometriosis, arteriosclerosis, scleroderma, pyogenic granuloma,
myocardial angiogenesis, Crohn's disease, plaque
neovascularisation, coronary collaterals, cerebral collaterals,
arteriovenous malformations, ischemic limb angiogenesis, corneal
diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,
retrolental fibroplasia, arthritis, diabetic neovascularisation,
macular degeneration, peptic ulcer, Helicobacter related diseases,
fractures, keloids, and vasculogenesis. Specific disorders that can
be treated, and compounds and compositions useful in these methods,
are described in more detail below.
Carcinomas/Tumors
[0223] Tumors that may be treated include those tumors whose growth
is promoted by angiogenesis. In one embodiment such tumors may
express CD44. Carcinomas that may be treated using the compounds,
compositions and methods of the invention may include colorectal
carcinoma, gastric carcinoma, signet ring type, oesophageal
carcinoma, intestinal type, mucinous type, pancreatic carcinoma,
lung carcinoma, breast carcinoma, renal carcinoma, bladder
carcinoma, prostate carcinoma, testicular carcinoma, ovarian
carcinoma, endometrial carcinoma, thyroid carcinoma, liver
carcinoma, larynx carcinoma, mesothelioma, neuroendocrine
carcinomas, neuroectodermal tumors, melanoma, gliomas,
neuroblastomas, sarcomas, leiomyosarcoma, MFII, fibrosarcoma,
liposarcoma, MPNT, and chondrosarcoma.
[0224] For treatment of cancer, FKBP-L may be administered with
other chemotherapeutic and/or chemopreventative agents known in the
art. Such agents may include, but are not limited to
antiangiogenics, endostatin, angiostatin and VEGF inhibitors,
thalidomide, and others, or cytotoxic drugs such as adriamycin,
daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids,
such as vincristine, farnesyl transferase inhibitors, and
antimetabolites such as methotrexate. In alternate embodiments,
FKBP-L peptides or polynucleotides encoding FKBP-L polypeptides may
be used with cancer therapeutics such as the following: (a) cancer
growth inhibitors including, but not limited to bortezomib,
erlotinib, gefitinib, imatinib and sorafenib; (b) gene therapy
approaches, e.g., using nucleic acid constructs that encode tumor
suppressor gene or siRNAs to oncogenes; (c) cancer vaccines; (d)
interferon; (e) Aldesleukin; (f) monoclonal antibodies including,
but not limited to 90Y-Ibritumomab tiuxetan, ADEPT, Alemtuzumab,
Bevacizumab, Cetuximab, Gemtuzumab, Iodine 131 tositumomab,
Panitumumab, Rituximab, Trastuzumab; (g) chemotherapy drugs
including, but not limited to Amsacrine, Bleomycin, Busulfan,
Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,
Cladribine, Crisantaspase, Cyclophosphamide, Cytarabine,
Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin,
Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,
Gliadel implants, Hydroxycarbamide, Idarubicin, Ifosfamide,
Irinotecan, Leucovorin, Liposomal doxorubicin, Liposomal
daunorubicin, Lomustine, Melphalan, Mercaptopurine, Mesna,
Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel,
Pemetrexed, Pentostatin, Procarbazine, Raltitrexed, Streptozocin,
Tegafur-uracil, Temozolomide, Teniposide, Thiotepa, Tioguanine,
Topotecan, Treosulfan, Vinblastine, Vincristine, Vindesine, and
Vinorelbine; (h) radiotherapy; (i) hormonal therapies including,
but not limited to Anastrozole, Bicalutamide, Buserelin,
Cyproterone, Diethylstilbestrol, Exemestane, Flutamide,
Fulvestrant, Goserelin (Breast), Goserelin (Prostate), Letrozole,
Leuprorelin, Medroxyprogesterone, Megestrol acetate, Tamoxifen,
Toremifene, and Triptorelin; (j) supportive therapies including,
but not limited to bisphosphonates, blood transfusions,
Erythropoietin, haematopoietic, growth factors, plasma exchange,
platelet transfusions and steroids; and (k) other treatments
including, but not limited to hyperbaric oxygen therapy,
hyperthermia treatment, and photodynamic therapy. Such therapies
may be used with FKBP-L treatment either alone or as complementary
therapies.
Ocular Disorders Mediated by Angiogenesis
[0225] Various ocular disorders are mediated by angiogenesis, and
may be treated using the active compounds, compositions and methods
described herein. One example of a disease mediated by angiogenesis
is ocular neovascular disease, which is characterized by invasion
of new blood vessels into the structures of the eye and is the most
common cause of blindness. In age-related macular degeneration, the
associated visual problems are caused by an ingrowth of chorioidal
capillaries through defects in Bruch's membrane with proliferation
of fibrovascular tissue beneath the retinal pigment epithelium. In
the most severe form of age-related macular degeneration (known as
"wet" ARMD) abnormal angiogenesis occurs under the retina resulting
in irreversible loss of vision. The loss of vision is due to
scarring of the retina secondary to the bleeding from the new blood
vessels. Current treatments for "wet" ARMD utilize laser based
therapy to destroy offending blood vessels. However, this treatment
is not ideal since the laser can permanently scar the overlying
retina and the offending blood vessels often re-grow. An
alternative treatment strategy for macular degeneration is the use
of anti-angiogenesis agents to inhibit the new blood vessel
formation or angiogenesis which causes the most severe visual loss
from macular degeneration.
[0226] Angiogenic damage is also associated with diabetic
retinopathy, retinopathy of prematurity, corneal graft rejection,
neovascular glaucoma and retrolental fibroplasia. Other diseases
associated with corneal neovascularisation include, but are not
limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,
atopic keratitis, superior limbic keratitis, pterygium keratitis
sicca, periphigoid radial keratotomy, and corneal graph rejection.
Diseases associated with retinal/choroidal neovascularization
include, but are not limited to, diabetic retinopathy, macular
degeneration, presumed myopia, optic pits, chronic retinal
detachment, hyperviscosity syndromes, trauma and post-laser
complications. Other diseases include, but are not limited to,
diseases associated with rubeosis (neovascularization of the angle)
and diseases caused by the abnormal proliferation of fibrovascular
or fibrous tissue including all forms of proliferative
vitreoretinopathy.
[0227] Thus, in certain embodiments of the invention, the active
compounds, compositions and methods of the invention may be used in
the treatment of angiogenesis-mediated ocular disorders, for
example, macular degeneration.
Inflammation
[0228] The FKBP-L polypeptides may also be used to treat
angiogenesis-mediated disorders, such as angiogenesis-associated
inflammation, including various forms of arthritis, such as
rheumatoid arthritis and osteoarthritis. In these methods,
treatment with combinations of the compounds described herein with
other agents useful for treating the disorders, such as
cyclooxygenase-2 (COX-2) inhibitors, which are well known to those
of skill in the art.
[0229] The blood vessels in the synovial lining of the joints can
undergo angiogenesis. The endothelial cells form new vascular
networks and release factors and reactive oxygen species that lead
to pannus growth and cartilage destruction. These factors are
believed to actively contribute to rheumatoid arthritis and also to
osteoarthritis. Chondrocyte activation by angiogenic-related
factors contributes to joint destruction, and also promotes new
bone formation. The methods described herein can be used as a
therapeutic intervention to prevent bone destruction and new bone
formation.
[0230] Pathological angiogenesis is also believed to be involved
with chronic inflammation. Examples of disorders that can be
treated using the compounds, compositions and methods described
herein include ulcerative colitis, Crohn's disease, bartonellosis,
and atherosclerosis.
Combination Therapies
[0231] In treating a specific disease using a polypeptide, nucleic
acid or method of the invention, in the treatment of a specific
disease, the peptides or nucleic acids may be combined with various
existing therapeutic agents used for that disease.
[0232] The combination of FKBP-L polypeptides as described herein
with an anti-histamine (H.sub.1 antagonist) can be particularly
favoured for use in the prophylaxis and treatment of asthma and
rhinitis. Examples of anti-histamines are chlorpheniramine,
brompheniramine, clemastine, ketotifen, azatadine, loratadine,
terfenadine, cetirizine, astemizole, tazifylline, levocabastine,
diphenhydramine, temelastine, etolotifen, acrivastine, azelastine,
ebastine, mequitazine, KA-398, FK-613, mizolastine, MDL-103896,
levocetirizine, mometasone furoate, DF-1111301, KC-11404,
carebastine, ramatroban, desloratadine, noberastine, selenotifen,
alinastine, E-4716, efletirizine, tritoqualine, norastemizole,
ZCR-2060, WY-49051, KAA-276, VUF-K-9015, tagorizine, KC-11425,
epinastine, MDL-28163 terfenadine, HSR-609, acrivastine and
BMY-25368.
[0233] Additionally or alternatively, the polypeptides of the
invention may advantageously be employed in combination with one or
more other therapeutic agents, including an antibiotic,
anti-fungal, anti-viral, anti-histamine, non-steroidal
anti-inflammatory drug or disease modifying anti-rheumatic
drug.
[0234] In other embodiments, for treating rheumatoid arthritis, the
FKBP-L polypeptides may be combined with agents such as TNF-alpha
inhibitors, for example, anti-TNF monoclonal antibodies (such as
Remicade, CDP-870 and D.sub.2 E.sub.7) and TNF receptor
immunoglobulin molecules (such as Enbrel.RTM.), COX-2 inhibitors
(such as meloxicam, celecoxib, rofecoxib, valdecoxib and
etoricoxib) low dose methotrexate, leflunomide, hydroxychloroquine,
d-penicillamine, auranofin or parenteral or oral gold.
[0235] In yet other embodiments, the FKBP-L polypeptides may also
be used in combination with existing therapeutic agents for the
treatment of osteoarthritis. Suitable agents to be used in
combination include standard non-steroidal anti-inflammatory agents
(hereinafter NSAID's) such as piroxicam, diclofenac, propionic
acids such as naproxen, flubiprofen, fenoprofen, ketoprofen and
ibuprofen, fenamates such as mefenamic acid, indomethacin,
sulindac, apazone, pyrazolones such as phenylbutazone, salicylates
such as aspirin, COX-2 inhibitors such as celecoxib, valdecoxib,
rofecoxib and etoricoxib, analgesics and intraarticular therapies
such as corticosteroids and hyaluronic acids such as hyalgan and
synvisc.
[0236] The FKBP-L polypeptides may also be used in combination with
anticancer agents such as antiangiogenics, endostatin, angiostatin
and VEGF inhibitors and others, or cytotoxic drugs such as
adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere
and alkaloids, such as vincristine, farnesyl transferase
inhibitors, and antimetabolites such as methotrexate. Other
anti-cancer agents and therapeutic methods such as a cancer growth
inhibitor, gene therapy, a cancer vaccine, interferon, Aldesleukin,
a monoclonal antibody, a chemotherapy drug, radiotherapy, hormonal
therapy or other supportive therapies that may be used with FKBP-L
are described herein.
[0237] Additionally or alternatively, the FKBP-L polypeptides may
also be used in combination with antiviral agents such as Viracept,
AZT, aciclovir and famciclovir, and antisepsis compounds such as
Zovant, tifacogin, NOX-100 and 13R270773.
[0238] The FKBP-L polypeptides may also be used in combination with
anti-osteoporosis agents such as roloxifene, droloxifene,
lasofoxifene or fosomax and immunosuppressant agents such as FK-506
and rapamycin.
[0239] The FKBP-L polypeptides may also be combined with one or
more of the following: (a) leukotriene biosynthesis inhibitors:
5-lipoxygenase (5-LO) inhibitors and 5-lipoxygenase activating
protein (FLAP) antagonists selected from the group consisting of
zileuton; ABT-761; fenleuton; tepoxalin; Abbott-79175;
Abbott-85761; N-(5-substituted)-thiophene-2alkylsulfonamides,
2,6-di-tert-butylphenol hydrazones; the class of
methoxytetrahydropyrans which includes Zeneca ZD-2138; the compound
SB-210661 and the class to which it belongs; the class of
pyridinyl-substituted 2-cyanonaphthalene compounds to which
L-739,010 belongs; the class of 2-cyanoquinoline compounds to which
L-746,530 belongs; the classes of indole and quinoline compounds to
which MK-591, MK-886, and BAY X 1005 belong; (b) receptor
antagonists for leukotrienes LTB.sub.4, LTC.sub.4, LTD.sub.4, and
LTE.sub.4 selected from the group consisting of the
phenothiazin-3-one class of compounds to which L-651,392 belongs;
the class of amidino compounds to which CGS-25019c belongs; the
class of benzoxaolamines to which ontazolast belongs; the class of
benzenacarboximidamides to which BIIL 2841260 belongs; and the
classes of compounds to which zafirlukast, ablukast, montelukast,
praniukast, verlukast (MK-679), RG-12525, Ro-2459913, iralukast
(CGP 45715A), and BAY X 7195 belong; (c) PDE4 inhibitors including
inhibitors of the isoform PDE4D; (d) 5-Lipoxygenase (5-LO)
inhibitors; or 5-lipoxygenase activating protein (FLAP)
antagonists; (e) dual inhibitors of 5-lipoxygenase (5-LO) and
antagonists of platelet activating factor (PAF); (f) leukotriene
antagonists (LTRAs) including antagonists of LTB.sub.4, LTC.sub.4,
LTD.sub.4, and LTE.sub.4; (g) antihistaminic H.sub.1 receptor
antagonists including cetirizine, loratadine, desloratadine,
fexofenadine, astemizole, azelastine, and chlorpheniramine; (h)
gastroprotective H.sub.2 receptor antagonists; (i) alpha.sub.1- and
alpha.sub.2-adrenoceptor agonist vasoconstrictor sympathomimetic
agents administered orally or topically for decongestant use,
including propylhexedrine, phenylephrine, phenylpropanolamine,
pseudoephedrine, naphazoline hydrochloride, oxymetazoline
hydrochloride, tetrahydrozoline hydrochloride, xylometazoline
hydrochloride, and ethylnorepinephrine hydrochloride; (j)
alpha.sub.1- and alpha.sub.2-adrenoceptor agonists in combination
with inhibitors of 5-lipoxygenase (5-LO); (k) anticholinergic
agents including ipratropium bromide; tiotropium bromide;
oxitropium bromide; pirenzepine; and telenzepine; (I) [3- to
beta.sub.4-adrenoceptor agonists including metaproterenol,
isoproterenol, isoprenaline, albuterol, salbutamol, formoterol,
salmeterol, terbutaline, orciprenaline, bitolterol mesylate, and
pirbuterol; (m) methylxanthanines including theophylline and
aminophylline; (n) sodium cromoglycate; (o) muscarinic receptor
(M1, M2, and M3) antagonists; (p) COX-1 inhibitors (NTHEs); COX-2
selective inhibitors including rofecoxib; and nitric oxide NTHEs;
(q) insulin-like growth factor type I (IGF-1) mimetics; (r)
ciclesonide; (s) inhaled glucocorticoids with reduced systemic side
effects, including prednisone, prednisolone, flunisolide,
triamcinolone acetonide, beclomethasone dipropionate, budesonide,
fluticasone propionate, and mometasone furoate; (t) tryptase
inhibitors; (u) platelet activating factor (PAF) antagonists; (v)
monoclonal antibodies active against endogenous inflammatory
entities; (w) IPL 576; (x) anti-tumor necrosis factor (TNF-alpha)
agents including Etanercept, Infliximab, and D2E7; (y) DMARDs
including Leflunomide; (z) TCR peptides; (aa) interleukin
converting enzyme (ICE) inhibitors; (bb) IMPDH inhibitors; (cc)
adhesion molecule inhibitors including VLA-4 antagonists; (dd)
cathepsins; (ee) MAP kinase inhibitors; (ff) glucose-6 phosphate
dehydrogenase inhibitors; (hh) gold in the form of an aurothio
group together with various hydrophilic groups; (ii)
immunosuppressive agents, e.g., cyclosporine, azathioprine, and
methotrexate; (jj) anti-gout agents, e.g., colchicine; (kk)
xanthine oxidase inhibitors, e.g., allopurinol; (ll) uricosuric
agents, e.g., probenecid, sulfinpyrazone, and benzbromarone; (mm)
antineoplastic agents, especially antimitotic drugs including the
vinca alkaloids such as vinblastine and vincristine; (nn) growth
hormone secretagogues; (oo) inhibitors of matrix metalloproteases
(MMPs), i.e., the stromelysins, the collagenases, and the
gelatinases, as well as aggrecanase; especially collagenase-1
(MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13),
stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3
(MMP-11); (pp) transforming growth factor (TGFP); (qq)
platelet-derived growth factor (PDGF); (rr) fibroblast growth
factor, e.g., basic fibroblast growth factor (bFGF); (ss)
granulocyte macrophage colony stimulating factor (GM-CSF); (tt)
capsaicin cream; (uu) Tachykinin NK, and NK.sub.3 receptor
antagonists selected from the group consisting of NKP-608C;
SB-233412 (talnetant); and D-4418; and (vv) elastase inhibitors
selected from the group consisting of UT-77 and ZD-0892.
Wound Healing
[0240] Angiogenesis is an important step in wound healing. Use of
antisense and/or siRNA and/or inhibitory antibodies to the FKBP-L
polypeptide of the invention as described hereinbefore may be used
either on its own or in combination with other therapies to promote
wound healing.
[0241] Thus, embodiments of the invention also encompasses
combinations of at least one of the FKBP-L compound described
herein with at least one other agent useful for treating wounds.
