U.S. patent application number 11/880766 was filed with the patent office on 2008-02-28 for pharmaceutical compositions comprising fragments and homologs of troponin subunits.
This patent application is currently assigned to Children's Medical Center Corporation. Invention is credited to Marsha A. Moses, Dmitri G. Wiederschain.
Application Number | 20080051345 11/880766 |
Document ID | / |
Family ID | 27402051 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051345 |
Kind Code |
A1 |
Moses; Marsha A. ; et
al. |
February 28, 2008 |
Pharmaceutical compositions comprising fragments and homologs of
troponin subunits
Abstract
The present invention relates to pharmaceutical compositions
comprising therapeutically effective amounts of troponin C, I or T
subunits, fragments or homologs for the treatment of diseases or
disorders involving abnormal angiogenesis and methods of use
thereof.
Inventors: |
Moses; Marsha A.;
(Brookline, MA) ; Wiederschain; Dmitri G.;
(Cambridge, MA) |
Correspondence
Address: |
DAVID S. RESNICK
100 SUMMER STREET
NIXON PEABODY LLP
BOSTON
MA
02110-2131
US
|
Assignee: |
Children's Medical Center
Corporation
Boston
MA
|
Family ID: |
27402051 |
Appl. No.: |
11/880766 |
Filed: |
July 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10192806 |
Jul 9, 2002 |
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11880766 |
Jul 24, 2007 |
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09612421 |
Jul 7, 2000 |
6589936 |
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10192806 |
Jul 9, 2002 |
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09268274 |
Mar 15, 1999 |
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09612421 |
Jul 7, 2000 |
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08961264 |
Oct 30, 1997 |
6025331 |
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09268274 |
Mar 15, 1999 |
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08602941 |
Feb 16, 1996 |
5837680 |
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08961264 |
Oct 30, 1997 |
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Current U.S.
Class: |
514/9.1 ;
514/13.3; 514/19.3; 514/20.8 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 27/02 20180101; C07K 14/4716 20130101; A61K 38/1709
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 27/02 20060101 A61P027/02; A61P 35/00 20060101
A61P035/00 |
Claims
1. A pharmaceutical composition comprising an amount of a peptide
that is effective to inhibit angiogenesis, in which the peptide is:
a. an inhibitor of bFGF-stimulated bovine endothelial cell
proliferation having an IC.sub.50 of 10 .mu.M or less; b. greater
than 75 amino acids in length; and C. greater than 80% homologous
with a subunit selected from the group consisting of human
fast-twitch troponin subunit C (SEQ ID NO:1), human fast-twitch
troponin subunit I (SEQ ID NO: 2), and human fast-twitch troponin
subunit T (SEQ ID NO: 3); and a pharmaceutically acceptable
carrier.
2. The composition of claim 1, in which the subunit is human
fast-twitch troponin C or human fast-twitch tropinin I.
3. The composition of claim 1, in which the subunit is human
fast-twitch troponin C.
4. The composition of claim 1, in which the subunit is human
fast-twitch troponin I.
5. The composition of claim 1, in which the peptide is greater than
95% homologous with a human troponin subunit.
6. The composition of claim 5, in which the subunit is human
fast-twitch troponin C.
7. The composition of claim 5, in which the subunit is human
fast-twitch troponin I.
8. The composition of claim 1, in which the peptide is a mammalian
troponin subunit.
9. The composition of claim 8, in which the peptide is a mammalian
troponin C or troponin I subunit.
10. The composition of claim 9, in which the peptide is a troponin
subunit selected from the group consisting of bovine, rabbit, mouse
and rat troponin subunits.
11. The composition of claim 8, in which the peptide is a human
troponin C or troponin I subunit.
12. The composition of claim 8, in which the peptide is a troponin
subunit selected from the group consisting of bovine, rabbit, mouse
and rat troponin subunits.
13. The composition of claim 1, in which the peptide is a fragment
of a mammalian troponin subunit.
14. The composition of claim 13, in which the peptide is a fragment
of a human troponin C or troponin I subunit.
15. The composition of claim 13, in which the peptide is a fragment
of a troponin I or troponin C subunit selected from the group
consisting of bovine, rabbit, mouse and rat troponin C and I
subunits.
16. The composition of claim 1, wherein the carrier is acceptable
for topical application to the eye.
17. The composition of claim 1, wherein the carrier is acceptable
for topical application to the skin.
18. The composition of claim 1, wherein the angiogenesis inhibitor
is in a biodegradable, biocompatible polymeric delivery device.
19. A method of inhibiting atopic angiogenesis in a subject, having
a disease or disorder causing atopic angiogenesis requiring such
inhibition, which comprising the step of applying to a site of
atopic angiogenesis an amount of a peptide that is effective to
inhibit angiogenesis, in which the peptide is: a. an inhibitor of
bFGF-stimulated bovine endothelial cell proliferation having an
IC.sub.50 of 10 .mu.M or less; b. greater than 75 amino acids in
length; and c. greater than 80% homologous with a subunit selected
from the group consisting of human fast-twitch troponin I subunit,
human fast-twitch troponin C subunit and human fast-twitch troponin
T subunit.
20. The method of claim 19, in which the subunit is human
fast-twitch troponin C or human fast-twitch troponin I.
21. The method of claim 20, in which the disease or disorder is a
solid tumor.
22. The method of claim 20, in which the tumor is a tumor of the
central nervous system.
23. The method according to claim 20, in which the disease or
disorder is an ophthalmologic disease or disorder.
Description
[0001] This is a continuation-in-part of copending U.S. application
Ser. No. 08/961,264, filed Oct. 30, 1997 which is a continuation of
U.S. application Ser. No. 08/602,941, filed Feb. 16, 1996, now U.S.
Pat. No. 5,837,680.
1. INTRODUCTION
[0002] The present invention provides for novel pharmaceutical
compositions, and methods of use thereof for the treatment of
diseases or disorders involving abnormal angiogenesis.
[0003] More particularly, the present invention is based, in part,
on the discovery that troponin subunits C, I and T and fragments
thereof inhibit stimulated endothelial cell proliferation.
Pharmaceutical compositions containing therapeutically effective
amounts of troponin C, I, or T, subunits, fragments, or homologs
and methods of therapeutic use thereof are provided.
2. BACKGROUND
[0004] Angiogenesis, the process of new blood vessel development
and formation, plays an important role in numerous physiological
events, both normal and pathological. Angiogenesis occurs in
response to specific signals and involves a complex process
characterized by infiltration of the basal lamina by vascular
endothelial cells in response to angiogenic growth signal(s),
migration of the endothelial cells toward the source of the
signal(s), and subsequent proliferation and formation of the
capillary tube. Blood flow through the newly formed capillary is
initiated after the endothelial cells come into contact and connect
with a preexisting capillary.