Such agents can be selected amongst bioactive compounds involved in
wound healing such as growth factors, cytokines inhibitors,
proteases and adhesion molecules which are well known to those of
skill in the art and described for example in Kumar et al. Turk J
Med Sci, 34 (2004) 147-160. For example suitable growth factors can
be chosen in group consisting of TGFP and its isoforms, PDGF, KGF,
VEGF and EGF which are factors known for their importance in wound
healing. FKBP-L polypeptides and derivatives can also be associated
with matrix metalloproteases or adhesion molecules like the
immunoglobulin-like superfamilly, the cadherins, the integrins, the
receptor protein tyrosine phosphatases, the selectins and the
hyaluronate receptors.
[0242] Alternatively or in combination with any of the wound
healing compositions described above, other agents known to promote
wound healing such as disinfectants, antibiotics and the like may
also be used with the compounds of the invention.
[0243] In certain embodiments, anti-sense oligonucleotides, as
described in more detail herein, may be used in the methods and
compositions for wound healing.
[0244] Also, anti-sense FKBP-L oligonucleotides, FKBP-L siRNAs or
antibodies to FKBP-L may be applied alone or in combination with
the above active ingredients may be applied topically as a powder
or as a solution or dispersion and use for the manufacture of a
wide variety of dressings. Such dressings may be classical
dressings such as cotton or cellulosic fibres and deposited as a
coating or coatings on base materials such as cellulose or
cellulose acetate or nylon or regenerated cellulose, or plastic
base materials, either woven or non-woven in sheet form, perforated
or imperforate. The antibodies to FKBP-L polypeptide may be bonded
to a suitable base material, e.g., cotton gauze, plastic sheet,
etc, using an appropriate adhesive formulation, e.g., pectin,
gelatin, starch, innocuous vegetable gums, etc according to known
procedures like that disclosed in U.S. Pat. No. 3,194,732.
Alternatively the FKBP-L antibodies of the invention can be
associated to more elaborate types of wounds dressings like
moisture-retaining and semi-occlusive dressings which promote a
moist environment beneficial to wound healing.
Anti-Sense and siRNA Oligonucleotide Therapeutics
[0245] A. Antisense RNA
[0246] As described above, the present invention may comprise an
antisense nucleic acid molecule or an antisense oligonucleotide as
therapeutic agents. In an embodiment, the antisense oligonucleotide
may comprise an inhibitor RNA (e.g., RNAi or siRNA).
[0247] Antisense oligonucleotides are short fragments of DNA or RNA
that have complementary sequences to a portion of, or to all of, an
mRNA. Being complementary to a particular target mRNA, antisense
oligonucleotides bind specifically to that mRNA. It is known to
chemically modify such antisense molecules to facilitate tight
binding. When binding occurs, the ability of the mRNA to be read by
the cell's translational machinery is inhibited, and synthesis of
the protein that it encodes is blocked. Unlike a gene knockout,
this inhibition may require the continuous presence of the
antisense molecule; thus, it is reversible and portion can design
specific inhibitors of a gene of interest based only on knowledge
of the gene sequence. In one embodiment, the invention provides an
isolated nucleic acid molecule which is antisense to the coding
strand of a nucleic acid of the invention. In yet another
embodiment, it is provided a nucleic acid molecule having a
nucleotide sequence that is antisense to the coding strand of an
mRNA encoding a polypeptide of the invention.
[0248] The antisense nucleic acid can be complementary to an entire
coding strand, or to only a portion thereof, e.g., all or part of
the protein coding region (or open reading frame). An antisense
nucleic acid molecule can be antisense to all or part of a
non-coding region of the coding strand of a nucleotide sequence
encoding a polypeptide of the invention. The non-coding regions
("5' and 3' untranslated regions") are the 5' and 3' sequences
which flank the coding region and are not translated into amino
acids.
[0249] An antisense oligonucleotide can be, for example, about 5,
10, 15, 18, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in
length. An antisense nucleic acid of the invention can be
constructed using chemical synthesis and 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. Examples of modified nucleotides which 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-thiouridine,
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-thiouracil,
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-predicted N-2-carboxyp 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). The antisense
nucleic acid molecules of the invention can typically be
administered to a patient. Alternatively it could be generated in
situ such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a selected polypeptide of the invention to
thereby inhibit expression, e.g., by inhibiting transcription
and/or translation.
[0250] 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 which 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 which bind to cell surface receptors or antigens. To
achieve sufficient intracellular concentrations of the 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 preferred.
[0251] Oligonucleotides containing natural sugars (D-ribose and
D-2-deoxyribose) and phosphodiester (PO) linkages are rapidly
degraded by serum and intracellular nucleases, which limits their
utility as effective therapeutic agents. Chemical strategies to
improve nuclease stability include modification of the sugar
moiety, the base moiety, and/or modification or replacement of the
internucleotide phosphodiester linkage. To date, the most widely
studied analogues are the phosphorothioate (PS)
oligodeoxynucleotides, in which one of the non-bridging oxygen
atoms in the phos-phodiester backbone is replaced with a sulfur
(Eckstein, F. Ann. Rev. Biochem. 1985, 54, 367). An exemplary
antisense targeting FKBP-L suitable for use in the methods of the
invention is described by Robson et al. (See Robson et al., (1999)
Radiation Research 152, 451-461; Robson, T., et al., (2000) Int. J.
Radiat).
[0252] B. siRNAs
[0253] In certain embodiments, siRNAs to FKBP-L may be used as
therapeutic agents. Small interfering RNAs (siRNAs) are powerful
tools for directed post-transcriptional gene expression knockdown
in mammalian cells (Elbashir et al., Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells. Nature. 2001,
411: 494-8).
[0254] siRNAs typically comprise a double-stranded target-specific
region which corresponds to the target gene to be down-regulated
(i.e. FKBP-L). This double-stranded target-specific region
typically has a length in the range of from 19 to 25 base pairs. In
specific, non-limiting embodiments, siRNAs having a double-stranded
target-specific region of 19, 20, 21, 22, 23, 24 or 25 base pairs
corresponding to the target gene to be down-regulated (FKBP-L) may
be used.
[0255] The target-specific region typically has a sequence 100%
complementary to a portion of the target gene (FKBP-L). However, it
will be appreciated that 100% sequence identity is not essential
for functional RNA inhibition. RNA sequences with insertions,
deletions, and single point mutations relative to the target
sequence have also been found to be effective for RNA inhibition.
The term "corresponding to", when used to refer to sequence
correspondence between the target-specific part of the siRNA and
the target region of the target gene (FKBP-L), is therefore to be
interpreted accordingly as not absolutely requiring 100% sequence
identity. However, the % sequence identity between the
double-stranded RNA and the target region will generally be at
least 90%, or at least 95% or at least 99%.
[0256] Therefore, in embodiments of the invention, siRNAs capable
of specifically down-regulating expression of FKBP-L may include a
double-stranded portion which comprises or consists of 19, 20, 21,
22, 23, 24 or 25 consecutive base pairs of the nucleotide sequence
shown in SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:29, or a
double-stranded portion of 19, 20, 21, 22, 23, 24 or 25 consecutive
bases which is at least 90%, or at least 95%, or at least 99%
identical to a portion of the nucleotide sequence shown in SEQ ID
NO:1, SEQ ID NO:2, or SEQ ID NO:29, or which include one or two
single nucleotide mismatches in comparison to a portion of the
nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2.
[0257] The siRNA can be designed to target any suitable region of
the FKBP-L mRNA transcript. Algorithms are available for siRNA
design, based essentially on the characteristics of the primary
sequence of the siRNA (eg Reynolds A, et al. Nat Biotechnol. 2004
March; 22(3):326-30. Epub 2004 Feb. 1.). An exemplary siRNA
targeting FKBP-L suitable for use in the methods of the invention
is described by Jascur et al. 2006, Molecular Cell, 17:237-239.
[0258] The term "down-regulation of gene expression" refers to a
measurable or observable reduction in gene expression or a complete
abolition of detectable gene expression, at the level of protein
product and/or mRNA product from the target gene (e.g. FKBP-L).
Down-regulation of gene expression is "specific" when
down-regulation of the target gene (e.g. FKBP-L) occurs without
manifest effects on other genes.
[0259] siRNAs may include single-stranded overhangs at one or both
ends, flanking the double-stranded target-specific region
corresponding to FKBP-L. In a particular embodiment, the siRNA may
contain 3' overhanging nucleotides, such as two 3' overhanging
thymidines (dTdT) or uridines (UU). 3' TT or UU overhangs may be
included in the siRNA if the sequence of the target gene
immediately upstream of the sequence included in double-stranded
part of the dsRNA is AA. This allows the TT or UU overhang in the
siRNA to hybridise to the target gene. Although a 3' TT or UU
overhang may also be included at the other end of the siRNA it is
not essential for the target sequence downstream of the sequence
included in double-stranded part of the siRNA to have AA.
[0260] The double-stranded target-specific portion of the siRNA is
typically formed from two annealed RNA strands comprised entirely
of ribonucleotides in phosphodiester linkage. However, siRNAs which
are RNA/DNA chimeras are also contemplated. These chimeras include,
for example, the siRNAs comprising a double-stranded RNA with 3'
overhangs of DNA bases (e.g. dTdT), as discussed above, and also
double-stranded "RNAs" which are polynucleotides in which one or
more of the RNA bases or ribonucleotides, or even all of the
ribonucleotides on an entire strand, are replaced with DNA bases or
deoxynucleotides. In other embodiments the backbone of the "RNA"
strands in the siRNA may be modified, by inclusion of non-natural
nucleobases and/or non-natural backbone linkages (see for example
Soutschek et al. Nature. 2004 Nov. 11; 432(7014):173-8; Zimmermann
T S, et al. Nature 441, 111-4). By way of example, 2-O-methyl
modifications may be included to stabilised the siRNAs (as
described by Soutschek et al. ibid.).
[0261] The siRNA may be prepared in a manner known per se in the
art. For example, siRNAs may be synthesised in vitro using chemical
or enzymatic polynucleotide synthesis techniques well known in the
art. In one approach the two separate strands of the siRNA may be
synthesised separately and then annealed to form
double-strands.
[0262] Unmodified "exogenous" siRNAs are known to be effective in
gene silencing in vivo without the need for additional reagents
(Filleur S, et al. Cancer Res 63, 3919-22; Duxbury M S, et al.
Oncogene 23, 465-73). In other embodiments, siRNAs can be used in
conjunction with carriers or delivery vehicles such as
atelocollagen (Nozawa H, et al. Cancer Sci. 2006 October;
97(10):1115-24; Takeshita F, et al. Proc Natl Acad Sci USA. 2005
Aug. 23; 102(34):12177-82. Epub 2005 Aug. 9) or nanoparticles
(Schiffelers R M, et al. Nucleic Acids Res. 2004 Nov. 1;
32(19):e149) or lipid-based carriers including, for example, oil-in
water emulsions, micelles, and liposomes which promote uptake.
Delivery vehicles (e.g. liposomes and nanoparticles) may be
targeted to a particular tissue by coupling the vehicle to a
specific ligand, such as a monoclonal antibody, sugar, glycolipid
or protein.
[0263] In a further embodiment, rather than being formed of two
separate RNA strands annealed together, the "siRNA" may have a
foldback stem-loop or hairpin structure, wherein the annealed
sequences forming the target-specific part of the siRNA are
covalently linked. In one embodiment the annealed sequences may be
present on a single RNA strand. RNAs having this structure are
typical if the dsRNA is synthesised by expression in vivo or by in
vitro transcription. The precise nature and sequence of the "loop"
linking the two RNA strands is generally not material to the
invention, except that it should not impair the ability of the
double-stranded part of the molecule to mediate RNAi. The "loop"
structure need not necessarily be formed from nucleic acid.
[0264] In one embodiment, siRNAs (or precursor structures which can
be processed to produce siRNAs, for example by the action of the
endogenous enzyme "dicer") may be synthesised by intracellular
expression in a host cell or organism from a suitable expression
vector.
[0265] A number of non-viral (e.g. plasmid) or viral expression
vector systems for in vivo expression of siRNAs are known in the
art. Generally, siRNAs are expressed as stem-loops, which may be
rapidly processed within the cell to produce the "free" siRNA (see
review by Tuschl, Nature Biotechnology, Vol. 20(5), 446-448, 2002).
Vector systems for expression of siRNAs are often based on RNA Pol
III promoters, since these are particularly suited to accurate
expression of very short RNA sequences. Suitable vector systems are
described in, for example, Brummelkamp, T. R. et al., Science, Vol.
296, 550-553, 2002; Lee, N. S. et al., Nature Biotechnology, Vol.
20, 500-505, 2002; Miyagashi, M & Taira, K. Nature
Biotechnology, Vol. 20, 497-500, 2002; Paul, C. P. et al., Nature
Biotechnology, Vol. 20, 505-508, 2002, the contents of which are
incorporated herein by reference.
[0266] siRNAs may be formulated into pharmaceutical compositions
comprising a therapeutically effective amount of the nucleic acid
in combination with any standard physiologically and/or
pharmaceutically acceptable carriers known in the art.
Targeting
[0267] Targeting therapies may be used to deliver the active agent
e.g. polypeptide more specifically to particular tissues or cells,
by using targeting systems such as antibody or cell specific
ligands. These targeting systems can be covalently linked to the
peptide sequence, or to a drug delivery vehicle (such as a
liposome, microsphere, microparticle, microcapsule and the like).
The polypeptides can also be targeted to growing tumor beds (which
are associated with attached capillary beds) by incorporating the
peptides into microparticles or other drug delivery vehicles that
are suitably sized so that they pass through the veins but lodge in
capillary beds. When lodged in the beds, the polypeptides can be
locally released (rather than systemically released) at a location
where they are most useful. As described above, the present
invention further extends to methods of gene therapy using
nucleotides of the invention.
[0268] In another embodiment, the FKBP-L peptides may be used to
target cytotoxic agents to tumor cells. Thus, in an embodiment, the
FKBP-L peptide may be conjugated to a cytotoxic agent using methods
known in the art. The FKBP-L peptide may then, by interaction with
CD44, deliver the cytotoxic agent to cells that express CD44. Where
the cytotoxic agent is an agent that is able to preferentially
inhibit tumor cell growth, the agent may be active againts CD44 +ve
tumor cells.
Anti- or Pro-Angiogenesis Activity
[0269] Certain embodiments of the present invention may comprise
assessment of angiogenic activity of the compositions of the
invention. Angiogenic activity may be assessed by any means known
in the art or as described herein. For example, angiogenesis
activity may be assayed using any standard assays, such as the
Matrigel assay and the assays used in the Examples.
EXAMPLES
[0270] The invention may be better understood by reference to the
following non-limiting Examples. The designation "N" provides the
number of individual experiments performed for the particular
example.
Example 1
Transient Transfection of FKBP-L Inhibits Wound Closure (N=3)
[0271] Experiments were done to determine the effect of FKBP-L (SEQ
ID NO: 1; FIG. 1) on wound closure. The in vitro migration assay
used in these studies is a modified version of the method described
by Ashton et al (1999) The J. of Biol. Chem., 1999, 274: 50,
35562-35570. Human Microvascular Endothelial Cells (HMEC1) were
plated into individual chambers on a glass slide and grown to 90%
confluence.
[0272] The monolayer was transfected with a FKBP-L/pcDNA mammalian
expression construct having an insert with the nucleotide sequence
of SEQ ID NO: 31, in the presence of lipofectin (Invitrogen, UK).
To make the expression construct, the nucleic acid fragment of SEQ
ID NO: 31 was excised from a recombinant pUC18 construct using
BamH1 and ligated into the BamH1 restriction site of pcDNA3.1
(Invitrogen).
[0273] Expression of the FKBP-L insert generates the full-length
recombinant polypeptide in SEQ ID NO: 2. After 6 hours the
transfection reagents were removed and the monolayer wounded with a
pipette tip and re-supplemented with MCDB-131 and incubated for 7
hours.
[0274] The monolayer was fixed in 4% PBS buffered paraformaldehyde
solution for 10 minutes. The extent of "wound" closure was blindly
assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50). The extent
of closure in the FKBP-L treated slides was compared to the wound
size at time zero.
[0275] The results of these experiments are shown in FIG. 3. It was
found that the transiently transfected FKBP-L produces a peptide
equivalent to SEQ ID NO: 2 and significantly inhibited the ability
of the HMEC-1 to migrate compared to lipofectin only and empty
vector control. FKBP-L inhibits HMEC-1 migration by 50% compared to
controls (Lipo--lipofectin reagients; pcDNA-vector only) at 7 hr
following wounding. This data suggests that the FKBP-L protein is a
potential anti-migratory protein.
Example 2
Full Length Recombinant FKBP-L Protein Inhibits Endothelial Cell
Migration in the Wound Closure Assay (N=3)
[0276] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al (1999).
HMEC-1 were plated into individual chambers on a glass slide and
grown to 90% confluence overnight. The medium was removed and the
monolayer wounded. The monolayer was re-supplemented with fresh
medium and the required volume of recombinant full length
his-tagged FKBP-L protein (SEQ ID NO: 1) was added to give the
required final concentration.
[0277] In order to generate the recombinant full length FKBPL
protein, the FKBPL cDNA (polynucleotide SEQ ID:31; polypeptide
variant variant Thr182, Gly186; SEQ ID NO:1) was subcloned from
pcDNA3.1/FKBPL into the BamHI and PstI sites of the pRSET-A vector
(Invitrogen) and was expressed in BL21 (DE3) to give the
corresponding N-terminal poly-histidine tagged (his-tag) protein
(SEQ ID NO: 1). Expression was induced at OD 0.6 with 0.2 mM IPTG,
growing cells overnight at 15.degree. C. Cells were pelleted by
centrifugation and stored at -20.degree. C. The protein was
purified using standard IMAC purification followed by desalting to
remove any contaminating E. coli proteins (See example 32 for full
description). The expressed recombinant protein has a calculated
molecular weight of 38 kDa; the His-tagged FKBP-L which has a
calculated molecular weight of 42220 Da was found to have a
molecular weight of 42 kDa as ascertained by SDS polyacrylamide gel
electrophoresis (SDS-PAGE).
[0278] The monolayers were incubated for 7 hours after exposure to
recombinant FKBP-L protein and then fixed in 4% PBS buffered
paraformaldehyde. The extent of "wound" closure was blindly
assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50). The extent
of closure in the FKBP-L treated slides was compared to time
matched sham treated controls and the % inhibition of wound closure
compared to time matched controls calculated.
[0279] The results of these experiments are shown in FIG. 4. It can
be seen that treatment with FKBP-L recombinant protein resulted in
a significant inhibition of migration, with an optimum
concentration of 750 ngml.sup.-1 inducing a 60% inhibition of
HMEC-1 migration into the denuded area of the monolayer compared to
time matched controls. The findings from this experiment support
the results observed with transiently transfected FKBP-L (FIG.
3).
[0280] The results also suggest that FKBP-L can inhibit endothelial
cell migration when expressed intercellularly (as in the previous
FIG. 3 using an expression construct) or extracellularly (i.e., by
addition of recombinant protein to the tissue culture medium). This
implied that either FKBP-L is inhibiting endothelial cell migration
by two different mechanisms or that FKBP-L is secreted from the
cell. As shown herein, FKBP-L is indeed secreted.
Example 3
FKBP-L Protein is Secreted from HMEC-1 Cells (N=1)
[0281] Human Microvascular Endothelial Cells (HMEC1) were plated
onto 35 mm plastic culture plates and grown to 100% confluence. The
monolayer was transfected with an haemagglutanin(HA)-tagged
FKBP-L/pcDNA mammalian expression construct in the presence of
lipofectin (Invitrogen, UK). This would result in expression of SEQ
ID NO: 2 with a HA tag.
[0282] In order to generate the HA-tagged FKBPL plasmid, the FKBPL
cDNA (polynucleotide SEQ ID NO:31; polypeptide variant variant
Thr182, Gly186; SEQ ID NO:2) was excised from pUC18 by digestion
with BamH1, blunt ended and directionally cloned into a blunt ended
Sal1 site of pCMV-HA mammalian expression vector (Clontech, U.K.).
This results in expression of SEQ ID NO: 2, with an N-terminal
HA-tag to produce a 44 kDa protein.
[0283] After 6 hours the transfection reagents were removed and the
monolayer wounded (controls were unwounded) with a pipette tip and
re-supplemented with MCDB-131 and incubated for a further 7 hours.
The medium was collected for analysis and the cells were then
washed twice with PBS and harvested into 100 .mu.l of 2.times.
Laemmli buffer (Sigma) and heated to 100.degree. C. for 10 minutes.
Both cell lysates and culture medium were slot blotted onto
nitrocellulose membrane and probed with monoclonal anti HA antibody
(Clontech) (1:1000 dilution) in order to detect the HA-tagged
FKBP-L protein, and then probed with rabbit Ig HRP-linked secondary
antibody (1:7500 dilution) (Amersham Biosciences). Antibody binding
was detected using SuperSignal.RTM. West Pico Chemiluminescent
Substrate detection reagent (Pierce).
[0284] The results are shown in FIG. 5. FIG. 5 is a slot/Western
blot showing that transfection of an HA-tagged FKBP-L cDNA
construct into either normal HMEC-1 monolayers or wounded monlayers
results in secretion, into the medium, of the HA-tagged FKBP-L
protein 24 h after transfection. Western blots were probed with an
HA antibody.
[0285] These data indicate that under normal growth conditions
FKBP-L protein is actively secreted, supporting the hypothesis that
FKBP-L may be mediating its anti-angiogenic effects via receptor
activation. The data also provides an explanation as to why either
recombinant protein or over-expression using a cDNA construct are
both able to exert anti-angiogenic effects observed both in vitro
and in vivo.
Example 4
The Effect of the Full Length Recombinant Protein FKBP-L on the
Wound Closure Assay Measured as a Function of Time (N=3)
[0286] The following studies were performed to determine the effect
of a full length His-tagged recombinant FKBP-L protein on the wound
closure assay measured as a function of time. Again, the in vitro
migration assay used in these studies is a modified version of the
method described by Ashton et al (1999). HMEC-1 were plated into
individual chambers on a glass slide and grown to 90% confluence
overnight. The medium was removed and the monolayer wounded. The
monolayer was re-supplemented with fresh medium and the required
volume of full length his-tagged recombinant FKBP-L (i.e., SEQ ID
NO: 1) added to give the required final concentration 750
ngml.sup.-1.
[0287] Slides were removed at fixed time points until complete
closure of the wound, then fixed in 4% PBS buffered
paraformaldehyde. The extent of "wound" closure was blindly
assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50). The extent
of closure in the FKBP-L treated slides was compared to time
matched sham treated controls and the % inhibition of wound closure
compared to time matched controls calculated.
[0288] The results of these experiments are shown in FIG. 6. It can
be seen that wound closure was overall significantly inhibited in
the FKBP-L treated (750 ngml.sup.-1) HMEC-1 cells compared to
controls. 50% wound closure was observed in the control at 7 hours,
whereas 50% wound closure in the FKBP-L treated monolayer was not
observed until 16 hours after initial wounding, resulting in a
significant delay of 9 hours. Total wound closure was observed at
16 hours in control experiments, in contrast to FKBP-L treated
monolayers which remained open until 34 hours. These results
indicate that the effect of a single administration of FKBP-L may
be an extremely effective method of delaying wound closure in this
in vitro model.
Example 5
The Effect of the Full-Length Recombinant Protein FKBP-L on the
Formation of Endothelial Cell-to-Cell Contacts on the Synthetic
Basement Membrane Matrigel (N=3)
[0289] In this experiment, the effect of the full length His-tagged
recombinant FKBP-L protein (SEQ ID NO: 1) on the formation of
endothelial cell-to-cell contacts was assessed. Samples were run in
triplicate.
[0290] The in vitro tubule formation assay used in these studies is
a modified version of the method described by Ashton et al (1999).
In brief, assays were conducted using BD BioCoat.TM. Matrigel.TM.
Matrix Thin Layer 24-well Multiwell Plates (BD Discovery Labware,
Oxford, UK). The Matrigel.TM. was rehydrated with 500 .mu.l
MCDB-131 serum free medium and incubated at 37.degree. C. for 30
minutes. Excess medium was removed and HMEC-1 were seeded at a
density of 1.times.10.sup.5 and the plates incubated at 37.degree.
C. under 5% CO.sub.2/95% air for 1 hour.
[0291] Increasing concentrations of the recombinant FKBP-L protein
(SEQ ID NO: 1) were added to each individual well in triplicate
(250-1000 ngml.sup.-1) and the plate was incubated for a further 18
hours. The degree of tubule formation between adjacent HMEC-1 cells
was assessed in each well in five fields of view, by counting the
number of cell to cell contacts between different HMEC-1 cells in
the designated area. An independent investigator assessed each well
and the FKBP-L treated wells were compared to sham treated
controls.
[0292] The results are shown in FIG. 7. It was found that
recombinant FKBP-L protein inhibited the ability of the HMEC-1's to
form cell to cell contacts or tubules on Matrigel in a dose
dependent manner. The optimum concentration for this effect was 750
ngml.sup.-1, with an efficacy of 80% and an EC50 potency of 314
ngml.sup.-1. These results indicate that at these doses, FKBP-L is
anti-angiogenic, preventing tube formation by HMEC-1 cells.
Example 6
The Effect of the Full Length Recombinant FKBP-L Polypeptide on
Angiogenesis In Vivo Using the Mouse Sponge Assay (N=1; Two Mice
Per Group)
[0293] This experiment measured the effects of FKBP-L on
angiogenesis using two other in vitro models, the mouse sponge
assay, and the aortic ring model.
[0294] In these experiments, polyether sponges were subcutaneously
implanted in C57 black mice and injected on alternate days with 10
ng bovine fibroblast growth factor (bFGF) or 10 ng bFGF+ 5 .mu.g
full length His-tagged recombinant FKBP-L polypeptide (SEQ ID NO:
1). After 14 days of treatment, sponges were harvested, sectioned
and stained with heamatoxylin and eosin.
[0295] The results are shown in FIGS. 8 and 9. In FIG. 8A,
erythrocytes, which appear as dark gray cells and are indicated by
arrows, can be seen within the microvessels of bFGF treated
sponges. Also, it can be seen that there are large amounts of
cellular ingrowth (appearing as light gray). Both the erythrocytes
and cellular ingrowth are much less obvious in sponges also treated
with FKBP-L (FIG. 8B). Vessel counts in sponges from 2 mice per
group, counted in a blind fashion at 40.times. magnification are
shown in FIG. 9. FKBP-L treated sponges had significantly fewer
vessels than those treated with bFGF alone (p=0.0008).
[0296] The results indicate that the full-length recombinant FKBP-L
polypeptide can inhibit angiogenesis in vivo, and that this
polypeptide may have potential therapeutic value in a clinical
setting.
Example 7
The Effect of Full-Length Recombinant FKBP-L Polypeptide on the
Ex-Vivo Aortic Ring Explant Model of Angiogenesis, Investigating
the Effect on Mean Length, Maximum Length and Number of Vessels
Formed (N=3)
[0297] Male Wistar rats were euthanised and the thoracic aorta was
aseptically removed and sectioned into 1 cm thick rings. The rings
were washed ten times in sterile medium to remove any bacteria and
embedded into Matrigel on 24 well plates. The wells were
supplemented with 2 ml of medium and increasing concentrations of
full-length His-tagged recombinant FKBP-L protein (SEQ ID 1). The
plate was incubated for 8 days and post incubation the Matrigel and
rings were fixed in 4% PBS buffered paraformaldehyde and stored in
PBS. The extent of vessel development was blindly assessed
microscopically by an independent investigator and quantified using
a calibrated eyepiece graticule (1 mm/100 .mu.m graduation) at
20.times. magnification (Olympus BX 50). The extent of vessel
length, maximum vessel length and number of vessels in each field
of view was measured and compared to time matched sham controls and
the percent (%) inhibition calculated.
[0298] The results of these experiments are shown in FIG. 10.
FKBP-L was seen to be a potent dose dependent inhibitor of
angiogenesis in this ex-vivo model. The mean vessel length and
maximum vessel length formed were significantly inhibited at 1000
ngml.sup.-1 exhibiting 63% and 70% inhibition respectively compared
to time matched controls. The number of vessels formed from the
aortic explant was optimally inhibited by 65% following treatment
with FKBL-L protein at 500 ngml.sup.-1.
Example 8
The Effect of the Full Length Recombinant FKBP-L Polypeptide on the
Viability or Proliferation of HMEC-1 Using the MTT Assay (N=3)
[0299] These experiments assessed whether the antiangiogenic
effects of full length FKBP-L protein were due to toxicity of the
polypeptide. An MTT assay was used to measure cell
viability/proliferation. Briefly, HMEC-1 cells were seeded
(2.5.times.10.sup.3) in 96 well plates and allowed to attach for 5
hours. The cells were treated with increasing concentrations of
recombinant His-tagged FKBP-L protein (SEQ ID NO: 1) and incubated
for 24 (FIG. 11A) and 48 hours (FIG. 11B). Post incubation the
cells were exposed to a 5 mgml.sup.-1 solution of
3-(-4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium (MTT) for 4
hours. The cells were aspirated and 200 .mu.l of DMSO added to
reduce the salt and induce a colour change. The wells were analyzed
colorimetrically at 550 nm and the results compared to untreated
control cells. The experiment was repeated three times.
[0300] The results are shown in FIGS. 11A and 11B. It was found
that FKBP-L had no significant effect on the proliferation of
HMEC-1 cells compared to time matched controls at any of the time
points measured, suggesting that the antiangiogenic effects
observed in the previous assays were not caused by inhibition of
cell growth or by FKBP-L-mediated toxicity.
Example 9
Changes in Cytoskeletal Morphology of Migrating Endothelial Cells
on Exposure to 750 Mgml.sup.-1 Full Length Recombinant FKBP-L
Polypeptide (N=2)
[0301] Immunohistochemical analysis was carried out to assess
cytoskeletal morphology upon treatment with FKBP-L by staining for
tubulin and vimentin. HMEC-1 were seeded in four well chamber
slides and incubtated overnight until confluent monolayers had
formed. Media was removed from each well and the monolayer wounded
as previously described. The cells were re-supplemented with medium
containing 750 ngml.sup.-1 recombinant His-tagged FKBP-L protein
(i.e., SEQ ID NO: 1). The cells were incubated for 5 hours and the
chambers were removed from the slides and the cells washed four
times in PBS followed by fixation in 4% PBS buffered
paraformaldehyde treated with 0.1% Triton X for 20 minutes. The
cells were washed three times in PBS, and blocked for 20 minutes in
2% BSA containing 0.1% Triton X. Blocked cells were washed in PBS
and incubated with one of the following monoclonal primary
antibodies: (A) anti a tubulin (1:500); and (B) anti-vimentin
(1:200), for 90 minutes. The cells were washed in PBS followed by a
1 hour incubation with FITC conjugated anti-mouse secondary (1:30)
at room temperature. The cells were mounted with Vectashield
containing propidium iodide and sealed to prevent dehydration. The
slides were covered in tinfoil and stored at 4.degree. C. for
analysis using fluorescence confocal microscopy.
[0302] The results are shown as FIG. 12 (anti-tubulin staining of
cells) and FIG. 13 (anti-vimentin staining of cells). In the
control migrating HMEC-1, the microtubules (stained using anti a
tubulin) (FIG. 12: control) have a regular linear structure running
in the direction of the wound thus helping the process of
directional migration. Dense regions of staining can be observed at
the front of the nucleus, and this microtubule organizational
center (MTOC) is a good indicator that directional migration is
occurring at the time of fixation. In contrast, in the FKBP-L
treated cells (FIG. 12: FKBP-L) the microtubules appear to have
little alignment into the wound. It can be seen that the
microtubules appear slightly tortuous or wispy and aligned from
left to right, the MTOC appears to sit at the side of the cell,
indicating no active directional migration.
[0303] FIG. 13 shows the intermediate filaments of the HMEC-1's
stained using anti-vimentin. The control (untreated) cells again
appear to be organized, elongated and pointing in the direction of
the wound again suggesting the cells are actively migrating (FIG.
13: control). In contrast, the intermediate filaments in the FKBP-L
treated non-migrating HMEC-1's appear highly disorganized, even
clumped and showing no indication that they are actively migrating
into the wound (FIG. 13: FKBP-L).
[0304] The results of this confocal investigation suggest that the
mechanism of FKBP-L mediated inhibition of migration may be
directed at the cytoskeleton.
Example 10
The Effect of Full Length Recombinant FKBP-L on PC3, HT29 and MDA
Tumor Cell Migration (N=3)
[0305] In these experiments, the effect of recombinant FKBP-L on
tumor cell migration was assessed. The in vitro migration assay
used in these studies is a modified version of the method described
by Ashton et al (1999) see supra. PC3, MDA231, and HT29 tumor cells
were plated into individual chambers on a glass slide and grown to
90% confluence overnight. The medium was removed and the monolayers
wounded. The monolayer was re-supplemented with fresh medium and
the required volume of His-tagged recombinant FKBP-L protein (SEQ
ID NO: 1) was added to give the final concentrations shown. The
monolayers were incubated for 24 hours and then fixed in 4% PBS
buffered paraformaldehyde.
[0306] The extent of "wound" closure was blindly assessed
microscopically by an independent investigator and quantified using
a calibrated eyepiece graticule (1 mm/100 .mu.m graduation) at
20.times. magnification (Olympus BX 50). The extent of closure in
the FKBP-L treated slides was compared to time matched sham-treated
controls and the percent inhibition of wound closure compared to
time matched controls calculated.
[0307] The results are shown in FIG. 14, panels A, B, and C. It was
found that recombinant FKBP-L polypeptide inhibits tumor cell
migration in a dose-dependent manner. These finding indicate that
FKBP-L may be useful as a therapeutic to reduce tumor cell invasion
and metastasis.
Example 11
The Effect of Direct Injection of a FKBP-L cDNA Construct on DU145
Human Prostate Tumor Cell Growth In Vivo. (N=1, 4-7 Mice Per
Treatment Group)
[0308] Experiments were conducted to determine the effect of direct
intra-tumoral injection of a FKBP-L cDNA construct on DU145 human
prostate tumor cell growth in vivo.
[0309] Cell Culture
[0310] Du145 (prostate carcinoma) cells were obtained from Cancer
Research UK and cultured in RPMI 1640 medium (Invitrogen)
supplemented with 10% foetal calf serum. All cell lines were grown
as monolayers, incubated at 37.degree. C. under 5% CO.sub.2.
[0311] DNA Plasmid Construction
[0312] The FKBP-L/pcDNA3.1 plasmid was constructed by excision of
the FKBPL cDNA using BamH1 (polynucleotide SEQ ID NO:31) from pUC18
and then directional ligation of FKBP-L into the BamHI restriction
site of pcDNA3.1 (Invitrogen) as described in Example 1. The
endostatin plasmid (for use as a positive anti-angiogenic control)
hEndo XV/pcDNA3.1, was constructed by digesting the pBLAST hENDO XV
plasmid (InVivoGen) with Hpa1 (Promega) and EcoV (Invitrogen) to
release the hEndo XV insert. The hEndo XV insert was ligated
directionally into the ECoRV restriction site of pcDNA3.1
(Invitrogen).