[0005] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinejad et al., 1989, Cell
56:345-355. In those rare instances in which neovascularization
occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female
reproductive processes, angiogenesis is stringently regulated and
spatially and temporally delimited. Under conditions of
pathological angiogenesis such as that characterizing solid tumor
growth, these regulatory controls fail.
[0006] Unregulated angiogenesis becomes pathologic and sustains
progression of many neoplastic and non-neoplastic diseases. A
number of serious diseases are dominated by abnormal
neovascularization including solid tumor growth and metastases,
arthritis, some types of eye disorders, and psoriasis. See, e.g.,
reviews by Moses et al., 1991, Biotech. 9:630-634; Folkman et al.,
1995, N. Engl. J. Med., 333:1757-1763; Auerbach et al., 1985, J.
Microvasc. Res. 29:401-411; Folkman, 1985, Advances in Cancer
Research, eds. Klein and Weinhouse, Academic Press, New York, pp.
175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; and Folkman et
al., 1983, Science 221:719-725. In a number of pathological
conditions, the process of angiogenesis contributes to the disease
state. For example, significant data have accumulated which suggest
that the growth of solid tumors is dependent on angiogenesis.
Folkman and Klagsbrun, 1987, Science 235:442-447.
[0007] The maintenance of the avascularity of the cornea, lens, and
trabecular meshwork is crucial for vision as well as to ocular
physiology. There are several eye diseases, many of which lead to
blindness, in which ocular neovascularization occurs in response to
the diseased state. These ocular disorders include diabetic
retinopathy, neovascular glaucoma, inflammatory diseases and ocular
tumors (e.g., retinoblastoma). There are also a number of other eye
diseases which are also associated with neovascularization,
including retrolental fibroplasia, uveitis, retinopathy of
prematurity, macular degeneration, and approximately twenty eye
diseases which are associated with choroidal neovascularization and
approximately forty eye diseases associated with iris
neovascularization. See, e.g., reviews by Waltman et al., 1978, Am.
J. Ophthal. 85:704-710 and Gartner et al., 1978, Surv. Ophthal.
22:291-312. Currently, the treatment of these diseases, especially
once neovascularization has occurred, is inadequate and blindness
often results. Studies have suggested that vaso-inhibitory factors
which are present in normal ocular tissue (cornea and vitreous) are
lost in the diseased state.
[0008] An inhibitor of angiogenesis could have an important
therapeutic role in limiting the contributions of this process to
pathological progression of the underlying disease states as well
as providing a valuable means of studying their etiology. For
example, agents that inhibit tumor neovascularization could play an
important role in inhibiting metastatic tumor growth.
[0009] The components of angiogenesis relating to vascular
endothelial cell proliferation, migration and invasion, have been
found to be regulated in part by polypeptide growth factors.
Experiments in culture, indicate that endothelial cells exposed to
a medium containing suitable growth factors can be induced to evoke
some or all of the angiogenic responses. Several polypeptides with
in vitro endothelial growth promoting activity have been
identified. Examples include acidic and basic fibroblast growth
factors, transforming growth factors .alpha. and .beta.,
platelet-derived endothelial cell growth factor, granulocyte
colony-stimulating factor, interleukin-8, hepatocyte growth factor,
proliferin, vascular endothelial growth factor and placental growth
factor. See, e.g., review by Folkman et al., 1995, N. Engl. J.
Ned., 333:1757-1763.
[0010] Although extracts from several different tissue sources have
been shown to contain anti-angiogenic activity, several molecules
such as platelet factor-4, thrombospondin, protamine, and
transforming growth factor B, have been found to negatively
regulate different aspects of angiogenesis, such as cell
proliferation or cell migration. No single tissue-derived
macromolecule capable of inhibiting angiogenesis has been
identified in the prior art. See, e.g., reviews by Folkman, J.,
1995, N. Engl. J. Med. 333:1757-1763 and D'Amore, 1985, Prog. Clin.
Biol. Res. 221:269-283. There is therefore a great need for the
further identification and characterization of chemical agents
which can prevent the continued deregulated spread of
vascularization and which would potentially have broad
applicability as a therapy for those diseases in which
neovascularization plays a prominent role.
[0011] Capillary endothelial cells ("EC") proliferate in response
to an angiogenic stimulus during neovascularization. Ausprunk and
Folkman, 1977, J. Microvasc. Res. 14:153-65. An in vitro assay
assessing endothelial cell proliferation in response to known
angiogenesis simulating factors, such as acidic or basic fibroblast
growth factor (aFGF and bFGF, respectively), has been developed to
mimic the process of neovascularization in vitro. This type of
assay is the assay of choice to demonstrate the stimulation of
capillary EC proliferation by various angiogenic factors. Shing et
al., 1984, Science 223:1296-1298.
[0012] The process of capillary EC migration through the
extracellular matrix towards an angiogenic stimulus is also a
critical event required for angiogenesis. See, e.g., review by
Ausprunk et al., 1977, J. Microvasc. Res. 14:53-65. This process
provides an additional assay by which to mimic the process of
neovascularization in vitro. A modification of the Boyden chamber
technique has been developed to monitor EC migration. Boyden et
al., 1962, J. Exptl. Med. 115:453-456, Example 4. To date, only a
few tissue-derived EC cell migration inhibitors are known. See,
e.g., review by Langer et al., 1976, Science 193:70-72.
[0013] In the early 1970's, a number of in vivo angiogenesis model
bioassays were widely used. These model systems included rabbit
corneal pocket, chick chorioallantoic membrane ("CAM"), rat dorsal
air sac and rabbit air chamber bioassays. For review, see, Blood et
al., 1990, Biochem. et Biophys. Acta 1032:89-118. The development
of controlled release polymers capable of releasing large molecules
such as angiogenesis stimulators and inhibitors was critical to the
use of these assays. Langer et al., 1976, Nature 263:797-800.
[0014] In the CAM bioassay, fertilized chick embryos are cultured
in Petri dishes on day 6 of development, a disc of a release
polymer, such as methyl cellulose, impregnated with the test sample
or an appropriate control substance is placed onto the vascular
membrane at its advancing edge. On day 8 of development, the area
around the implant is observed and evaluated. Avascular zones
surrounding the test implant indicate the presence of an inhibitor
of embryonic neovascularization. Moses et al., 1990, Science,
248:1408-1410 and Taylor et al., 1982, Nature, 297:307-312. The
reported doses for previously described angiogenesis inhibitors
tested alone in the CAM assay are 50 .mu.g of protamine (Taylor et
al. (1982)), 200 .mu.g of bovine vitreous extract (Lutty et al.,
1983, Invest. Opthalmol. Vis. Sci. 24:53-56), and 10 .mu.g of
platelet factor IV (Taylor et al. (1982)). The lowest reported
doses of angiogenesis inhibitors effective as combinations include
heparin (50 .mu.g) and hydrocortisone (60 .mu.g), and
B-cyclodextrin tetradecasulfate (14 .mu.g) and hydrocortisone (60
.mu.g), reported by Folkman et al., 1989, Science 243:1490.