[0313] Prostate Cancer Xenograft Model
[0314] Nineteen (19) male immunocompromised (severe combined
immunodeficient) mice were used (Harlan). The mice were aclimatized
and caged in groups of 5 or less in a barrier care facility. Du145
(prostate carcinoma) cells were cultured as previously described.
Subconfluent cells were harvested and the cell concentration was
adjusted to 5.times.10.sup.7 cells/ml in PBS. The dorsum of each
mouse was shaved. After administrating aesthetic, each mouse
received intra-dermal injections of 5.times.10.sup.6 Du145 tumor
cells (100 .mu.l) bilaterally into the rear dorsum with a 26-gauge
needle. The tumors were allowed to grow until they reached a volume
of 100-125=.sup.3. The mice were randomly divided into four
treatment regimens: (a) untreated (4 mice); (b) empty vector
(pcDNA3.1) (4 mice); (c) hEndo XV/pcDNA3.1 (4 mice); and (d)
FKBP-L/pcDNA3.1 (7 mice). The mice received intratumoral injections
of Lipofectamine 2000 (Invitrogen): plasmid complexes, twice
weekly, every 3 or 4 days. Briefly the Lipofectamine 2000: plasmid
complexes were made as for each injection per animal as follows:
25p1 of plasmid (1 .mu.g/.mu.l) was added to 25p1 of optimem
(Invitrogen) and 10p1 of Lipofectamine 2000 (Invitrogen) was added
to 40p1 of optimem. The two solutions were incubated at room
temperature for 5 minutes. The 2 solutions were combined and
allowed to incubate at room temperature for a further 20 minutes
before tumor intra-tumor injection. The tumors were measured before
each treatment. Tumor volume was calculated as: 4/3.pi.r.sup.3
(where r=1/2 GMP and GMP=.sup.3 {square root over
(Length.times.Breadth.times.Height)}).
[0315] The results are shown in FIG. 15. Both FKBP-L and endostatin
treated tumors showed evidence of a necrotic center, i.e. they
looked donut shaped. This is typical of the effects seen with
anti-angiogenics. Controls reached their volume quadrupling time by
.about.35 days, however growth of FKBP-L treated tumors was for
inhibited over 3 months (100 days) after initial treatment, with
tumors only 10% of their initial volume.
[0316] Thus, it was found that intratumoral injection of a FKBP-L
expression construct inhibits DU145 human tumor xenograft growth
and is comparable, if not superior, to the effects seen with
endostatin currently approved in at least some countries (e.g.,
China) for treatment of lung cancer. Again this shows the potential
therapeutic value of FKBP-L gene therapy in a clinical setting.
Example 12
Genes Regulated in L132 Cells by FKBP-L Antisense Oligonucleotides
Associated with Angiogenesis/Migration
[0317] cDNA Microarray Analysis
[0318] Total RNA was isolated from L132 cells, 8 h after exposure
to FKBP-L antisense (FKBP-L antisense: 5' ATG GCC AGG CTC CCG CTC
3') (SEQ ID NO: 40) or lipofectin only as a control. Poly A+ mRNA
was extracted from total RNA samples using the Qiagen Oligotex kit
(Qiagen, UK), according to the manufacturer's instructions. These
mRNA samples (800 ng per sample) were sent to Incyte Genomics, USA
where a UniGEM 2.0 microarray analysis was conducted. Incyte's
Lifearray chips enable the interrogation of up to 10,000 genes
simultaneously, resulting in the comparison of gene expression
levels in two different samples. Briefly, a standard reverse
transcription reaction was carried out to convert both mRNA samples
to cyanine-dye-labelled cDNA. mRNA from L132 cells treated with
FKBP-L antisense oligonucleotides after 8 h was used to generate a
Cyanine 3 (green) labelled probe and mRNA from L132 cells treated
with lipofectin only after 8 hours was used to produce a Cyanine 5
(red) labelled probe. The two fluorescent probe samples were
simultaneously applied to a single microarray chip containing
numerous cDNA probes immobilised on a solid support in specific
locations, where they competitively reacted with the arrayed cDNA
molecules. Following incubation, the microarray was rinsed in a
series of baths to ensure the removal of any unhybridised sample.
The microarray was then captured as an image that was acquired
using a scanner for fluorescent signal detection. This scanner
generated data on the intensity of each spot by excitation of the
fluorochromes on the array. Each element of the chip was scanned
for the Cy3 (green) and then the Cy5 (red) fluorescent label to
create electronic images for both dye channels. The final array
images were analysed using the Incyte GEMTools software
package.
[0319] Genes that are up-regulated are associated with an increase
in angiogenesis (Table 1). Elevated RhoA, RhoC, ROCK I, and ROCK II
expression is known to be associated with tumor progression to more
advanced stages and it has been suggested that Rho and ROCK
signalling contribute to the morphologic changes and metastatic
behaviour of some tumor cells. This is consistent with the
hypothesis that overexpression of FKBP-L inhibits angiogenesis and
FKBP-L repression using antisense oligonucleotides could promote
angiogenesis by activation of Rho and ROCK. The data imply that
knock-down of FKBPL with antisense or a targeted siRNA could
promote angiogeneis and could be used to promote healing of chronic
wounds.
TABLE-US-00001 TABLE 1 Genes differentially expressed following
exposure to FKBP-L anti-sense oligonucleotides Fold Increase
(.uparw.) Genes or Decrease (.dwnarw.) PI3K .uparw. 3.1 Rho GTPase
activating protein- .uparw. 2.0 oligophrenin 1 ROCK .uparw. 1.7
Microtubule associated protein 1B .uparw. 1.6 MMP-like 1 .uparw.
1.6 TNF ligand superfamily member 1 .uparw. 1.5 CYR61 .dwnarw. 2.4
Tubulin .gamma. .dwnarw. 1.6 Annexin 2 .dwnarw. 1.6 Tubulin .beta.
.dwnarw. 1.5 Tubulin .alpha. .dwnarw. 1.5
Example 13
Inhibition of Cell Migration is Dependent on CD44 in HMEC-1 and
Tumor Cell Lines DU145, PC3, HT29, MCF-7, MDA-231
[0320] RT-PCR to Detect CD74 mRNA Expression
[0321] Du145, HMEC-1, HT29, PC3, MCF-7 and MDA-231 cells were
seeded into T25 tissue culture flasks and allowed to grow until
they reached 70% confluency. RNA was isolated from the cells using
the RNAqueous kit (Ambion, Cat #AM1912), according to
manufacturer's instructions. The RNA was treated with Turbo
DNA-Free.TM. (Ambion, Cat #1906) according to manufacturers'
instructions in order to remove contaminating DNA. cDNA was
prepared from the RNA samples using the ImProm II.TM. Reverse
Transcription Kit (Promega, Cat A3800). Briefly, 0.5 .mu.g of RNA,
0.5 .mu.g of oligo dT primer was made up to 5 .mu.l with
nuclease-free water, incubated at 70.degree. C. for 5 min before
incubating on ice for 5 min. The following reagents were then
added: nuclease-free water (5.3 .mu.l), 5.times. ImProm II.TM.
Reaction Buffer (4 .mu.l), 25 mM MgCl.sub.2 (3.2 .mu.l) 10 mM dNTP
mixture (1 .mu.l) ImProm II.TM. Reverse Transcriptase (1 .mu.l) and
Recombinant RNasin ribonuclease inhibitor (0.5 .mu.l). The reverse
transcription reactions were incubated at 25.degree. C. for 5 min,
42.degree. C. for 1 h and finally 70.degree. C. for 15 min.
[0322] For each PCR reaction: cDNA(2 .mu.l), 10.times.PCR buffer (5
.mu.l), 10 mM dNTP mix (2 .mu.l), 50 mM MgCl.sub.2 (2 .mu.l), Taq
DNA polymerase (5 U/.mu.l) (0.25 .mu.l) (Invitrogen Cat
#18038-018), molecular grade water (34.75 .mu.l), and 2 .mu.l of
the appropriate forward and reverse primers (10 .mu.M) (see Table
2) were combined. The samples were amplified using the following
temperature program: 1 cycle of 94.degree. C. for 1 min, 40 (CD74)
or 25 (GAPDH) cycles of 94.degree. C. for 45 s, 55.degree. C. for
30 s and 72.degree. C. for 90 s; followed by 1 cycle of 72.degree.
C. for 10 min.
TABLE-US-00002 TABLE 2 CD74 and GAPDH primer sequences Primer
Sequence SEQ ID NO: CD74 5'-CTTCCCAAGCCTCCCAAG-3' 41
5'-AGAAGACGGGTCCTCCAGTT-3' 42 GAPDH 5'-GAGTCAACGGATTTGGTCGT-3' 43
5'-TTGATTTTGGAGGGATCTCG-3' 44
Western Blot to Detect MIF and CD44
[0323] All cell lines including the mouse endothelial cell line
2H-11 (for MIF testing only) were assessed for their CD44 and MIF
status using western blot analysis. Cells were harvested in laemmli
buffer (Sigma) and heated to 90.degree. C. for 10 min. Samples were
subjected to SDS-PAGE electrophoresis using the Xcell SureLock
Mini-cell system (Invitrogen), transferred to nitrocellulose
membranes, blocked for 1 h at room temperature in 1% milk solution
and probed with either monoclonal anti-CD44H antibody (R&D
Systems, Cat #BBA10) at dilution 1:500, or anti-MIF antibody
(R&D Systems, Cat #AF-289-PB) at dilution 1:500 and
anti-.beta.-Actin antibody (Sigma, Cat #A 4700) at 1:5000 dilution.
Blots were then probed with mouse Ig HRP-linked secondary antibody
(GE Healthcare, UK, Cat NA931V) at 1:3500 dilution when probing for
CD44 or .beta.-actin or goat Ig HRP-linked secondary antibody
(Santa Cruz Biotechnology, Cat #sc-2020) when probing for MIF.
Antibody binding was detected using the SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Cat #34080).
[0324] The results are shown in FIG. 16. It was found that CD74 and
MIF were expressed in all cell lines previously evaluated for
FKBP-L-mediated inhibition of wound closure. However, CD44 was
present in PC3, MDA-231, HT29 and HMEC-1 but absent in Du145 and
MCf-7. The absence of CD44 correlated with the inability of FKBP-L
to inhibit wound closure in DU145 and MCF-7 (shown in Example 14
below). The data support the hypothesis that FKBP-L binds to CD44
and interferes with the CD74/MIF binding resulting in inhibition of
the angiogenic signalling responses from these receptors.
Example 14
The Effect of Full Length Recombinant FKBP-L Polypeptide on PC3
(CD44 +ve), MDA (CD44 +ve), HT29 (CD44 +ve), MCF-7(CD44 -ve) and
DU145 (CD44 -ve) Tumor Cell Migration (N=3)
[0325] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al. (1999)
see supra. PC3 (prostate tumor cell line; CD44 positive; CD44 +ve),
MDA231 (breast tumor cell line; CD44 +ve), HT29 (Colorectal tumor
cell line; CD44 +ve), MCF-7 (breast tumor cell line; CD44 negative;
CD44 -ve) and DU145 (prostate tumor cell line; CD44 -ve) were
plated into individual chambers on a glass slide and grown to 90%
confluence overnight. The medium was removed and the monolayers
wounded. The monolayer was re-supplemented with fresh medium and
the required volume of recombinant His-tagged FKBP-L protein (SEQ
ID NO: 1) was added to give the required final concentration. The
monolayers were incubated for 24 h and then fixed in 4% PBS
buffered paraformaldehyde.
[0326] The extent of "wound" closure was blindly assessed
microscopically by an independent investigator and quantified using
a calibrated eyepiece graticule (1 mm/100 .mu.m graduation) at
20.times. magnification (Olympus BX 50). The extent of closure in
the FKBP-L treated slides was compared to time matched sham treated
controls and the % inhibition of wound closure compared to time
matched controls calculated.
[0327] Cell lines were also assessed for their CD44 status using
western blot analysis (FIG. 16). Cells were harvested in laemmli
buffer (Sigma) and heated to 90.degree. C. for 10 min. Samples were
subjected to SDS-PAGE electrophoresis using the Xcell SureLock
Mini-cell system (Invitrogen), transferred to nitrocellulose
membranes, blocked for 1 h at room temperature in 1% milk solution
and probed with either monoclonal anti-CD44H antibody (R&D
Systems, Cat #BBA10) at dilution 1:500 and anti-.beta.-Actin
antibody (Sigma, Cat #A 4700) at 1:5000 dilution then probed with
mouse Ig HRP-linked secondary antibody (GE Healthcare, UK, Cat
NA931V) at 1:3500 dilution when probing for CD44 or .beta.-actin or
goat Ig HRP-linked secondary antibody (Santa Cruz Biotechnology,
Cat #sc-2020) when probing for MIF. Antibody binding was detected
using the SuperSignal West Pico Chemiluminescent Substrate (Pierce,
Cat #34080).
[0328] Results of the wound closure assay are shown in FIG.
17A-17E. It can be seen that recombinant FKBP-L can inhibit tumor
cell migration in CD44 +ve tumor cell lines, but not in CD44 -ve
tumor cell lines. The data suggest that FKBP-L could inhibit tumor
metastases in a subset of CD44 +ve tumor cell lines.
Example 15
Knock-Down of CD44 in PC3 Cells Via an siRNA Targeted Approach
Inhibits the FKBP-L-Mediated Inhibition of PC3 Migration (N=2)
[0329] PC3 cells were transfected for 72 h with either sicontrol
non-targeting siRNA (SCR siRNA) (Dharmacon, Cat #D-001210-01-05) or
CD44 targeted siRNA (CD44siRNA) (Dharmacon, Cat #009999). Briefly,
1.2.times.10.sup.6 PC3 cells were seeded into two P90 dishes and
incubated at 37.degree. C. for 24 h. To transfect, 150 .mu.l of the
either sicontrol non-targeting siRNA or CD44 targeting siRNA (2
.mu.M) was added to 450 .mu.l of serum free medium (Tube 1). 18
.mu.l of Dharmafect 2 transfection reagent (Dharmacon, Cat
#T-2002-03) was added to 582 .mu.l of serum free medium in
duplicate (Tube 2). All tubes were incubated at room temperature
for 5 min. The contents of the tubes 1 and 2 were mixed and
incubated for a further 20 min at room temperature. During this
incubation period, the two P90 dishes of PC3 cells were washed and
4.8 ml of complete medium was added to each dish. The appropriate
siRNA transfection mix was then added dropwise and the dishes were
incubated for 72 h at 37.degree. C. The transfected cells were then
seeded into chamber slides (1.25.times.10.sup.5 cells/chamber) and
incubated for a further 24 h at 37.degree. C. The monolayers were
wounded and full length recombinant His-tagged FKBP-L (SEQ ID NO:
1) (1500 ng/ml) or complete medium was added to the monolayers. The
monolayer was fixed after a further 24 h and the extent of wound
closure was blindly assessed using a calibrated graticule. Percent
inhibition of wound closure in FKBP-L-treated monolayers compared
to untreated monolayers was calculated. FKBP-L inhibited the
migration of the SCR siRNA treated cells by 21.7%, but had no
effect on CD44 siRNA treated cells.
[0330] Western blot analysis was carried out to confirm knock-down
of CD44 in PC3 cells. 144 h post-transfection with either sicontrol
non-targeting siRNA (50 nM) or CD44 targeted siRNA (50 nM) cells
were harvested in laemmli buffer (Sigma) and heated to 90.degree.
C. for 10 min. Samples were subjected to SDS-PAGE electrophoresis
using the Xcell SureLock Mini-cell system (Invitrogen), transferred
to nitrocellulose membranes, blocked for 1 h at room temperature in
1% milk solution and probed with monoclonal anti-CD44H antibody
(R&D Systems, Cat #BBA10) at dilution 1:500, and
anti-.beta.-Actin antibody (Sigma, Cat #A 4700) at 1:5000 dilution.
The blot was then probed with mouse Ig HRP-linked secondary
antibody (GE Healthcare, UK, Cat NA931V) at 1:3500 dilution.
Antibody binding was detected using the SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Cat #34080).
[0331] Results are shown in FIG. 18. It was found that FKBP-L can
inhibit migration in the CD44 +ve cell line, PC3, in the presence
of the control siRNA. By knocking down CD44 with CD44 siNA (see
CD44 siRNA lane), it was found that the FKBP-L-mediated inhibition
of migration is dependent on the presence of CD44. These data also
correlate with the need for endogenous CD44 in cell lines such as
HMEC-1, PC3, MDA-231 and HT29 in order to promote FKBP-L-mediated
inhibition of migration. Such FKBP-L mediated inhibition of
migration is not detected in cell lines lacking CD44 i.e MCF-7 and
DU145.
Example 16
FKBP-L Interacts with Endogenous CD44 in Wounded HMEC-1
Monolayers
[0332] Four P90 tissue culture dishes were seeded with HMEC-1
cells, so that they were 90% confluent 24 h later. The four P90
dishes of HMEC-1 cells were transfected with the FKBP-L/pcDNA3.1
DNA construct. Briefly the Lipofectin: FKBP-L/pcDNA3.1 plasmid
complexes were made up for each p90 dish as follows: 4 .mu.g of
plasmid was added to optimem (Invitrogen) to a final volume of 400
.mu.l and 40 .mu.l of Lipofectin (Invitrogen) was added to 360
.mu.l of optimem. The two solutions were incubated at room
temperature for 45 min. The 2 solutions were combined and allowed
to incubate at room temperature for a further 15 min. During this
incubation period, the P90 dishes were washed twice with PBS and
3.2 ml of Optimem was added to each dish. The Lipofectin/plasmid
complexes were gently added to the dishes and incubated at
37.degree. C. for 6 h. The transfection medium was then removed
from the cells and replaced with complete medium. The cells were
incubated for a further 18 h at 37.degree. C. The HMEC-1 monolayers
were wounded (3 wounds per P90 dish) and incubated at 37.degree. C.
for 7 h. The cells were then washed twice in ice-cold PBS and
harvested in Cell Lysis buffer (PBS, 1% Igepal, 0.5% sodium
deoxycholate, 0.1% SDS, 10 mM sodium molybdate, 1 EDTA-free
tablet); 300 .mu.l per P90 dish. The cell lysate was incubated at
4.degree. C. with rotation for 30 min. The cell lysate was
centrifuged at 13000 rpm for 20 min at 4.degree. C., in order to
remove cell debris. The supernatant was then pre-cleared by
incubating with pre-washed agarose G beads for 1 h at 4.degree. C.
with rotation. The pre-cleared cell lysate was split into 3, 1/3
was added to agarose G-CD44 antibody conjugate, 1/3 was added to
agarose G-FKBP-L antibody conjugate and 1/3 was added to prewashed
beads (negative control). The antibody-agarose G/cell lysate
mixtures were incubated overnight at 4.degree. C. with rotation.
The beads were then washed 3 times with ice-cold cell lysis buffer
and 3 times with ice-cold PBS. The beads were then reconstituted in
60 .mu.l of laemmli buffer.
[0333] Western blot analysis of immunoprecipitated samples was
carried out to confirm interactions between FKBP-L and CD44.
Samples were heated to 90.degree. C. for 10 min. Samples were
subjected to SDS-PAGE electrophoresis using the Xcell SureLock
Mini-cell system (Invitrogen), transferred to nitrocellulose
membranes blocked for 1 h at room temperature in 1% milk solution
and probed with monoclonal anti-CD44H antibody (R&D Systems,
Cat #BBA10) at dilution 1:500 and anti-FKBP-L antibody
(Proteintech) at dilution 1:1000 and then probed with either mouse
(CD44) or rabbit (FKBP-L) Ig HRP-linked secondary antibody (GE
Healthcare, UK, Cat NA931V) at 1:3500. Antibody binding was
detected using the SuperSignal West Pico Chemiluminescent Substrate
(Pierce, Cat #34080).
[0334] The results are shown in FIG. 19. Thus, it was found using
immunoprecipitation that exogenously overexpressed FKBP-L interacts
with endogenous CD44 in wounded monolayers. An interaction between
endogenous FKBP-L and CD44 could only be detected in wounded, but
not in non-wounded monolayers (data not shown). This suggests that
a critical level of FKBP-L needs to be expressed before the
interaction with CD44 can be detected. Furthermore, this
interaction only occurs in endothelial cells that are primed for
migration i.e. in wounded monolayers.
Example 17
The N-Terminal Domain of FKBP-L is Important for the
Anti-Angiogenic Properties of FKBP-L (N-3)
[0335] Preparation of the truncated FKBP-L mutant constructs To
construct the 5 FKBP-L truncated mutant plasmid constructs
(.DELTA.34FKBP-L/pcDNA3.1, .DELTA.40FKBP-L/pcDNA3.1,
.DELTA.48FKBP-L/pcDNA3.1, .DELTA.58FKBP-L/pcDNA3.1,
.DELTA.86FKBP-L/pcDNA3.1, .DELTA.151FKBP-L/pcDNA3.1 and
.DELTA.200FKBP-L/pcDNA3.1); stop codons were introduced at amino
acid position 34, 40, 48, 58, 86, 151 or 200 by site directed
mutagenesis (Quikchange kit, Stratagene).
[0336] For each site directed mutagenesis reaction:
pcDNA3.1/FKBP-L/DIR1 (10 ng), 10.times. reaction buffer (5 .mu.l),
10 mM dNTPs (2 .mu.l), Pfu Turbo DNA polymerase (2.5 U/.mu.l) (1
.mu.l) molecular grade water (37 .mu.l), QuikSolution (3 .mu.l) and
1 .mu.l of the appropriate forward and reverse primers (125
ng/.mu.l) were combined. The samples were amplified using the
following temperature program: 1 cycle of 95.degree. C. for 1
minute, 18 cycles of 95.degree. C. for 50 seconds, 60.degree. C.
for 50 seconds and 68.degree. C. for 16 minutes; followed by 1
cycle of 68.degree. C. for 7 minutes.
TABLE-US-00003 TABLE 3 Primers used to prepare FKBP-L truncated
FKBP-L mutant constructs FKBP-L SEQ Truncated ID Mutant Primer
Sequence NO: .DELTA.34FKBP-
5'-GAACCTTGATTCAGTTATTTAGATTAGGCAGCAGCCCCG-3' 45 L/pcDNA3.1
5'-CGGGGCTGCTGCCTAATCTAAATAACTGAATCAAGGTTC-3' 46 .DELTA.40FKBP-
5'-CAGATTAGGCAGCAGCCCTGAGACCCTCCTACCGAAAC-3' 47 L/pcDNA3.1
5'-GTTTCGGTAGGAGGGTCTCAGGGCTGCTGCCTAATCTG-3' 48 .DELTA.48FKBP-
5'-CCTACCGAAACGCTTTAGCTGGAAGTAAGCC-3' 49 L/pcDNA3.1
5'-GGCTTACTTCCAGCTAAAGCGTTTCGGTAGG-3' 50 .DELTA.58FKBP-
5'-CCCAGATCCAGCCAGCTAAATTCTAGAGCATAC-3' 51 L/pcDNA3.1
5'-GTATGCTCTAGAATTTAGCTGGCTGGATCTGGG-3' 52 .DELTA.86FKBP-
5'-CATGGATCAACCAGTTAGATGCCAGAGGCCC-3' 53 L/pcDNA3.1
5'-GGGCCTCTGGCATCTAACTGGTTGATCCATG-3' 54 .DELTA.151FKBP-
5'-GGCGTAGGGCCATGAAGGGAGGAAACTTG-3' 55 L/pcDNA3.1
5'CAAGTTTCCTCCCTTCATGGCCCTACGCC-3' 56 .DELTA.200FKBP-
5'-CCGAGACTCCTGGTAGCTGGAGACTAGC-3' 57 L/pcDNA3.1
5'-GCTAGTCTCCAGCTACCAGGAGTCTCGG-3' 58
[0337] The restriction endonuclease Dpn I (10 U/.mu.l) (1 .mu.l)
was added directly to each amplification reaction and incubated at
37.degree. C. for 1 hour to digest the parental (non-mutated) DNA.
The digested amplification reactions were transformed into
XL-10-Gold Ultracompetent cells and plated onto LB agar plates
containing ampicillin (100 .mu.g/ml). One colony was picked and
grown in 200 ml of LB broth containing ampicillin (100 .mu.g/ml).
Each truncated FKBP-L mutant DNA construct was purified using the
Qiagen Plasmid Maxi Kit. Sequence changes in the mutated constructs
were confirmed by automated DNA sequencing (Fusion Antibodies Ltd)
(see e.g., FIGS. 20A and 20B).
[0338] The seven FKBP-L truncated mutant constructs were
transfected to express the polypeptides (SEQ ID NOS: 3-9) shown in
FIG. 1.
[0339] In Vitro Migration Assay
[0340] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al (1999).
HMEC-1 were plated into individual chambers on a glass slide and
grown to 90% confluence. The monolayer was transfected with either
1 .mu.g wild-type FKBP-L/pcDNA (to express the polypeptide SEQ ID
1), .DELTA.34FKBP-L/pcDNA3.1, .DELTA.40FKBP-L/pcDNA3.1,
.DELTA.48FKBP-L/pcDNA3.1, .DELTA.58FKBP-L/pcDNA3.1,
.DELTA.86FKBP-L/pcDNA3.1, .DELTA.151FKBP-L/pcDNA3.1 or
.DELTA.200FKBP-L/pcDNA3.1 construct (to express the polypeptides
shown in FIG. 1) in the presence of lipofectin. After 6 hours the
transfection reagents were removed and the monolayer wounded with a
pipette tip and re-supplemented with MCDB-131 and incubated for 7
hours.
[0341] The monolayer was fixed in 4% PBS buffered paraformaldehyde
solution for 10 minutes. The extent of "wound" closure was blindly
assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50).
[0342] The results are shown in FIG. 20C. It was found that full
length wild-type FKBP-L and the truncated mutants, .DELTA.48,
.DELTA.58, .DELTA.86, .DELTA.151, .DELTA.200 inhibited wound
closure. WT-FKBP-L and .DELTA.58 inhibited wound closure by 36.2.6%
and 48.8% respectively. .DELTA.34 and .DELTA.40 failed to
significantly inhibit wound closure, suggesting that the active
domain was deleted in these mutants and that the active
anti-angiogenic domain resides between amino acids 34 and 57 of
full-length FKBP-L.
Example 18
Evaluation of Candidate Peptides Spanning the Active Domain of
FKBP-L Using the Wound Scrape Assay: Comparison with Recombinant
FKBP-L (N=3)
[0343] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al. (1999)
see supra. HMEC-1 were plated into individual chambers on a glass
slide and grown to 90% confluence overnight. The medium was removed
and the monolayer wounded. The monolayer was re-supplemented with
fresh medium and the required volume of the following peptides was
added to achieve a dose range from 10.sup.-14-10.sup.-6M.
TABLE-US-00004 FKBP-L 24 mer NH.sub.2-QIRQQPRDPPTETLELEVSPDPAS-COOH
(aa-34-57) SEQ ID NO: 10 FKBP-L 1-57 NH.sub.2 SEQ ID NO: 6
METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQI RQQPRDPPTETLELEVSPDPAS-COOH
[0344] The monolayers were incubated for 7 h and then fixed in 4%
PBS buffered paraformaldehyde. The extent of "wound" closure was
blindly assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50). The extent
of closure in the FKBP-L treated slides was compared to time
matched sham treated controls and the % inhibition of wound closure
compared to time matched controls calculated.
[0345] The results of these experiments are shown in FIG. 21. In
the lower dose range (10.sup.-14-10.sup.-9M) the FKBP-L 24mer and
1-57mer were potent inhibitors of wound closure. Maximal inhibition
was observed between 10.sup.-10 and 10.sup.-9 M, and the EC50 was
very similar for each peptide. Both of these peptides showed
increased potency compared with the full length recombinant protein
on a mole/mole basis. In conclusion, the 24mer and 1-57mer are
potent inhibitors of endothelial cell migration.
Example 19
Evaluation of Candidate Peptides Spanning the Active Domain of
FKBP-L on the Formation of Endothelial Cell-to-Cell Contacts Using
the Synthetic Basement Membrane Matrigel in the Tube Formation
Assay: Comparison with Recombinant FKBP-L (N=3)
Methods:
[0346] The in vitro tubule formation assay used in these studies is
a modified version of the method described by Ashton et al. (1999).
In brief, assays were conducted using BD BioCoat.TM. Matrigel.TM.
Matrix Thin Layer 24-well Multiwell Plates (BD Discovery Labware,
Oxford, UK). The Matrigel.TM. was rehydrated with 500 .mu.l
MCDB-131 serum free medium and incubated at 37.degree. C. for 30
min. Excess medium was removed and HMEC-1 were seeded at a density
of 1.times.10.sup.5 and the plates incubated at 37.degree. C. under
5% CO.sub.2/95% air for 1 h. Increasing concentrations of FKBP-L
24mer (SEQ ID NO: 10) and 1-57 mer (SEQ ID NO: 6) from
10.sup.-14-10.sup.-6 M were used.
[0347] The plate was incubated for a further 18 h. The degree of
tubule formation between adjacent HMEC-1 cells was assessed in each
well in five fields of view, by counting the number of cell to cell
contacts between different HMEC-1 cells in the designated area. An
independent investigator assessed each well and the FKBP-L treated
wells were compared to sham treated controls.
[0348] The results are shown in FIG. 22. Both the FKBP-L 24mer and
1-57mer inhibited the ability of the HMEC-1's to form cell to cell
contacts or tubules on Matrigel in a dose dependent manner. The
1-57mer was more effective in this assay with an EC50=0.7 .mu.M
compared to 30 .mu.M for the 24mer. In conclusion the data suggest
that the FKBP-L 24mer and the FKBP-L 1-57 mer are potent inhibitors
of endothelial tube formation.
Example 20
The Effect of Candidate Peptides Spanning the Active Domain of
FKBP-L on Angiogenic Sprouting Using the Rat Aortic Ring Assay. The
Effect on Mean Length, Maximum Length and Number of Vessels Formed
(n=3); Comparison to Full Length Recombinant Protein
[0349] Male Wistar rats were euthanised and the thoracic aorta was
aseptically removed and sectioned into 1 cm thick rings. The rings
were washed ten times in sterile medium to remove any bacteria and
embedded into Matrigel on 24 well plates. The wells were
supplemented with 2 ml of medium and increasing concentrations of
FKBP-L 24 mer (SEQ ID NO: 10) and FKBP-L 1-57mer (SEQ ID NO: 6) and
recombinant FKBP-L (SEQ ID NO:1).
[0350] The plate was blindly assessed by an independent
investigator and quantified using a calibrated eyepiece graticule
(1 mm/100 .mu.m graduation) at 20.times. magnification (Olympus BX
50). The extent of vessel length, maximum vessel length and number
of vessels in each field of view was measured and compared to time
matched sham controls and the % inhibition calculated.
[0351] The results of these experiments are shown in FIGS. 23-24.
It was found that both the FKBP-L 24mer and the FKBP-L 1-57mer were
active in this assay when assessed by all three parameters i.e.
extent of vessel length, maximum vessel length and number of
vessels (FIGS. 23A and 23B, respectively). However, in this assay
the 24mer was most potent especially in terms of number of vessels,
with an IC50:0.2 .mu.M compared to 0.53 nM for the 1-57mer and 1.56
nM for the full length recombinant FKBP-L (FIGS. 24A and 24B). The
24mer also shows some biphasic activity. These data suggest that
the 24mer may be most potent at inhibiting initial vessel sprouting
and hence the decrease in the number of vessels. In summary, the
FKBP-L 24mer, 1-57mer, and the recombinant FKBP-L are potent
inhibits of angiogenesis.
Example 21
The Effect of the FKBP-L 24Mer on Cell Invasion in a Modified
Boyden Chamber System (N=3)
[0352] This assay measures the ability of cells to migrate and
invade. Microvascular endothelial cells need to migrate and invade
the extracellular matrix (ECM) after angiogenic stimuli.
Furthermore, tumor cells need to migrate and invade the ECM in
order to spread/metastasize to other sites. Both HMEC-1
(microvascular endothelial cells; CD44 +ve) and two tumor cell
lines, MDA-231 (breast; CD44 +ve) and PC3 (Prostate; CD44 +ve) were
evaluated for their invasive potential in the presence of the
FKBP-L 24 mer.
[0353] Twelve well plate polycarbonate inserts were divided into
two groups with half remaining uncoated and half coated with 100
.mu.g/cm.sup.2 of Matrigel. The coated inserts were allowed to dry
overnight at room temperature in a sterile tissue culture hood. The
required cell line; HMEC-1, PC3 or MDA231 was trypsinised,
re-suspended in fresh medium and the cell number calculated.
5.times.10.sup.5 cells, in a total volume of 500 .mu.l, were added
to the insert (top chamber) and 1.5 ml of complete medium added to
the bottom chamber of the plate as a stimulus for invasion. FKBP-L
24mer was added to both the upper and lower chamber of the plate at
the required concentration in the experimental wells. The plate was
incubated for 24 h (PC3 or MDA231) or 48 h (HMEC-1).
[0354] The inserts were carefully removed from their 12 well plate
and inserts without Matrigel coating were placed directly into
Carnoys fixative. Inserts, which were coated with Matrigel, had the
top surface of the insert wiped three times with a cotton bud to
remove non-invading cells. The inserts were then placed in Carnoys
and left for 10 min.
[0355] The inserts were removed from the Carnoys solution and
allowed to air dry for 20 min. The dried inserts were stained in
Hoescht (50 ngml.sup.-1) for 30 min before washing in distilled
water.
[0356] The polycarbonate inserts were cut from the holders and
placed on to mounting medium on a microscope slide. A cover-slip
was applied and sealed with nail varnish. The slides were stored at
4.degree. C. until analysed.
[0357] Ten images from each insert were captured and the number of
fluorescent cells per image was analysed by Lucia Imaging software.
The ratio of cells visible on non-coated inserts compared to cells
visible on Matrigel coated inserts was expressed as % invasion. The
percent (%) invasion in the control was then compared to 24mer
treated cells.
[0358] The results are shown in FIG. 25. It can be seen that the
FKBP-L 24mer (SEQ ID NO: 10) is a potent inhibitor of HMEC-1, PC3
and MDA-231 cell invasion. As well as providing further data to
support the inhibition of HMEC-1 migration, the data indicate that
the FKBP-L 24mer can also inhibit invasion through Matrigel; an
important step in the angiogenic process. The data also indicate
that metastasis of CD44 +ve tumors could be inhibited in a clinical
setting.
Example 22
The Effect of the FKBP-L 24Mer on Cell Adhesion (N=3)
[0359] This assay measures the ability of cells to adhere. This is
an important feature of angiogenesis and metastasis. Important
mediators of leukocyte recruitment and adherence to the endothelium
include E-selectin, VCAM-1, and ICAM-1 which are upregulated during
inflammation, initiating leukocyte adhesion to the endothelium, and
ultimately contributing to disease progression or tissue
damage.