[0015] According to the rabbit corneal pocket assay, polymer
pellets of ethylene vinyl acetate copolymer ("EVAC") are
impregnated with test substance and surgically implanted in a
pocket in the rabbit cornea approximately 1 mm from the limbus.
Langer et al., 1976, Science 193:707-72. To test for an
angiogenesis inhibitor, either a piece of carcinoma or some other
angiogenic stimulant is implanted distal to the polymer 2 mm from
the limbus. In the opposite eye of each rabbit, control polymer
pellets that are empty are implanted next to an angiogenic
stimulant in the same way. In these control corneas, capillary
blood vessels start growing towards the tumor implant in 5-6 days,
eventually sweeping over the blank polymer. In test corneas, the
directional growth of new capillaries from the limbal blood vessel
towards the tumor occurs at a reduced rate and is often inhibited
such that an avascular region around the polymer is observed. This
assay is quantitated by measurement of the maximum vessel lengths
with a stereospecific microscope.
[0016] Troponin, a complex of three polypeptides is an accessory
protein that is closely associated with actin filaments in
vertebrate muscle. The troponin complex, acts in conjunction with
the muscle form of tropomyosin to mediate the Ca.sup.2+ dependency
of myosin ATPase activity and thereby regulate muscle contraction.
The troponin polypeptides T, I, and C, are named for their
tropomyosin binding, inhibitory, and calcium binding activities,
respectively. Troponin T binds to tropomyosin and is believed to be
responsible for positioning the troponin complex on the muscle thin
filament. Troponin I binds to actin, and the complex formed by
troponins I and T, and tropomyosin, inhibits the interaction of
actin and myosin. Troponin C is capable of binding up to four
calcium molecules. Studies suggest that when the level of calcium
in the muscle is raised, troponin C causes troponin I to loose its
hold on the actin molecule, causing the tropomyosin molecule shift,
thereby exposing the myosin binding sites on actin and stimulating
myosin ATPase activity.
[0017] The citation of a reference herein shall not be construed as
an admission that such reference is prior art to the present
invention.
3. SUMMARY OF THE INVENTION
[0018] The present invention relates to pharmaceutical compositions
containing troponin subunits C, I, or T, or fragments thereof, in
therapeutically effective amounts that are capable of inhibiting
angiogenesis, for example, by inhibiting endothelial cell
proliferation. The invention also relates to pharmaceutical
compositions containing homologs of troponin subunits C, I, or T
and homologs of their fragments, in therapeutically effective
amounts that are capable of inhibiting angiogenesis, for example,
by inhibiting endothelial cell proliferation. The invention further
relates to treatment of neovascular disorders by administration of
a therapeutic compound of the invention. Such therapeutic compounds
(termed herein "Therapeutics"), include: troponin subunits C, I,
and T, and fragments and homologs thereof, in particular, fragments
of troponin subunit I comprising the inhibitory (I') and carboxy
terminal (C') regions. In one embodiment, a Therapeutic of the
invention is administered to treat a cancerous condition, for
example, to inhibit the growth or reduce the volume of a solid
tumor, or to prevent progression from the pre-neoplastic or
pre-malignant state into a neoplastic or a malignant state or to
inhibit metastasis. In other specific embodiments, a Therapeutic of
the invention is administered to treat ocular disorders associated
with neovascularization. As used herein, the term "troponin
subunit", when not preceding the terms C, I or T, means generically
any of troponin subunits C, I, or T. The amino-terminal, inhibitory
and carboxy-terminal regions of troponin I are designated N', I',
and C', respectively.
4. BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1. Inhibition of bovine capillary Endothelial Cell
(BCE) proliferation by troponin C. Percent inhibition of
bFGF-stimulated BCE proliferation is shown as a function of
troponin C concentration (.mu.g/well). Percent inhibition was
determined by comparing results obtained for cells treated with
stimulus alone with those obtained for samples exposed to both
stimulus and inhibitor. Well volume was 200 .mu.l.
[0020] FIG. 2. Inhibition of capillary BCE proliferation by
troponin I. Percent inhibition of bFGF-stimulated BCE proliferation
is shown as a function of troponin I concentration (.mu.g/well).
Percent inhibition was determined as described in FIG. 1. Well
volume was 200 .mu.l.
[0021] FIG. 3. Inhibition of capillary BCE proliferation by
troponin T. Percent inhibition of bFGF-stimulated BCE proliferation
is shown as a function of troponin T concentration (.mu.g/well).
Percent inhibition was determined as described in FIG. 1. Well
volume was 200 .mu.l.
[0022] FIG. 4. Inhibition of BCE proliferation by troponins C and
I. Percent inhibition of bFGF-stimulated BCE proliferation is shown
as a function of troponin I and C concentration (.mu.g/well).
Percent inhibition was determined as described in FIG. 1. Well
volume was 200 .mu.l.
[0023] FIG. 5. Inhibition of capillary BCE proliferation by
troponin C, I and T. Percent inhibition of bFGF-stimulated BCE
proliferation is shown as a function of troponin C, I, and T
concentration (.mu.g/well). Percent inhibition was determined as
described in FIG. 1. Well volume was 200 .mu.l.
[0024] FIG. 6. Schematic representation of amino acid sequences of
tryptic peptides purified from cartilage as described in Methods.
Sequence similarity to human TnI is indicated by alignment with the
amino acid sequence of the human isoform.
[0025] FIG. 7. (A) RT-PCR products amplified from total RNA
purified from two separate human intercostal cartilage specimens.
Gene-specific primers were designed based on the cDNA sequence of
human fast-twitch skeletal muscle TnI. (B) Nucleotide sequence of
these PCR products showing identity to the cDNA sequence of human
fast-twitch skeletal muscle TnI (nt 189-nt 384) (SEQ ID NO:16). (C)
RT-PCR amplification, from total RNA (20 ng each lane) purified
from rat skeletal muscle (lane 1), xyphoid (lane 2), chondrosarcoma
(lane 3) and liver (lane 4). Gene-specific primers were designed
based on the cDNA sequence of rat fast-twitch skeletal muscle TnI
as described in Methods.