[0360] A 96-well plate was pre-coated with a thin layer of Matrigel
which was allowed to set overnight. The plate wells were blocked
with 0.5% BSA for 1 h at 37.degree. C. in a 95% air/5% CO.sub.2
incubator. Human microvascular endothelial cells (HMEC-1) were
trypsinised and re-suspended in fresh medium and seeded at a
density of 20000 cells per well. The plates were placed at
4.degree. C. for 10 min to allow the cells to sediment to the
bottom of the wells. The required amount of medium supplemented
with the FKBP-L 24mer was added to each well and the plate
incubated for 1 h at 37.degree. C. The excess medium and unattached
cells were removed and the wells washed three times with sterile
PBS. The wells were supplemented with fresh medium and MTT added (5
mgml.sup.-1). The plate was incubated for a further 4 h at
37.degree. C. DMSO was added to each well to solubilise the MTT to
formazen and the plate read at 540 nm, with the relative absorbance
of control wells compared to FBKP-L 24mer-supplemented wells.
[0361] The results are shown in FIG. 26. It can be seen that the
FKBP-L 24mer is a potent inhibitor of HMEC-1 adhesion. As well as
providing further data to support the inhibition of HMEC-1
migration and invasion, this assay also indicates that the FKBP-L
24mer can inhibit adhesion, an important step in the angiogenic
process and other disease states.
Example 23
The Effect of the FKBP-L 24Mer on MDA-231 and PC3 Tumor Cell
Migration (N=3)
[0362] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al. (1999)
see supra. MDA231 (breast tumor cell line; CD44 +ve) and PC3
(prostate tumor cell line; CD44 +ve) cells were plated into
individual chambers on a glass slide and grown to 90% confluence
overnight. The medium was removed and the monolayers wounded. The
monolayer was re-supplemented with fresh medium and the required
volume of FKBP-L 24mer (SEQ ID NO: 10) was added to give the
required final concentration (10.sup.-14-10.sup.-7 M). The
monolayers were incubated for 24 h and then fixed in 4% PBS
buffered paraformaldehyde.
[0363] The extent of "wound" closure was blindly assessed
microscopically by an independent investigator and quantified using
a calibrated eyepiece graticule (1 mm/100 .mu.m graduation) at
20.times. magnification (Olympus BX 50). The extent of closure in
the FKBP-L treated slides was compared to time matched sham treated
controls and the % inhibition of wound closure compared to time
matched controls calculated.
[0364] The results are shown in FIG. 27A (MDA-23 cells) and 27B
(PC3 cells). It was found that the FKBP-L 24 mer can inhibit
MDA-231 and PC3 tumor cell migration. These are CD44 +ve tumor cell
lines, again indicating that FKBP-L may act via CD44, similar to
what was observed with the full length recombinant protein (FIG.
17). The data suggest that the FKBP-L 24mer could inhibit tumor
metastases in a subset of CD44 +ve tumor cell lines.
Example 24
The FKBP-L 24 Mer is an Angiostatic Inhibitor (N=3)
[0365] In order to determine whether the FKBP-L 24mer exerted a
permanent or static effect on endothelial cell sprouting the rat
aortic ring assay was used. Male Wistar rats were euthanised and
the thoracic aorta was aseptically removed and sectioned into 1 cm
thick rings. The rings were washed ten times in sterile medium to
remove any bacteria and embedded into Matrigel on 24 well plates.
The wells were supplemented with 2 ml of medium. The plates were
incubated for up to 15 days. Each day the Matrigel rings were
photographed and returned to their incubator. Two further
experiments were carried out: (A) addition of FKBP-L 24mer to the
medium after the vessels had grown for seven days; and (B) addition
of FKBP-L 24mer to the medium at the initial embedding stage, with
subsequent removal after seven days and replacement with fresh
medium for a further seven days. The extent of vessel development
was quantified using a calibrated eyepiece graticule (1 mm/100
.mu.m graduation) at 20.times. magnification (Olympus BX 50), and
measured electronically using Lucia imaging software. Vessel length
was measured and compared to time matched sham controls and the
percent (%) inhibition calculated.
[0366] The results are shown in FIGS. 28A and 28B. In control
conditions, vessel development was observed between days 3 and
reaching a maximum of 1400 .mu.m at day 14. In a parallel
experiment vessels were allowed to develop for seven days (approx.
800 .mu.m) and the medium removed and re-supplemented with medium
that contained 10.sup.-9 M FKBP-L 24mer. The addition of 24mer
caused complete inhibition of vessel development when compared to
time matched controls (FIG. 28A).
[0367] In a reversed experiment (FIG. 28B), the aortic rings were
initially exposed to medium supplemented with the FKBP-L 24mer and
incubated for seven days. The FKBP-L 24mer almost completely
inhibited vessel development. The FKBP-L 24mer supplemented medium
was removed from the rings and fresh medium added, resulting in the
continued growth of vessels.
[0368] These experiments suggest that the FKBP-L 24mer inhibits
vessel development in an angiostatic manner and when the vessels
are either mature or freshly embedded.
Example 25
The FKBPL 24Mer (SEQ IN NO:10) Inhibits Angiogenesis In Vivo Using
the Sponge Assay; Comparison to Full Length Recombinant FKBPL (N=1,
3 Mice Per Group)
[0369] This experiment evaluated the ability of the FKBP-L 24mer to
inhibit angiogenesis using the mouse sponge assay. Polyether
sponges were subcutaneously implanted in C57 black mice on day 0
and injected on alternate days with (a) 10 ng bFGF control (3 mice)
(b) 10 ng bovine fibroblast growth factor (bFGF)+5 .mu.g
full-length his-tagged recombinant FKBPL (equivalent to
3.2.times.10.sup.6 M in vitro) (3 mice) (c) 10 ng bFGF+0.35 .mu.g
FKBPL 24mer (molar equivalent of 5 .mu.g full-length recombinant
FKBPL) (3 mice) or (d) 0.11 ng FKBPL 24 mer (equivalent to
10.sup.-9 M in vitro) (3 mice).
[0370] All mice were sacrificed on day 21. Sponges were removed,
fixed and paraffin embedded. Five micron sections were stained with
haematoxylin and eosin. Vessels were blindly counted by 3
independent assessors using .times.40 magnification in 10 fields
per section. The average count per sponge/mouse was then plotted
for each assessor.
[0371] The results are shown in FIG. 29. It can be seen that
injection of bFGF alone resulted in a significant number of vessel
growth into the sponge (mean no of vessels/.times.40 field=10). A
50% reduction in vessel number was observed in those sponges
treated with both bFGF and 5 .mu.g recombinant full length FKBPL.
An 80% reduction in vessel number was observed in those sponges
treated with both bFGF and 0.35 .mu.g FKBPL 24mer. Even the lowest
dose of FKBPL 24mer reduced vessel number by 70% compared to the
bFGF alone treated sponges. These results show that the FKBPL 24mer
can inhibit angiogenesis in vivo, suggesting potential therapeutic
value in a clinical setting. The data also indicate that the FKBPL
24 mer may be more potent than the full length FKBPL protein in
inhibiting angiogenesis.
Example 26
Evaluation of the FKBPL 24Mer Peptide (SEQ ID NO: 10) in a Mouse
Endothelial Cell Line Using the Wound Scrape Assay
[0372] This experiment evaluated the ability of the FKBP-L 24mer to
inhibit endothelial cell migration over a dose ranges spanning from
10.sup.-14 M to 10.sup.-7 M. The in vitro migration assay used in
these studies is a modified version of the method described by
Ashton et al (1999) see supra. In this assay mouse endothelial
cells, 2H-11, were obtained from the American Tissue Culture
Collection and were grown in D-MEM containing 10% FCS. They were
plated into individual chambers on a glass slide and grown to 90%
confluence overnight. The medium was removed and the monolayer
wounded. The monolayer was re-supplemented with fresh medium and
the required volume of the FKBPL 24mer peptide was added to achieve
a dose range from 10.sup.-14-10.sup.-7 M. The monolayers were
incubated for 7 hours and then fixed in 4% PBS buffered
paraformaldehyde.
[0373] The extent of "wound" closure was blindly assessed
microscopically by an independent investigator and quantified using
a calibrated eyepiece graticule (1 mm/100 .mu.m graduation) at
20.times. magnification (Olympus BX 50). The extent of closure in
the FKBP-L 24mer treated slides was compared to time matched sham
treated controls and the % inhibition of wound closure compared to
time matched controls calculated.
[0374] The results of these experiments are shown in FIG. 30. It
can be seen that the FKBPL 24 mer inhibited wound closure in mouse
endothelial cells. Maximal inhibition was observed between
10.sup.-9 and 10.sup.-11 M. The data demonstrate that the FKBPL
24mer inhibits migration of mouse endothelial cells and as such,
may be an inhibitor of cell migration, angiogenesis and metastasis.
The data support the in vivo experiments carried out in mice
described herein (e.g. FIGS. 8,9,15,29 and 31)
Example 27
The FKBP-L 24Mer Peptide (QIRQQPRDPPTETLELEVSPDPAS) is a Potent
Inhibitor of DU145 Tumor Growth In Vivo after Daily IP Injection
(N=1, 6 Mice Per Treatment Group)
[0375] Cell Culture
[0376] Du145 (prostate carcinoma) cells were obtained from Cancer
Research UK and cultured in RPMI 1640 medium (Invitrogen)
supplemented with 10% foetal calf serum. All cell lines were grown
as monolayers, incubated at 37.degree. C. under 5% CO.sub.2.
[0377] Prostate Cancer Xenograft Model
[0378] 24 male immunocompromised (severe combined immunodeficient)
mice were used (Harlan). The mice were acclimatised and caged in
groups of 5 or less in an isolator. Du145 (prostate carcinoma)
cells were cultured as previously described. Sub-confluent cells
were harvested and the cell concentration was adjusted to
5.times.10.sup.7 cells/ml in PBS. The dorsum of each mouse was
shaved. After administrating aesthetic, each mouse received
intra-dermal injections of 5.times.10.sup.6 Du145 tumour cells (100
.mu.l) bilaterally into the rear dorsum with a 26-gauge needle. The
tumours were allowed to grow until they reached a volume of 150-175
mm.sup.3. The mice were randomly divided into four treatment
groups: (a) Control: PBS only (8 mice); (b) 24mer FKBPL peptide:
0.3 mg/kg/day (6 mice); (c) 24mer FKBPL peptide: 3.times.10.sup.-3
mg/kg/day (6 mice); and (d) 24mer FKBPL peptide: 3.times.10.sup.-4
mg/kg/day (5 mice).
[0379] The mice received daily IP injections (100 .mu.l) of the
above treatments. The weight and the tumour volume of each mouse
were recorded every 2 days. Tumour volume was calculated as:
Length.times.Breadth.times.Height.times.0.5236. Twenty-one days
after initial treatment the following animals were sacrificed: 0.3
mg/kg/day 24mer FKBPL (2 mice), 3.times.10.sup.-3 mg/kg/day (2
mice), 3.times.10.sup.-4 mg/kg/day (1 mouse) and PBS (2 mice). The
tumours were excised and stored in saline formalin solution for
future histopathological analysis.
[0380] The results are shown in FIG. 31. It can be seen that
treatment by i.p. injection with the 24mer FKBPL peptide at doses
of either 0.3 mg/kg/day or 3.times.10.sup.-3 mg/kg/day
significantly slowed the growth of DU145 tumours in SCID mice
compared to vehicle only treated tumours (FIG. 31A). A number of
tumours treated with the most effective doses of 24mer FKBPL
peptide showed evidence of a necrotic centre, i.e. they looked
donut in shape. This is typical of the effects seen with
anti-angiogenics.
[0381] A complete data set is shown in (FIG. 31A). Note that two
PBS control-treated animals were excluded from the data shown in
FIG. 31A. The first control animal was excluded because its tumor
was eaten by another animal; the other control animal was excluded
because its tumor was implanted too close to the tail in error,
which is known to restrict growth.
[0382] Kaplan-Meier survival curves were drawn using the time for
tumours to reach 3.times. their treatment volume as the criterion
for sacrifice(FIGS. 31B-D). It can be clearly seen that the tumours
of FKBPL 24 mer treated animals at both 0.3 mg/kg/day (FIG. 31B)
and 0.003 mg/kg/day (FIG. 31D) reached 3.times. their treatment
volume significantly later than controls. All but two tumours (of
6) from the 0.3 mg/kg/day treatment group and one (of 6) from the
0.003 mg/kg/day treatment group failed to reach their volume
tripling within the duration of the experiment. However, those
tumors which did reach 3.times. treatment volume were clearly
necrotic following gross examination. These tumors therefore were
also responding but their larger size was caused by massive
necrosis rather than viable tumour cells. Tumors in animals treated
with the lowest dose of 0.0003 mg/kg/day were not significantly
different from controls.
[0383] None of the animals lost weight after daily treatment with
the 24 mer suggesting that it is well-tolerated and not grossly
toxic (FIG. 31E).
Example 28
The Effect of Candidate Peptides Spanning Active Domain of FKBP-L
on the Viability or Proliferation of HMEC-1 Using the MTT Assay
(N=3)
[0384] An MTT assay was used to measure cell viability and/or
proliferation. Briefly, HMEC-1 cells were seeded
(2.5.times.10.sup.3) in 96 well plates and allowed to attach for 5
h. The cells were treated with FKBP-L 24 mer (SEQ ID NO: 10)
(10.sup.-5-10.sup.-10 M), 1-57mer (SEQ ID NO: 6) (10.sup.-9M and
10.sup.-10M) or medium (control).
[0385] Post incubation the cells were exposed to a 5 mgml.sup.-1
solution of 3-(-4,5-dimethylthiazol-2-yl) 2,5 diphenyl tetrazolium
(MTT) for 4 h. The cells were aspirated and 200 .mu.l of DMSO added
to reduce the salt and induce a colour change. The wells were
analysed colourimetrically at 550 nm and the results compared to
untreated control cells.
[0386] The results are shown in FIGS. 32 and 33. FIG. 32 shows a
dose range for treatment of cells with the FKBP-L 24mer and FIGS.
33A and 33B show the effect of the FKBP-L 24mer and FKBP-L 1-57
(57mer) after 24 hours and 48 hours, respectively. It can be seen
that neither of the peptides had any significant effect on the
proliferation of HMEC-1 cells compared to time-matched controls at
any of the time points measured, suggesting that the antiangiogenic
effects observed in the previous assays were not caused by
inhibition of cell growth or by peptide-mediated toxicity.
Example 29
Analysis of Truncated 24 Mer Based Peptides in Order to Assess the
Importance of Each Peptide in Terms of Inhibition of Cell Migration
Using the Wound Scrape Assay
[0387] The in vitro migration assay used in these studies is a
modified version of the method described by Ashton et al. (1999)
see supra. HMEC-1 were plated into individual chambers on a glass
slide and grown to 90% confluence overnight. The medium was removed
and the monolayers wounded. The monolayer was re-supplemented with
fresh medium and the required volume of each peptide (i.e.,
peptides 1-17, SEQ ID NOS: 12-28; Table 4 below) was added to give
the required final concentration (10.sup.-14-10.sup.-6 M).
[0388] To make Peptide 1, the fluorophore Alexa488 (Invitrogen) was
attached to the side-chain sulfhyrdryl functionality of a cysteine
reside which was placed at the C-terminus of the 24mer sequence. A
-PEG-spacer was used to link this C-terminal cysteine residue to
the C-terminus of the 24 mer sequence. This was done during the
synthesis of the peptide by incorporating the commercially
available building block Fmoc-8-amino-3,6-dioxaoctanoic acid, a
polyethylene glycol spacer (NeoMPS) to give a -PEG spacer between
the 24mer sequence and the C-terminal Alexa labeled cysteine. The
PEG spacer/fluorophore has the structure:
--NH--(CH.sub.2).sub.2O--(CH.sub.2).sub.2O--(CH.sub.2)--CO-Cys-(Alexa488)-
. The other peptides were also made by incorporating commercially
available building blocks to generate the peptides 2-17 below.
TABLE-US-00005 TABLE 4 FKBP-L Peptdes SEQ ID Peptide Sequence NO: 1
QIRQQPRDPPTETLELEVSPDPAS-PEG-C(Alexa488) 12 2
PyroGlu-IRQQPRDPPTETLELEVSPDPAS-OH 13 3 IRQQPRDPPTETLELEVSPDPAS-OH
14 4 QIRQQPRDPPTETLELEVSPD-OH 15 5 QIRQQPRDPPTETLELEV-OH 16 6
QIRQQPRDPPTETLE-OH 17 7 QIRQQPRDPPTE-OH 18 8
QQPRDPPTETLELEVSPDPAS-OH 19 9 RDPPTETLELEVSPDPAS-OH 20 10
PTETLELEVSPDPAS-OH 21 11 TLELEVSPDPAS-OH 22 12
RQQPRDPPTETLELEVSPD-OH 23 13 RQQPRDPPTETLELEVSP-OH 24 14
RQQPRDPPTETLELEVS-OH 25 15 PRDPPTETLELEVSPD-OH 26 16
RDPPTETLELEVSPD-OH 27 17 Ac-QIRQQPRDPPTETLELEVSPDPAS-NH.sub.2
28
[0389] The monolayers were incubated for 24 h and then fixed in 4%
PBS buffered paraformaldehyde. The extent of "wound" closure was
blindly assessed microscopically by an independent investigator and
quantified using a calibrated eyepiece graticule (1 mm/100 .mu.m
graduation) at 20.times. magnification (Olympus BX 50). The extent
of closure in the FKBP-L treated slides was compared to time
matched sham treated controls and the % inhibition of wound closure
compared to time matched controls calculated.