[0026] FIG. 8. SDS-PAGE of recombinant human TnI before (lane A)
and after (lane B) purification. In both cases, approximately 1
.mu.g of total protein was electrophoresed, followed by silver
staining as described in Methods. Recombinant TnI migrates at a
molecular weight of approximately 21,000 Da.
[0027] FIG. 9. Inhibition of capillary EC proliferation by rTnI.
Percent inhibition was determined by comparing wells exposed to the
angiogenic stimulus bFGF (A) and VEGF (B) with those exposed to
stimulus and inhibitor. Each point represents the mean of duplicate
control and inhibitor wells. This is a representative experiment of
four different EC proliferation assays, each testing different TnI
preparations.
[0028] FIG. 10. Inhibition of embryonic angiogenesis in vivo by
rTnI. After a 48 h exposure to rTnI as described in Methods,
avascular zones, free of capillaries and small vessels were
observed using a binocular dissecting microscope at .times.7-10
magnification. This zone was produced by approximately 380 pmoles
of TnI (A). A normal chorioallantoic membrane (CAM) implanted with
a methylcellulose disk containing buffer alone is shown in (B).
[0029] FIG. 11. Inhibition of FGF-induced angiogenesis by systemic
administration of TnI. TnI (50 mg/kg) was administered systemically
every 12 hours to mice whose corneas had been implanted with
pellets containing bFGF (40 ng/ml) on Day 1. After-six days of
treatment, significant inhibition of FGF-induced neovascularization
was observed in TnI-treated corneas (B) as compared to control
corneas (A).
[0030] FIG. 12. (A) Derived amino acid sequence of recombinant
human TnI (Hu) (SEQ ID NO:17) and its sequence comparison with
recombinant rabbit TnI (Rb) (SEQ ID NO:10). Identical residues are
shown by dashes. (B) Schematic representation of various
recombinant TnI deletion fragments based on rabbit TnI and
wild-type rabbit TnI.sub.w (SEQ ID NO:10). The troponin I
inhibitory region is designated I', and the sequences located on
amino- and carboxy-terminal sides of this region are designated N'
and C', respectively. TnI.sub.1-120, TnI.sub.1-94, TnI.sub.96-181,
TnI.sub.122-181 contain the N' and I', N', I' and C', and C'
regions, respectively. The number of amino acids at the beginning
and end of each fragment is indicated. TnI.sub.98-114 containing
amino acid residues 98-114 is a synthetic peptide representing the
I region.
5. DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention relates to therapeutic methods and
compositions based on troponin subunits. The invention provides for
treatment of neovascular disorders by, for example, inhibiting
angiogenesis, comprising administration of a therapeutic compound
of the invention. Such therapeutic compounds (termed herein
"Therapeutics") include: troponin C, I, and T subunits, fragments
and homologs thereof (collectively "peptides of the invention").
The peptides of the invention are characterized by the property of
inhibiting bovine endothelial cell (EC) proliferation in culture
preferably with an IC.sub.50 of about 10 .mu.M or less, more
preferably with an IC.sub.50 of about 5 .mu.M or less, most
preferably with an IC.sub.50 of about 1 .mu.M or less. In a
preferred embodiment, a Therapeutic of the invention is
administered to treat a cancerous condition, for example, to
inhibit the growth or reduce the volume of a solid tumor, or to
prevent progression from a pre-neoplastic or non-malignant state
into a neoplastic or a malignant state or to inhibit metastases. In
another specific embodiment, a Therapeutic of the invention is
administered to treat an ocular disorder associated with
neovascularization.
[0032] In a preferred aspect, a Therapeutic of the invention is a
peptide consisting of at least a fragment of troponin C, troponin
I, troponin T, or combinations thereof which is effective to
inhibit angiogenesis. More preferably, the Therapeutic is a peptide
consisting of the inhibitory (I') and carboxy terminal (C') region
(C'+I') (SEQ ID NO:14) of troponin subunit I or a fragment
thereof.
[0033] In specific embodiments, the peptides of the invention are
troponin C, troponin I and troponin T subunits, or fragments
thereof of the fast twitch, slow twitch and cardiac isoforms from
mammalian species, e.g., human, rabbit, rat, mouse, bovine, ovine
and porcine.
[0034] In other embodiments, the peptides of the invention are
troponin C, troponin I and troponin T subunits, or fragments
thereof from nonmuscle tissues, e.g., cartilage, preferably from
mammalian species, e.g., human, rabbit, rat, mouse, bovine, ovine
and porcine.
[0035] Examples of the troponin subunits that can be utilized in
accordance with the invention, include but are not limited to the
subunits of troponin from human fast twitch skeletal muscle, the
sequences of which are given below: TABLE-US-00001 Fast Twitch
Skeletal Muscle Troponin C (SEQ ID NO: 1) 1 M T D Q Q A E A R S Y L
S E E M I A E F 21 K A A F D M F D A D G G G D I S V K E L 41 G T V
M R M L G Q T P T K E E L D A I I 61 E E V D E D G S G T I D F E E
F L V M M 81 V R Q M K E D A K G K S E E E L A E C F 101 R I F D R
N A D G Y I D P E E L A E I F 121 R A S G E H V T D E E I E S L M K
D G D 141 K N N D G R I D F D E F L K M M E G V Q Fast Twitch
Skeletal Muscle Troponin I (SEQ ID NO: 2) 1 M G D E E K R N R A I T
A R R Q H L K S 21 V M L Q I A A T E L E K E E S R R E A E 41 K Q N
Y L A E H C P P L H I P G S M S E 61 V Q E L C K Q L H A K I D A A
E E E K Y 81 D M E V R V Q K T S K E L E D M N Q K L 101 F D L R G
K F K R P P L R R V R M S A D 121 A M L K A L L G S K H K V C M D L
R A N 141 L K Q V K K E D T E K E R D L R D V G D 161 W R K N I E E
K S G M E G R K K M F E S 181 E S Fast Skeletal Beta Troponin T
(SEQ ID NO: 3) 1 M S D E E V E Q V E E Q Y E E E E E A Q 21 E E E E
V Q E D T A E E D A E E E K P R 41 P K L T A P K I P E G E K V D F
D D I Q 61 K K R Q N K D L M E L Q A L I D S H F E 81 A R K K E E E
E L V A L K E R I E K R R 101 A E R A E Q Q R I R A E K E R E R Q N
R 121 L A E E K A R R E E E D A K R R A E D D 141 L K K K K A L S S
M G A N Y S S Y L A K 161 A D Q K R G K K Q T A R E M K K K I L A
181 E R R K P L N I D H L G E D K L R D K A 201 K E L W E T L H Q L
E I D K F E F G E K 221 L K R Q K Y D I T T L R S R I D Q A Q K 241
H S K K A G T P A K G K V G G R W K
[0036] In another embodiment, the invention encompasses peptides
which are homologous to troponin C (SEQ ID NO:1) or fragments
thereof, troponin I (SEQ ID NOS:2, 10, or 15) or fragments thereof,
or troponin T (SEQ ID NO:3) or fragments thereof.