[0390] The results for Peptides 1-12 are shown in FIG. 34A-L,
respectively and Table 5.
TABLE-US-00006 TABLE 5 low dose high dose Peptide activity activity
24 mer +++ +++ Pep1 +++ +++ QIRQQPRDPPTETLELEVSPDPAS(488) Pep 2 - -
pQIRQQPRDPPTETLELEVSPDPAS Pep 3 +++ + IRQQPRDPPTETLELEVSPDPAS Pep 4
+++ - QIRQQPRDPPTETLELEVSPD Pep 5 ++ + QIRQQPRDPPTETLELEV Pep 6 -
++ QIRQQPRDPPTETLE Pep 7 + - QIRQQPRDPPTE Pep 8 ++++ -
QQPRDPPTETLELEVSPDPAS Pep 9 ++++ ++ RDPPTETLELEVSPDPAS Pep 10 - ++
PTETLELEVSPDPAS Pep 11 - ++ TLELEVSPDPAS Pep 12 +++ +++
RQQPRDPPTETLELEVSPD-OH
[0391] It was found that Peptide 12 showed activity that was about
the same as the FKBP-L 24 mer. These data suggest that some FKBP-L
derived peptides exhibit a biphasic dose response. The data also
suggest that the subregion -QQPRDPPTETLELEVSPD- (SEQ ID NO: 11) may
be a potent anti-angiogenic domain. The data further indicate that
a fragment of SEQ ID NO: 10 including 18 or more contiguous amino
acids (see e.g., Peptide 5, SEQ ID NO: 16; Peptide 12, SEQ ID NO:
23, and SEQ ID NO: 11) may be active as an anti-angiogenic agent.
Additional peptides including this domain are shown in FIG. 1.
Example 30
Analysis of Purified Recombinant FKBP-L
[0392] Recombinant FKBP-L Protein Expression
[0393] FKBP-L (variant Thr181, Gly186), cloned into the BamHI and
PstI sites of the pRSET-A vector, was expressed in BL21 (DE3) to
give the corresponding N-terminal poly-histidine tagged protein
(SEQ ID NO: 1). Expression was induced at OD 0.6 with 0.2 mM IPTG,
growing cells overnight at 15.degree. C. Cells were pelleted by
centrifugation and stored at -20.degree. C.
[0394] Recombinant FKBP-L Purification
[0395] Purification of protein was done under denaturing
conditions, with an on-the-column refolding step. Cells were lysed
in lysis buffer (100 mM NaH.sub.2PO.sub.4 pH 8.0, 10 mM Tris, 8 M
urea, 150 mM NaCl, 5 mM .beta.-mercoptoethanol) by sonicating on
ice for 3.times.2 mins with cooling. Cell debris and insoluble
material was removed by centrifugation at 31,100 rcf for 20 mins at
4.degree. C. The supernatant was syringe filtered through 0.45
.mu.m filters.
[0396] A 5 ml HisTrap HP column was equilibrated in binding buffer
(8 M urea, 0.5 M NaCl, 20 mM sodium phosphate buffer pH 8.0, 5 mM
.beta.-mercoptoethanol) and the cell lysate loaded onto the column.
The column was washed with 10 column volumes of wash buffer (8 M
urea, 0.5 M NaCl, 20 mM sodium phosphate buffer pH 8.0, 20 mM
imidazole, 5 mM .beta.-mercoptoethanol), then re-equilibrated in
the binding buffer.
[0397] Bound protein was refolded slowly in a 30 ml 0-100% linear
gradient of refold buffer (5 mM imidazole, 0.5 M NaCl, 20 mM sodium
phosphate buffer pH 7.4, 1 mM .beta.-mercoptoethanol), followed by
5 mins at 100% refold buffer.
[0398] Bound proteins were eluted in a 30 ml 0-100% linear gradient
of elution buffer (500 mM imidazole, 0.5 M NaCl, 20 mM sodium
phosphate buffer pH 7.4, 1 mM .beta.-mercoptoethanol). Fractions
were analysed by SDS PAGE and pooled accordingly. To reduce the
concentrations of imidazole, NaCl and .beta.-mercoptoethanol,
protein was either dialysed against 20 mM sodium phosphate buffer
pH 7.4 with 150 mM NaCl (FIG. 35A) or run through a HiLoad 26/60
Superdex75 26/60 prep column in 20 mM sodium phosphate buffer pH
7.4, 150 mM NaCl, 5 mM imidazole (FIG. 35C and FIG. 36).
Recombinant FKBP-L samples were compared by SDS PAGE (FIGS. 35 A
and 35B) and native PAGE (FIG. 35C, inset).
[0399] Analytical HPLC and Mass Spectrometry
[0400] 50 .mu.g samples of recombinant FKBP-L with and without 100
mM DTT were run on an analytical Jupiter 5u c5 column with a 0-73%
gradient of acetonitrile over 30 minutes. Peaks were collected and
analysed by electrospray mass spectrometry.
[0401] Gel Permeation Analyses
[0402] The following molecular weight standards were run on a
Superose12 10/300 GL column in buffer (20 mM NaH.sub.2PO.sub.4 pH
7.4, 150 mM NaCl, 5 mM imidazole): blue dextran, alcohol
dehydrogenase, bovine serum albumin, ovalbumin, carbonic anhydrase
and cytochrome c. The elution volumes of the peaks were used to
calculate Kav for full length recombinant FKBP-L, from which the
molecular mass could be calculated from the calibration curve. The
Kav was calculated as
Kav=(Ve-Vo)/(Vt-Vo)
where Ve is the elution volume, Vo is the void volume (elution
volume for blue dextran) and Vt is the total column volume. Kav was
plotted against log molecular weight to give a straight line from
which the equation was extracted and used to estimate the molecular
weight for a given Ve.
[0403] For analysis, 140 .mu.g samples of recombinant FKBP-L with
and without 100 mM DTT were run under the same conditions and the
estimated molecular masses estimated from the Ve as described
above. In addition the column was equilibrated in buffer +1 mM DTT
and a further sample of FKBP-L pretreated with DTT was run under
these conditions (FIG. 36).
[0404] Protein Cross-Linking Using Glutaraldehyde
[0405] A 1% final concentration of glutaraldehyde was added to 25
.mu.g recombinant FKBP-L (dialysed) in 500 .mu.l buffer (20 mM
NaH.sub.2PO.sub.4 pH 7.4, 150 mM NaCl, .about.5 mM imidazole) for
30 seconds. The reaction was quenched by adding NaBH.sub.4, the
protein precipitated with Na deoxycholate and TCA and analysed by
SDS PAGE under reducing conditions (FIG. 37).
[0406] These experiments show that the recombinant FKBP-L protein
expressed and purified and dialysed here showed single band purity
upon SDS PAGE analysis under reducing conditions (FIG. 35A). SDS
PAGE analysis and native PAGE analysis of FKBP-L (FIG. 35B and FIG.
35C respectively) under non-reducing conditions (FIG. 35B lane 3
and FIG. 35C) and reducing conditions (FIG. 35B lane 4 and FIG.
35C) shows that FKBP-L forms higher molecular weight multimeric
species through the formation of intermolecular disulphide bond
formation between cysteine residues within the protein.
[0407] Analytical HPLC analysis of recombinant FKBP-L followed by
electrospray mass spectrometry gave a mass of 42,257 (expected
42,220) for the reduced FKBP-L, confirming the identity of the
protein.
[0408] Gel permeation analysis was used to try to gain information
about the quaternary structure of recombinant FKBP-L (FIG. 36).
Under the conditions described, the reduced FKBP-L elutes with an
average elution volume 12 ml. From calibration of the column with a
series of molecular weight standards, an elution volume of 12 ml
corresponds to a mass of 99 KDa. Similarly glutaraldehyde
cross-linking of recombinant FKBP-L in the presence of DTT
consistently showed a band on SDS PAGE analysis running at 97 kDa
(FIG. 37). These results indicate that FKBP-L may form homodimeric
and/or homotrimeric species through noncovalent association. This
is consistent with the predicted presence of tetratricopeptide
repeats within the FKBP-L amino acid sequence, which are known to
induce trimerisation in other proteins.
Example 31
Generation of FKBP-L Antibodies
[0409] FKBP-L (variant Thr181, Gly186), cloned into the BamHI and
PstI sites of the pRSET-A vector, was expressed in BL21 (DE3) to
give the corresponding N-terminal poly-histidine tagged protein
(SEQ ID NO: 1). A sequence verified clone was transformed into
BL21(DE3) E. coli cells and cultured to log phase, and target
protein expression induced by addition of
isopropyl-b-D-thiogalactoside (IPTG, 1 mM) and incubated for a
further 4 hours at 37.degree. C. Cell pellets were resuspended and
lysed in 50 mM NaH.sub.2PO.sub.4, pH 8.0, containing 8 M urea, 300
mM NaCl and 10 mM imidazole. The crude denatured lysate was
clarified by centrifugation (10,000 g, 60 minutes at 4.degree. C.),
prior to application to a IMAC column charged with Ni.sup.2+ ions
HiTrap 1 ml column (GE Healthcare). Non-specifically bound material
was washed from the column using 50 mM NaH.sub.2PO.sub.4, pH 8.0,
containing 8 M urea, 300 mM NaCl and 20 mM imidazole, followed by
on-column refolding by reduction of the urea from 8 to 0 M over 200
column volumes. Refolded column bound material was washed with a
further 20 column volumes of 50 mM NaH.sub.2PO.sub.4, pH 8.0, 300
mM NaCl and 20 mM imidazole, then eluted with 50 mM
NaH.sub.2PO.sub.4, pH 8.0, 300 mM NaCl, and 250 mM imidazole.
Protein fractions were collected and desalted into PBS.
[0410] Rabbits were immunized (following standard UK Home Office
guidelines) with the recombinant protein and boosts were given
every 3 weeks until four boosts were completed. Serum was collected
and evaluated against recombinant FKPP-L (generated as the antigen)
by western blot analysis. An FKBPL band of approximately 39 kDa was
detected.
[0411] Embodiments of the present invention therefore provide
methods and compositions comprising FKBP-L. In certain embodiments,
FKBP-L and its peptide fragments are polypeptides with clinical
utility as anti-angiogenic and/or anti-metastatic agents for use in
treatment of cancer and/or other conditions where such therapy
would be expected to have a positive prognostic outcome. The
polypeptide has demonstrable growth-inhibiting effects on selected
cancer cells indicative of a potential secondary or primary
therapeutic effect on specified tumors.
[0412] All documents referred to in this specification are herein
incorporated by reference. Various modifications and variations to
the described embodiments of the inventions will be apparent to
those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described
in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes of carrying out the invention which are
obvious to those skilled in the art are intended to be covered by
the present invention.
Sequence CWU 1
1
581385PRTHomo sapiens 1Met Arg Gly Ser His His His His His His Gly
Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu
Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Arg Trp Gly Ser Met Glu Thr
Pro Pro Val Asn Thr Ile Gly Glu Lys 35 40 45 Asp Thr Ser Gln Pro
Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn 50 55 60 Leu Asp Ser
Val Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr 65 70 75 80 Glu
Thr Leu Glu Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu 85 90
95 Glu His Thr Gln Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp
100 105 110 Ser His Lys Ser His Gly Ser Thr Ser Gln Met Pro Glu Ala
Leu Gln 115 120 125 Ala Ser Asp Leu Trp Tyr Cys Pro Asp Gly Ser Phe
Val Lys Lys Ile 130 135 140 Val Ile Arg Gly His Gly Leu Asp Lys Pro
Lys Leu Gly Ser Cys Cys 145 150 155 160 Arg Val Leu Ala Leu Gly Phe
Pro Phe Gly Ser Gly Pro Pro Glu Gly 165 170 175 Trp Thr Glu Leu Thr
Met Gly Val Gly Pro Trp Arg Glu Glu Thr Trp 180 185 190 Gly Glu Leu
Ile Glu Lys Cys Leu Glu Ser Met Cys Gln Gly Glu Glu 195 200 205 Ala
Glu Leu Gln Leu Pro Gly His Thr Gly Pro Pro Val Gly Leu Thr 210 215
220 Leu Ala Ser Phe Thr Gln Gly Arg Asp Ser Trp Glu Leu Glu Thr Ser
225 230 235 240 Glu Lys Glu Ala Leu Ala Arg Glu Glu Arg Ala Arg Gly
Thr Glu Leu 245 250 255 Phe Arg Ala Gly Asn Pro Glu Gly Ala Ala Arg
Cys Tyr Gly Arg Ala 260 265 270 Leu Arg Leu Leu Leu Thr Leu Pro Pro
Pro Gly Pro Pro Glu Arg Thr 275 280 285 Val Leu His Ala Asn Leu Ala
Ala Cys Gln Leu Leu Leu Gly Gln Pro 290 295 300 Gln Leu Ala Ala Gln
Ser Cys Asp Arg Val Leu Glu Arg Glu Pro Gly 305 310 315 320 His Leu
Lys Ala Leu Tyr Arg Arg Gly Val Ala Gln Ala Ala Leu Gly 325 330 335
Asn Leu Glu Lys Ala Thr Ala Asp Leu Lys Lys Val Leu Ala Ile Asp 340
345 350 Pro Lys Asn Arg Ala Ala Gln Glu Glu Leu Gly Lys Val Val Ile
Gln 355 360 365 Gly Lys Asn Gln Asp Ala Gly Leu Ala Gln Gly Leu Arg
Lys Met Phe 370 375 380 Gly 385 2349PRTHomo sapiens 2Met Glu Thr
Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1 5 10 15 Pro
Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val 20 25
30 Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu
35 40 45 Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His
Thr Gln 50 55 60 Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp
Ser His Lys Ser 65 70 75 80 His Gly Ser Thr Ser Gln Met Pro Glu Ala
Leu Gln Ala Ser Asp Leu 85 90 95 Trp Tyr Cys Pro Asp Gly Ser Phe
Val Lys Lys Ile Val Ile Arg Gly 100 105 110 His Gly Leu Asp Lys Pro
Lys Leu Gly Ser Cys Cys Arg Val Leu Ala 115 120 125 Leu Gly Phe Pro
Phe Gly Ser Gly Pro Pro Glu Gly Trp Thr Glu Leu 130 135 140 Thr Met
Gly Val Gly Pro Trp Arg Glu Glu Thr Trp Gly Glu Leu Ile 145 150 155
160 Glu Lys Cys Leu Glu Ser Met Cys Gln Gly Glu Glu Ala Glu Leu Gln
165 170 175 Leu Pro Gly His Thr Gly Pro Pro Val Gly Leu Thr Leu Ala
Ser Phe 180 185 190 Thr Gln Gly Arg Asp Ser Trp Glu Leu Glu Thr Ser
Glu Lys Glu Ala 195 200 205 Leu Ala Arg Glu Glu Arg Ala Arg Gly Thr
Glu Leu Phe Arg Ala Gly 210 215 220 Asn Pro Glu Gly Ala Ala Arg Cys
Tyr Gly Arg Ala Leu Arg Leu Leu 225 230 235 240 Leu Thr Leu Pro Pro
Pro Gly Pro Pro Glu Arg Thr Val Leu His Ala 245 250 255 Asn Leu Ala
Ala Cys Gln Leu Leu Leu Gly Gln Pro Gln Leu Ala Ala 260 265 270 Gln
Ser Cys Asp Arg Val Leu Glu Arg Glu Pro Gly His Leu Lys Ala 275 280
285 Leu Tyr Arg Arg Gly Val Ala Gln Ala Ala Leu Gly Asn Leu Glu Lys
290 295 300 Ala Thr Ala Asp Leu Lys Lys Val Leu Ala Ile Asp Pro Lys
Asn Arg 305 310 315 320 Ala Ala Gln Glu Glu Leu Gly Lys Val Val Ile
Gln Gly Lys Asn Gln 325 330 335 Asp Ala Gly Leu Ala Gln Gly Leu Arg
Lys Met Phe Gly 340 345 3199PRTHomo sapiens 3Met Glu Thr Pro Pro
Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1 5 10 15 Pro Gln Gln
Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val 20 25 30 Ile
Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu 35 40
45 Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His Thr Gln
50 55 60 Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp Ser His