[0037] In a particular embodiment, the peptides of the invention
are fragments of troponin I (SEQ ID NOS:11-15) or homologous to
fragments of troponin I (SEQ ID NOS:11-15).
[0038] In a specific embodiment, a Therapeutic of the invention is
combined with a therapeutically effective amount of another
molecule which negatively regulates angiogenesis which may be, but
is not limited to, platelet factor 4, thrombospondin-1, tissue
inhibitors of metalloproteases (TIMP1 and TIMP2) prolactin (16-Kd
fragment), angiostatin (38-Kd fragment of plasminogen), bFGF
soluble receptor, transforming growth factor .beta., interferon
alfa, and placental proliferin-related protein.
[0039] Paradoxically, neovascularization gradually reduces a
tumor's accessibility to chemotherapeutic drugs due to increased
interstitial pressure within the tumor, which causes vascular
compression and central necrosis. In vivo results have demonstrated
that rodents receiving angiogenic therapy show increased delivery
of chemotherapy to a tumor. Teicher et al., 1994, Int. J. Cancer
57:920-925. Thus, in one embodiment, the invention provides for a
pharmaceutical composition of the present invention in combination
with a chemotherapeutic agent.
[0040] In another preferred aspect, a Therapeutic of the invention
is combined with chemotherapeutic agents or radioactive isotope
exposure.
[0041] The invention is illustrated by way of examples infra which
disclose, inter alia, the inhibition of capillary endothelial cell
proliferation by troponin subunits C, I, and T and the means for
determining inhibition of capillary endothelial cell migration and
inhibition of neovascularization in vivo by troponin subunits.
[0042] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections set forth below.
5.1. Troponin Subunits, Fragments and Romologs
[0043] The invention provides for pharmaceutical compositions
comprising troponin subunits, fragments, and homologs thereof. In
particular aspects, the subunits, fragments, or homologs are of
fly, frog, mouse, rat, rabbit, pig, cow, dog, monkey, or human
troponin subunits.
[0044] In another embodiment, the invention encompasses peptides
which are homologous to troponin C (SEQ ID NO:1) or fragments
thereof. In one embodiment, the amino acid sequence of the peptide
has at least 80% identity compared to the troponin C from which it
is derived. In another embodiment, this identity is greater than
85%. In a more preferred embodiment, this identity is greater than
90%. In a most preferred embodiment, the amino acid sequence of the
peptide has at least 95% identity with the troponin C or fragment
thereof. Fragments are generally at least 10 amino acids, and in
alternate embodiments at least 20, 30, 40, 50, 75, and 100 amino
acids in length.
[0045] In another embodiment, the invention encompasses a troponin
submit subunit or fragment thereof encoded by a nucleic acid
hybridizable to the complement of a nucleic acid encoding a
troponin subunit, preferably troponin C, under low stringency,
moderate stringency or high stringency conditions.
[0046] In another embodiment, the invention encompasses peptides
which are homologous to troponin I (SEQ ID NOS:2, 10 or 15) or
fragments thereof. In one embodiment, the amino acid sequence of
the peptide has at least 80% identity with the troponin I or
fragment thereof. In another embodiment, this identity is greater
than 85%. In a more preferred embodiment, this identity is greater
than 90%. In a most preferred embodiment, the amino acid sequence
of the peptide has at least 95% identity with the troponin I or
fragment thereof. Fragments are generally at least 10 amino acids,
and in alternate embodiments at least 20, 30, 40, 50, 75, and 100
amino acids in length.
[0047] In another embodiment, the invention encompasses a troponin
submit subunit or fragment thereof encoded by a nucleic acid
hybridizable to the complement of a nucleic acid encoding a
troponin subunit, preferably troponin I, under low stringency,
moderate stringency or high stringency conditions.
[0048] In another embodiment, the invention encompasses peptides
which are homologous to troponin T (SEQ ID NO:3) or fragments
thereof. In one embodiment, the amino acid sequence of the peptide
has at least 80% identity with the troponin T or fragment thereof.
In another embodiment, this identity is greater than 85%. In a more
preferred embodiment, this identity is greater than 90%. In a most
preferred embodiment, the amino acid sequence of the peptide has at
least 95% identity with the troponin T or fragment thereof.
Fragments are generally at least 10 amino acids, and in alternate
embodiments at least 20, 30, 40, 50, 75, 100, 150, and 200 amino
acids in length.
[0049] In another embodiment, the invention encompasses a troponin
submit subunit or fragment thereof encoded by a nucleic acid
hybridizable to the complement of a nucleic acid encoding a
troponin subunit, preferably troponin T, under low stringency,
moderate stringency or high stringency conditions.
[0050] In a preferred embodiment, the invention encompasses
peptides which are homologous to the Inhibitory (I') and carboxy
terminus (C') region (C'+I') (SEQ ID NO:14).] In other embodiments,
the invention encompasses peptides that are homologous to the C'+I'
region of human troponin I (huTnI) (SEQ ID NO:17) corresponding to
amino acid residues of SEQ ID NO:17, including but not limited to
residues: 94-123 (huTnI.sub.94-123), 104-133 (huTnI.sub.104-133),
114-143 (huTnI.sub.114-143), 129-153 (huTnI.sub.129-153), 134-173
(huTnI.sub.134-173), 144-182 (huTnI.sub.144-182), 93-112
(huTnI.sub.93-112), 98-117 (huTnI.sub.98-117), 103-122
(huTnI.sub.103-122), 108-127 (huTnI.sub.108-127), 113-132
(huTnI.sub.113-132), and 118-137 (huTnI.sub.118-137). Fragments are
generally at least 10 amino acids, and in alternate embodiments at
least 20, 30, 40, 50, and 75 amino acids in length.
[0051] "Homologous," as defined herein, refers to identity over an
amino acid sequence of identical size or when compared to an
aligned sequence in which the alignment is done by a computer
homology program known in the art or whose encoding nucleic acid is
capable of hybridizing to a coding gene sequence, under high
stringency, moderate stringency, or low stringency conditions.
[0052] Specifically, by way of example, computer programs for
determining homology may include but are not limited to TBLASTN,
BLASTP, FASTA, TEASTA, and CLUSTALW (Pearson and Lipman, 1988,
Proc. Natl. Acad. Sci. USA 85(8):2444-8; Altschul et al., 1990, J.