Lys Ser 65 70 75 80 His Gly Ser Thr Ser Gln Met Pro Glu Ala Leu Gln
Ala Ser Asp Leu 85 90 95 Trp Tyr Cys Pro Asp Gly Ser Phe Val Lys
Lys Ile Val Ile Arg Gly 100 105 110 His Gly Leu Asp Lys Pro Lys Leu
Gly Ser Cys Cys Arg Val Leu Ala 115 120 125 Leu Gly Phe Pro Phe Gly
Ser Gly Pro Pro Glu Gly Trp Thr Glu Leu 130 135 140 Thr Met Gly Val
Gly Pro Trp Arg Glu Glu Thr Trp Gly Glu Leu Ile 145 150 155 160 Glu
Lys Cys Leu Glu Ser Met Cys Gln Gly Glu Glu Ala Glu Leu Gln 165 170
175 Leu Pro Gly His Thr Gly Pro Pro Val Gly Leu Thr Leu Ala Ser Phe
180 185 190 Thr Gln Gly Arg Asp Ser Trp 195 4150PRTHomo sapiens
4Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1
5 10 15 Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser
Val 20 25 30 Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu
Thr Leu Glu 35 40 45 Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile
Leu Glu His Thr Gln 50 55 60 Gly Ala Glu Lys Leu Val Ala Glu Leu
Glu Gly Asp Ser His Lys Ser 65 70 75 80 His Gly Ser Thr Ser Gln Met
Pro Glu Ala Leu Gln Ala Ser Asp Leu 85 90 95 Trp Tyr Cys Pro Asp
Gly Ser Phe Val Lys Lys Ile Val Ile Arg Gly 100 105 110 His Gly Leu
Asp Lys Pro Lys Leu Gly Ser Cys Cys Arg Val Leu Ala 115 120 125 Leu
Gly Phe Pro Phe Gly Ser Gly Pro Pro Glu Gly Trp Thr Glu Leu 130 135
140 Thr Met Gly Val Gly Pro 145 150 585PRTHomo sapiens 5Met Glu Thr
Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1 5 10 15 Pro
Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val 20 25
30 Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu
35 40 45 Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His
Thr Gln 50 55 60 Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp
Ser His Lys Ser 65 70 75 80 His Gly Ser Thr Ser 85 657PRTHomo
sapiens 6Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr
Ser Gln 1 5 10 15 Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn
Leu Asp Ser Val 20 25 30 Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro
Pro Thr Glu Thr Leu Glu 35 40 45 Leu Glu Val Ser Pro Asp Pro Ala
Ser 50 55 747PRTHomo sapiens 7Met Glu Thr Pro Pro Val Asn Thr Ile
Gly Glu Lys Asp Thr Ser Gln 1 5 10 15 Pro Gln Gln Glu Trp Glu Lys
Asn Leu Arg Glu Asn Leu Asp Ser Val 20 25 30 Ile Gln Ile Arg Gln
Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu 35 40 45 839PRTHomo sapiens
8Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1
5 10 15 Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser
Val 20 25 30 Ile Gln Ile Arg Gln Gln Pro 35 933PRTHomo sapiens 9Met
Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln 1 5 10
15 Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val
20 25 30 Ile 1024PRTHomo sapiens 10Gln Ile Arg Gln Gln Pro Arg Asp
Pro Pro Thr Glu Thr Leu Glu Leu 1 5 10 15 Glu Val Ser Pro Asp Pro
Ala Ser 20 1118PRTHomo sapiens 11Gln Gln Pro Arg Asp Pro Pro Thr
Glu Thr Leu Glu Leu Glu Val Ser 1 5 10 15 Pro Asp 1225PRTHomo
sapiensBINDING(24)..(25)There is a PEG (polyethylene glycol)
connection between residue 24 (Serine) and 25 (cystine). 12Gln Ile
Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu Leu 1 5 10 15
Glu Val Ser Pro Asp Pro Ala Ser Cys 20 25 1324PRTHomo
sapiensMOD_RES(1)..(1)Glutamic acid is modified to be Pyroglutamic
Acid (PyroGlu) 13Glu Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu
Thr Leu Glu Leu 1 5 10 15 Glu Val Ser Pro Asp Pro Ala Ser 20
1423PRTHomo sapiens 14Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu
Thr Leu Glu Leu Glu 1 5 10 15 Val Ser Pro Asp Pro Ala Ser 20
1521PRTHomo sapiens 15Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr
Glu Thr Leu Glu Leu 1 5 10 15 Glu Val Ser Pro Asp 20 1618PRTHomo
sapiens 16Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu
Glu Leu 1 5 10 15 Glu Val 1715PRTHomo sapiens 17Gln Ile Arg Gln Gln
Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu 1 5 10 15 1812PRTHomo
sapiens 18Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu 1 5 10
1921PRTHomo sapiens 19Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu
Glu Leu Glu Val Ser 1 5 10 15 Pro Asp Pro Ala Ser 20 2018PRTHomo
sapiens 20Arg Asp Pro Pro Thr Glu Thr Leu Glu Leu Glu Val Ser Pro
Asp Pro 1 5 10 15 Ala Ser 2115PRTHomo sapiens 21Pro Thr Glu Thr Leu
Glu Leu Glu Val Ser Pro Asp Pro Ala Ser 1 5 10 15 2212PRTHomo
sapiens 22Thr Leu Glu Leu Glu Val Ser Pro Asp Pro Ala Ser 1 5 10
2319PRTHomo sapiens 23Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr
Leu Glu Leu Glu Val 1 5 10 15 Ser Pro Asp 2418PRTHomo sapiens 24Arg
Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu Leu Glu Val 1 5 10
15 Ser Pro 2517PRTHomo sapiens 25Arg Gln Gln Pro Arg Asp Pro Pro
Thr Glu Thr Leu Glu Leu Glu Val 1 5 10 15 Ser 2616PRTHomo sapiens
26Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu Leu Glu Val Ser Pro Asp 1
5 10 15 2715PRTHomo sapiens 27Arg Asp Pro Pro Thr Glu Thr Leu Glu
Leu Glu Val Ser Pro Asp 1 5 10 15 2824PRTHomo
sapiensMOD_RES(1)..(1)ACETYLATION 28Gln Ile Arg Gln Gln Pro Arg Asp
Pro Pro Thr Glu Thr Leu Glu Leu 1 5 10 15 Glu Val Ser Pro Asp Pro
Ala Ser 20 29349PRTHomo sapiens 29Met Glu Thr Pro Pro Val Asn Thr
Ile Gly Glu Lys Asp Thr Ser Gln 1 5 10 15 Pro Gln Gln Glu Trp Glu
Lys Asn Leu Arg Glu Asn Leu Asp Ser Val 20 25 30 Ile Gln Ile Arg
Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu 35 40 45 Leu Glu
Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His Thr Gln 50 55 60
Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp Ser His Lys Ser 65
70 75 80 His Gly Ser Thr Ser Gln Met Pro Glu Ala Leu Gln Ala Ser
Asp Leu 85 90 95 Trp Tyr Cys Pro Asp Gly Ser Phe Val Lys Lys Ile
Val Ile Arg Gly 100 105 110 His Gly Leu Asp Lys Pro Lys Leu Gly Ser
Cys Cys Arg Val Leu Ala 115 120 125 Leu Gly Phe Pro Phe Gly Ser Gly
Pro Pro Glu Gly Trp Thr Glu Leu 130 135 140 Thr Met Gly Val Gly Pro
Trp Arg Glu Glu Thr Trp Gly Glu Leu Ile 145 150 155 160 Glu Lys Cys
Leu Glu Ser Met Cys Gln Gly Glu Glu Ala Glu Leu Gln 165 170 175 Leu
Pro Gly His Ser Gly Pro Pro Val Arg Leu Thr Leu Ala Ser Phe 180 185
190 Thr Gln Gly Arg Asp Ser Trp Glu Leu Glu Thr Ser Glu Lys Glu Ala
195 200 205 Leu Ala Arg Glu Glu Arg Ala Arg Gly Thr Glu Leu Phe Arg
Ala Gly 210 215 220 Asn Pro Glu Gly Ala Ala Arg Cys Tyr Gly Arg Ala
Leu Arg Leu Leu 225 230 235 240 Leu Thr Leu Pro Pro Pro Gly Pro Pro
Glu Arg Thr Val Leu His Ala 245 250 255 Asn Leu Ala Ala Cys Gln Leu
Leu Leu Gly Gln Pro Gln Leu Ala Ala 260 265 270 Gln Ser Cys Asp Arg
Val Leu Glu Arg Glu Pro Gly His Leu Lys Ala 275 280 285 Leu Tyr Arg
Arg Gly Val Ala Gln Ala Ala Leu Gly Asn Leu Glu Lys 290 295 300 Ala
Thr Ala Asp Leu Lys Lys Val Leu Ala Ile Asp Pro Lys Asn Arg 305 310
315 320 Ala Ala Gln Glu Glu Leu Gly Lys Val Val Ile Gln Gly Lys Asn
Gln 325 330 335 Asp Ala Gly Leu Ala Gln Gly Leu Arg Lys Met Phe Gly
340 345 301050DNAHomo sapiens 30atggagacgc caccagtcaa tacaattgga
gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga accttcggga gaaccttgat
tcagttattc agattaggca gcagccccga 120gaccctccta ccgaaacgct
tgagctggaa gtaagcccag atccagccag ccaaattcta 180gagcatactc
aaggagctga aaaactggtt gctgaacttg aaggagactc tcataagtct
240catggatcaa ccagtcagat gccagaggcc cttcaagctt ctgatctctg
gtactgcccc 300gatgggagct ttgtcaagaa gatcgtaatc cgtggccatg
gcttggacaa acccaaacta 360ggctcctgct gccgggtact ggctttgggg
tttcctttcg gatcagggcc gccagagggc 420tggacagagc taactatggg
cgtagggcca tggagggagg aaacttgggg ggagctcata 480gagaaatgct
tggagtccat gtgtcaaggt gaggaagcag agcttcagct gcctgggcac
540tctggacctc ctgtcaggct cacactggca tccttcactc aaggccgaga
ctcctgggag 600ctggagacta gcgagaagga agccctggcc agggaagaac
gtgcaagggg cacagaacta 660tttcgagctg ggaaccctga aggagctgcc
cgatgctatg gacgggctct tcggctgctc 720ctgactttac ccccacctgg
ccctccagaa cgaactgtcc ttcatgccaa tctggctgcc 780tgtcagttgt
tgctagggca gcctcagttg gcagcccaga gctgtgaccg ggtgttggag
840cgggagcctg gccatttaaa ggccttatac cgaagggggg ttgcccaggc
tgcccttggg
900aacctggaaa aagcaactgc tgacctcaag aaggtgctgg cgatagatcc
caaaaaccgg 960gcagcccagg aggaactggg gaaggtggtc attcagggga
agaaccagga tgcagggctg 1020gctcagggtc tgcgcaagat gtttggctga
1050311050DNAHomo sapiens 31atggagacgc caccagtcaa tacaattgga
gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga accttcggga gaaccttgat
tcagttattc agattaggca gcagccccga 120gaccctccta ccgaaacgct
tgagctggaa gtaagcccag atccagccag ccaaattcta 180gagcatactc
aaggagctga aaaactggtt gctgaacttg aaggagactc tcataagtct
240catggatcaa ccagtcagat gccagaggcc cttcaagctt ctgatctctg
gtactgcccc 300gatgggagct ttgtcaagaa gatcgtaatc cgtggccatg
gcttggacaa acccaaacta 360ggctcctgct gccgggtact ggctttgggg
tttcctttcg gatcagggcc gccagagggc 420tggacagagc taactatggg
cgtagggcca tggagggagg aaacttgggg ggagctcata 480gagaaatgct
tggagtccat gtgtcaaggt gaggaagcag agcttcagct gcctgggcac
540actggacctc ctgtcgggct cacactggca tccttcactc aaggccgaga
ctcctgggag 600ctggagacta gcgagaagga agccctggcc agggaagaac
gtgcaagggg cacagaacta 660tttcgagctg ggaaccctga aggagctgcc
cgatgctatg gacgggctct tcggctgctc 720ctgactttac ccccacctgg
ccctccagaa cgaactgtcc ttcatgccaa tctggctgcc 780tgtcagttgt
tgctagggca gcctcagttg gcagcccaga gctgtgaccg ggtgttggag
840cgggagcctg gccatttaaa ggccttatac cgaagggggg ttgcccaggc
tgcccttggg 900aacctggaaa aagcaactgc tgacctcaag aaggtgctgg
cgatagatcc caaaaaccgg 960gcagcccagg aggaactggg gaaggtggtc
attcagggga agaaccagga tgcagggctg 1020gctcagggtc tgcgcaagat
gtttggctga 105032102DNAArtificialHomo Sapiens 32atggagacgc
caccagtcaa tacaattgga gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga
accttcggga gaaccttgat tcagttattt ag 10233119DNAArtificialHomo
Sapiens 33atggagacgc caccagtcaa tacaattgga gaaaaggaca cctctcagcc
gcaacaagag 60tgggaaaaga accttcggga gaaccttgat tcagttattc agattaggca
gcagccccg 11934143DNAArtificialHomo Sapiens 34atggagacgc caccagtcaa
tacaattgga gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga accttcggga
gaaccttgat tcagttattc agattaggca gcagccccga 120gaccctccta
ccgaaacgct tga 14335174DNAArtificialHomo sapiens 35atggagacgc
caccagtcaa tacaattgga gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga
accttcggga gaaccttgat tcagttattc agattaggca gcagccccga
120gaccctccta ccgaaacgct tgagctggaa gtaagcccag atccagccag ctaa
17436258DNAArtificialHomo sapiens 36atggagacgc caccagtcaa
tacaattgga gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga accttcggga
gaaccttgat tcagttattc agattaggca gcagccccga 120gaccctccta
ccgaaacgct tgagctggaa gtaagcccag atccagccag ccaaattcta
180gagcatactc aaggagctga aaaactggtt gctgaacttg aaggagactc
tcataagtct 240catggatcaa ccagttag 25837453DNAArtificialHomo sapiens
37atggagacgc caccagtcaa tacaattgga gaaaaggaca cctctcagcc gcaacaagag
60tgggaaaaga accttcggga gaaccttgat tcagttattc agattaggca gcagccccga
120gaccctccta ccgaaacgct tgagctggaa gtaagcccag atccagccag
ccaaattcta 180gagcatactc aaggagctga aaaactggtt gctgaacttg
aaggagactc tcataagtct 240catggatcaa ccagtcagat gccagaggcc
cttcaagctt ctgatctctg gtactgcccc 300gatgggagct ttgtcaagaa
gatcgtaatc cgtggccatg gcttggacaa acccaaacta 360ggctcctgct
gccgggtact ggctttgggg tttcctttcg gatcagggcc gccagagggc
420tggacagagc taactatggg cgtagggcca tga 45338600DNAArtificialHomo
sapiens 38atggagacgc caccagtcaa tacaattgga gaaaaggaca cctctcagcc
gcaacaagag 60tgggaaaaga accttcggga gaaccttgat tcagttattc agattaggca
gcagccccga 120gaccctccta ccgaaacgct tgagctggaa gtaagcccag
atccagccag ccaaattcta 180gagcatactc aaggagctga aaaactggtt
gctgaacttg aaggagactc tcataagtct 240catggatcaa ccagtcagat
gccagaggcc cttcaagctt ctgatctctg gtactgcccc 300gatgggagct
ttgtcaagaa gatcgtaatc cgtggccatg gcttggacaa acccaaacta
360ggctcctgct gccgggtact ggctttgggg tttcctttcg gatcagggcc
gccagagggc 420tggacagagc taactatggg cgtagggcca tggagggagg
aaacttgggg ggagctcata 480gagaaatgct tggagtccat gtgtcaaggt
gaggaagcag agcttcagct gcctgggcac 540tctggacctc ctgtcaggct
cacactggca tccttcactc aaggccgaga ctcctggtag
60039600DNAArtificialHomo sapiens 39atggagacgc caccagtcaa
tacaattgga gaaaaggaca cctctcagcc gcaacaagag 60tgggaaaaga accttcggga
gaaccttgat tcagttattc agattaggca gcagccccga 120gaccctccta
ccgaaacgct tgagctggaa gtaagcccag atccagccag ccaaattcta
180gagcatactc aaggagctga aaaactggtt gctgaacttg aaggagactc
tcataagtct 240catggatcaa ccagtcagat gccagaggcc cttcaagctt
ctgatctctg gtactgcccc 300gatgggagct ttgtcaagaa gatcgtaatc
cgtggccatg gcttggacaa acccaaacta 360ggctcctgct gccgggtact
ggctttgggg tttcctttcg gatcagggcc gccagagggc 420tggacagagc
taactatggg cgtagggcca tggagggagg aaacttgggg ggagctcata
480gagaaatgct tggagtccat gtgtcaaggt gaggaagcag agcttcagct
gcctgggcac 540actggacctc ctgtcgggct cacactggca tccttcactc
aaggccgaga ctcctggtag 6004018DNAArtificialHomo sapiens 40atggccaggc
tcccgctc 184118DNAArtificialHomo sapiens 41cttcccaagc ctcccaag
184220DNAArtificialHomo sapiens 42agaagacggg tcctccagtt
204320DNAArtificialHomo sapiens 43gagtcaacgg atttggtcgt
204420DNAArtificialHomo sapiens 44ttgattttgg agggatctcg
204539DNAArtificialHomo sapiens 45gaaccttgat tcagttattt agattaggca
gcagccccg 394639DNAArtificialHomo sapiens 46cggggctgct gcctaatcta
aataactgaa tcaaggttc 394738DNAArtificialHomo sapiens 47cagattaggc
agcagccctg agaccctcct accgaaac 384838DNAArtificialHomo sapiens
48gtttcggtag gagggtctca gggctgctgc ctaatctg 384931DNAArtificialHomo
sapiens 49cctaccgaaa cgctttagct ggaagtaagc c
315031DNAArtificialHomo sapiens 50ggcttacttc cagctaaagc gtttcggtag
g 315133DNAArtificialHomo sapiens 51cccagatcca gccagctaaa
ttctagagca tac 335233DNAArtificialHomo sapiens 52gtatgctcta
gaatttagct ggctggatct ggg 335331DNAArtificialHomo sapiens
53catggatcaa ccagttagat gccagaggcc c 315431DNAArtificialHomo
sapiens 54gggcctctgg catctaactg gttgatccat g
315529DNAArtificialHomo sapiens 55ggcgtagggc catgaaggga ggaaacttg
295629DNAArtificialHomo sapiens 56caagtttcct cccttcatgg ccctacgcc
295728DNAArtificialHomo sapiens 57ccgagactcc tggtagctgg agactagc
285828DNAArtificialHomo sapiens 58gctagtctcc agctaccagg agtctcgg
28
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