Mol. Biol. 215(3):403-l0; Thompson, et al., 1994, Nucleic Acids
Res. 22(22):4673-80; Higgins, et al., 1996, Methods Enzymol
266:383-402; Altschul, et al., 1990, J. Mol. Biol. 215(3):403-10).
Default parameters for each of these computer programs are well
known and should be utilized.
[0053] Specifically, Basic Local Alignment Search Tool (BLAST)
(www.ncbi.nlm.nih.gov; It is to be understood that for
determination of homology, the default parameters are set and
utilized with the most recent BLAST program version available at
this site.) (Altschul et al., 1990, J. of Molec. Biol.,
215:403-410, "The BLAST Algorithm; Altschul et al., 1997, Nuc.
Acids Res. 25:3389-3402) is a heuristic search algorithm tailored
to searching for sequence similarity which ascribes significance
using the statistical methods of Karlin and Altschul 1990, Proc.
Natl Acad. Sci. USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA
90:5873-77. Five specific BLAST programs perform the following
tasks: 1) The BLASTP program compares an amino acid query sequence
against a protein sequence database; 2) The BLASTN program compares
a nucleotide query sequence against a nucleotide sequence database;
3) The BLASTX program compares the six-frame conceptual translation
products of a nucleotide query sequence (both strands) against a
protein sequence database; 4) The TBLASTN program compares a
protein query sequence against a nucleotide sequence database
translated in all six reading frames (both strands); 5) The TBLASTX
program compares the six-frame translations of a nucleotide query
sequence against the six-frame translations of a nucleotide
sequence database.
[0054] Smith-Waterman (database: European Bioinformatics Institute
wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterman, 1981, J. of Molec. Biol.,
147:195-197) is a mathematically rigorous algorithm for sequence
alignments.
[0055] FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,
85:2444-2448) is a heuristic approximation to the Smith-Waterman
algorithm. For a general discussion of the procedure and benefits
of the BLAST, Smith-Waterman and FASTA algorithms see Nicholas et
al., 1998, "A Tutorial on Searching Sequence Databases and Sequence
Scoring Methods" (www.psc.edu) and references cited therein.
[0056] It is envisioned that troponin subunits and fragments can be
made by altering troponin sequences by substitutions, additions or
deletions that provide for functionally equivalent molecules
capable of displaying one or more functional activities associated
with a full-length wild-type troponin subunit. Such functional
activities include but are not limited to inhibition of
angiogenesis; inhibition of metastases; inhibition of tumor growth.
These include, but are not limited to, troponin subunits,
fragments, or homologs containing, as a primary amino acid
sequence, all or part of the amino acid sequence of a troponin
subunit including altered sequences in which functionally
equivalent amino acid residues are substituted for residues within
the sequence resulting in a silent change. For example, one or more
amino acid residues within the sequence can be substituted by
another amino acid of a similar polarity which acts as a functional
equivalent, resulting in a silent alteration. Substitutes for an
amino acid within the sequence may be selected from other members
of the class to which the amino acid belongs. For example, the
nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
[0057] One embodiment of the invention provides for molecules
consisting of or comprising a fragment of at least 10 (contiguous)
amino acids of a troponin subunit which is capable of inhibiting
endothelial cell proliferation as discussed above. In other
embodiments, this molecule consists of at least 20 or 50 amino
acids of the troponin subunit. In specific embodiments, such
molecules consist of or comprise fragments of a troponin subunit
that are at least 20, 30, 40, 50, 75, 100 and 150 amino acids in
length, including but not limited to, C'+I' (SEQ ID NO:14),
huTnI.sub.94-123, huTnI.sub.104-133, huTnI.sub.114-143,
huTnI.sub.129-153, huTnI.sub.134-173, huTnI.sub.144-182,
huTnI.sub.93-122, huTnI.sub.98-117, huTnI.sub.103-122,
huTnI.sub.108-127, huTnI.sub.113-132, and huTnI.sub.118-137.
[0058] In a preferred embodiment, the protein is a mammalian
troponin subunit. In more preferred embodiments, it is a mammalian
troponin C, I, or T subunit.
[0059] The troponin subunits, fragments and homologs of the
invention can be derived from tissue (see, for example, Section 6,
Examples 1 and 7; Ebashi et al., 1968, J. Biochem. 64:465; Yasui et
al., 1968, J. Biol. Chem. 243:735; Hartshorne et al., 1968,
Biochem. Biophye. Res. Commun. 31:647; Shaub et al., 1969, Biochem.
J. 115:993; Greater et al., 1971, J. Biol. Chem. 246:4226-4733;
Brekke et al., 1976, J. Biol. Chem. 251:866-871; and Yates et al.,
1983, J. Biol. Chem. 258:5770-5774) or produced by various methods
known in the art, for example, recombinant techniques (see, for
example, Section 6, Examples 1 and 7).
[0060] Manipulations of troponin subunits can occur at the gene or
protein level. For example, a cloned troponin gene sequence coding
for troponin subunits C, I, or T, can be modified by any of
numerous strategies known in the art. Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. The sequence can be cleaved at
appropriate sites with restriction endonuclease(s), followed by
further enzymatic modification if desired, isolated, and ligated in
vitro. In the production of the gene encoding a fragment or homolog
of a troponin subunit, care should be taken to ensure that the
modified gene remains within the same translational reading frame
as the troponin subunit gene, uninterrupted by translational stop
signals, in the gene region where the desired troponin activity is
encoded.
[0061] In a specific embodiment, a nucleic acid which is
hybridizable to the complement of a troponin nucleic acid (e.g.,
having a sequence as set forth in SEQ ID NOS:13-17), or to a
nucleic acid encoding a troponin fragment or derivative under
conditions of low stringency is provided. By way of example and not
limitation, procedures using such conditions of low stringency are
as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad.
Sci. U.S.A. 78, 6789-6792). Filters containing DNA are pretreated
for 6 h at 40.degree. C. in a solution containing 35% formamide,
5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%
Ficoll, 1% BSA, and 500 .mu.g/ml denatured salmon sperm DNA.
Hybridizations are carried out in the same solution with the
following modifications: 0.02% PVP, 0.02% Ficoll, 0.2t BSA, 100
.mu.g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and
5-20.times.10.sup.6 cpm .sup.32P-labeled probe is used. Filters are
incubated in hybridization mixture for 18-20 h at 40.degree. C.,
and then washed for 1.5 h at 55.degree. C. in a solution containing
2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The
wash solution is replaced with fresh solution and incubated an
additional 1.5 h at 60.degree. C. Filters are blotted dry and
exposed for autoradiography. If necessary, filters are washed for a
third time at 65-68.degree. C. and re-exposed to film. Other
conditions of low stringency which may be used are well known in
the art (e.g., as employed for cross-species hybridizations).
[0062] In another specific embodiment, a nucleic acid which is
hybridizable to a troponin nucleic acid under conditions of high
stringency is provided. By way of example and not limitation,
procedures using such conditions of high stringency are as follows.
Prehybridization of filters containing DNA is carried out for 8 h
to overnight at 65.degree. C. in buffer composed of 6.times.SSC, 50
mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%
BSA, and 500 .mu.g/ml denatured salmon sperm DNA. Filters are
hybridized for 48 h at 65.degree. C. in prehybridization mixture
containing 100 .mu.g/ml denatured salmon sperm DNA and
5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Washing of
filters is done at 37.degree. C. for 1 h in a solution containing
2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is
followed by a wash in 0.1.times.SSC at 50.degree. C. for 45 min
before autoradiography. Other conditions of high stringency which
may be used are well known in the art.
[0063] In another specific embodiment, a nucleic acid which is
hybridizable to a troponin nucleic acid under conditions of
moderate stringency is provided. Selection of appropriate
conditions for such stringencies is well known in the art (see
e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; see also, Ausubel et al., eds., in the Current
Protocols in Molecular Biology series of laboratory technique
manuals, .COPYRGT. 1987-1997 Current Protocols, .COPYRGT. 1994-1997
John Wiley and Sons, Inc.).
[0064] Additionally, the troponin subunit encoding nucleic acid
sequence can be mutated in vitro or in vivo, to create and/or
destroy translation, initiation, and/or termination sequences, or
to create variations in coding regions and/or form new restriction
endonuclease sites or destroy preexisting ones, to facilitate
further in vitro modification. Any technique for mutagenesis known
in the art can be used, including, but not limited to, in vitro
site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.
253:6551), use of TAB.RTM. linkers (Pharmacia), etc.
[0065] Manipulations of troponin subunit C, I, or T sequence may
also be made at the protein level. Included within the scope of the
invention are troponin subunit fragments or other fragments or
homologs which are differentially modified during or after
translation, e.g., by acetylation, phosphorylation, carboxylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to an antibody molecule or other
cellular ligand, etc. Any of numerous chemical modifications may be
carried out by known techniques, including, but not limited to,
specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH.sub.4, acetylation,
formylation, oxidation, reduction, etc.
[0066] In addition, fragments and homologs of troponin subunits can
be chemically synthesized. For example, a peptide corresponding to
a portion of a troponin subunit which comprises the desired domain,
or which mediates the desired activity in vitro, can be synthesized
by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino acids or chemical amino acid homologs can be
introduced as a substitution or addition into the troponin subunit
sequence. Non-classical amino acids include, but are not limited
to, the D-isomers of the common amino acids, .alpha.-amino
isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine,
citrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, .beta.-alanine, designer amino
acids such as .beta.-methyl amino acids, C.alpha.-methyl amino
acids, and N.alpha.-methyl amino acids.
[0067] In a specific embodiment, the invention encompasses a
chimeric, or fusion, protein comprising a troponin subunit or
fragment thereof (consisting of at least a domain or motif of the
troponin subunit that is responsible for inhibiting endothelial
cell proliferation) joined at its amino or carboxy-terminus via a
peptide bond to an amino acid sequence of a different protein. Such
a chimeric product can be made by ligating the appropriate nucleic
acid sequences encoding the desired amino acid sequences to each
other by methods known in the art, in the proper coding frame, and
expressing the chimeric product by methods commonly known in the
art. Alternatively, such a chimeric product may be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
[0068] In another embodiment, the invention encompasses combination
of the troponin subunits, fragments, or homologs of the present
invention to inhibit angiogenesis. Another embodiment provides for
the combination of troponin subunits, fragments, or homologs with
other angiogenesis inhibiting factors. Such angiogenesis inhibiting
factors include, but are not limited to: angiostatic steroids,
thrombospondin, platelet factor IV, transforming growth factor
.beta., interferons, tumor necrosis factor a, bovine vitreous
extract, protamine, tissue inhibitors of metalloproteinases (TIMP-1
and TIMP-2), prolactin (16-kd fragment), angiostatin (38-kd
fragment of plasminogen), bFGF soluble receptor, and placental
proliferin-related protein. See, e.g., reviews by Folkman et al.,
1995, N. Engl. J. Med. 333:1757-1763 and Klagsbrun et al., 1991,
Annu. Rev. Physiol. 53:217-239.
5.2. Assays of Troponin Proteins Fragments and Homologs
[0069] The functional activity and/or therapeutically effective
dose of troponin subunits, fragments and homologs, can be assayed
in vitro by various methods. These methods are based on the
physiological processes involved in angiogenesis and while they are
within the scope of the invention, they are not intended to limit
the methods by which troponin subunits, fragments and homologs
inhibiting angiogenesis are defined and/or a therapeutically
effective dosage of the pharmaceutical composition is
determined.
[0070] For example, where one is assaying for the ability of
troponin subunits, fragments, and homologs, to inhibit or interfere
with the proliferation of capillary endothelial cells (EC) in
vitro, various bioassays known in the art can be used, including,
but not limited to, radioactive incorporation into nucleic acids,
colorimetric assays and cell counting.
[0071] Inhibition of endothelial cell proliferation may be measured
by colorimetric determination of cellular acid phosphatase activity
or electronic cell counting. These methods provide a quick and
sensitive screen for determining the number of endothelial cells in
culture after treatment with the troponin subunit, fragment, or
homolog of the invention, and an angiogenesis stimulating factor
such as aFGF. The calorimetric determination of cellular acid
phosphatase activity is described by Connolly et al., 1986, J.
Anal. Biochem. 152:136-140. According to this method, described in
Example 9, capillary endothelial cells are treated with
angiogenesis stimulating factors, such as aFGF, and a range of
potential inhibitor concentrations. These samples are incubated to
allow for growth, and then harvested, washed, lysed in a buffer
containing a phosphatase substrate, and then incubated a second
time. A basic solution is added to stop the reaction and color
development is determined at 405 .lamda.. According to Connolly et
al., a linear relationship is obtained between acid phosphatase
activity and endothelial cell number up to 10,000 cells/sample.
Standard curves for acid phosphatase activity are also generated
from known cell numbers in order to confirm that the enzyme levels
reflect the actual EC numbers. Percent inhibition is determined by
comparing the cell number of samples exposed to stimulus with those
exposed to both stimulus and inhibitor.
[0072] Colorimetric assays to determine the effect of troponin
subunits C, I, and T on endothelial cell proliferation demonstrate
that all three troponin subunits interfere with bFGF-stimulated
endothelial cell proliferation but have no detectable inhibitory
effect on the growth of Balb/c 3T3 cells, a non-endothelial cell
line. For an illustrative example, see Section 6, Examples 3 and 8,
infra.
[0073] The incorporation of radioactive thymidine by capillary
endothelial cells represents another means by which to assay for
the inhibition of endothelial cell proliferation by a potential
angiogenesis inhibitor. According to this method, a predetermined
number of capillary endothelial cells are grown in the presence of
.sup.3H-Thymidine stock, an angiogenesis-stimulator such as for
example, bFGF, and a range of concentrations of the angiogenesis
inhibitor to be tested. Following incubation, the cells are
harvested and the extent of thymidine incorporation is determined.
See, e.g., Section 6, Example 3.
[0074] The ability of varying concentrations of troponin subunits,
fragments or homologs to interfere with the process of capillary
endothelial cell migration in response to an angiogenic stimulus
can be assayed using the modified Boyden chamber technique. See,
e.g., Section 6, Example 4, infra.
[0075] Another means by which to assay the functional activity of
troponin subunits, fragments and homologs, involves examining the
ability of the compounds to inhibit the directed migration of
capillary endothelial cells which ultimately results in capillary
tube formation. This ability may be assessed for example, using an
assay in which capillary endothelial cells plated on collagen gels
are challenged with the inhibitor, and determining whether
capillary-like tube structures are formed by the cultured
endothelial cells.
[0076] Assays for the ability to inhibit angiogenesis in vivo
include the chorioallantoic membrane assay and corneal pocket
assays (see, e.g., Section 6, infra, Example 10, and Example 11,
respectively). See also, Polverini et al., 1991, Methods Enzymol.
198:440-450. According to the corneal pocket assay, a tumor of
choice is implanted into the cornea of the test animal in the form
of a corneal pocket. The potential angiogenesis inhibitor is
applied to the corneal pocket and the corneal pocket is routinely
examined for neovascularization. See, e.g., Example 11 infra.
[0077] The therapeutically effective dosage for inhibition of
angiogenesis In vivo, defined as inhibition of capillary
endothelial cell proliferation, migration, and/or blood vessel
ingrowth, may be extrapolated from in vitro inhibition assays using
the compositions of the invention above or in combination with
other angiogenesis inhibiting factors. The effective dosage is also
dependent on the method and means of delivery. For example, in some
applications, as in the treatment of psoriasis or diabetic
retinopathy, the inhibitor is delivered in a topical-ophthalmic
carrier. In other applications, as in the treatment of solid
tumors, the inhibitor is delivered by means of a biodegradable,
polymeric implant.
5.3. Therapeutic Uses
[0078] The invention provides for compositions and methods for
inhibition of angiogenesis. The invention further provides for
compositions and methods for treatment or prevention of diseases or
disorders associated with neovascularization by administration of a
therapeutic compound of the invention. Such compounds (termed
herein "Therapeutics") include troponin subunits and fragments and
homologs thereof (e.g., as described supra).
5.3.1. Malignancies
[0079] Malignant and metastatic conditions which can be treated
with the Therapeutic compounds of the present invention include,
but are not limited to, the solid tumors listed in Table 1 (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia) and blood-borne tumors
such as leukemias. TABLE-US-00002 TABLE 1 MALIGNANCIES AND RELATED
DISORDERS Solid tumors sarcomas and carcinomas fibrosarcoma
myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma
angiosarcoma endotheliosarcoma lymphangiosarcoma
lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor
leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer
breast cancer ovarian cancer prostate cancer squamous cell
carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma
sebaceous gland carcinoma papillary carcinoma papillary
adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic
carcinoma renal cell carcinoma hepatoma bile duct carcinoma
choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical
cancer testicular tumor lung carcinoma small cell lung carcinoma
bladder carcinoma epithelial carcinoma glioma astrocytoma
medulloblastoma craniopharyngioma ependymoma Kaposi's sarcoma
pinealoma hemangioblastoma acoustic neuroma ooligodendroglioma
menangioma melanoma neuroblastoma retinoblastoma
5.3.2. Ocular Disorders
[0080] Ocular disorders associated with neovascularization which
can be treated with the Therapeutic compounds of the present
invention include, but are not limited to:
[0081] neovascular glaucoma
[0082] diabetic retinopathy
[0083] retinoblastoma
[0084] retrolental fibroplasia
[0085] uveitis
[0086] retinopathy of prematurity
[0087] macular degeneration
[0088] corneal graft neovascularization
as well as other eye inflammatory diseases, ocular tumors and
diseases associated with choroidal or iris neovascularization. See,
e.g., reviews by Waltman et al., 1978, Am. J. Ophthal. 85:704-710
and Gartner et al., 1978, Surv. Ophthal. 22:291-312.
5.3.3. Other Disorders
[0089] Other disorders which can be treated with the Therapeutic
compounds of the present invention include, but are not limited to,
hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic
plaques, delayed wound healing, granulations, hemophilic joints,
hypertrophic scars, nonunion fractures, Osler-Weber syndrome,
pyogenic granuloma, scleroderma, trachoma, and vascular
adhesions.
5.4. Demonstration of Therapeutic or Propryiactic Utility
[0090] The Therapeutics of the invention can be tested in vivo for
the desired therapeutic or prophylactic activity as well as for
determination of therapeutically effective dosage. For example,
such compounds can be tested in suitable animal model systems prior
to testing in humans, including, but not limited to, rats, mice,
chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to
administration to humans, any animal model system known in the art
may be used.
5.5. Therapeutic/Prophylactic Administration and Compositions
[0091] The invention provides methods of inhibition of angiogenesis
and method of treatment (and prophylaxis) by administration to a
subject an effective amount of a Therapeutic of the invention. In a
preferred aspect, the Therapeutic is substantially purified as set
forth in Examples 1 and 7. The subject is preferably an animal,
including, but not limited to, animals such as cows, pigs,
chickens, etc., and is more preferably a mammal, and most
preferably a human.
[0092] The invention also provides for methods of treatment and
prevention by administration of an effective amount of a
Therapeutic of the invention to an immunocompromised patient, for
example, a patient having cancer or infected with human
immunodeficiency virus (HIV). In particular, the invention may be
used to treat or prevent secondary infections or diseases
associated with HIV infection or cancers.
[0093] The invention further provides methods of treatment and
prevention by administration to a subject, an effective amount of a
Therapeutic of the invention combined with a chemotherapeutic agent
and/or radioactive isotope exposure.
[0094] The invention also provides for methods of treatment and
prevention of a Therapeutic of the invention for patients who have
entered a remission in order to maintain a dormant state.
[0095] Various delivery systems are known and can be used to
administer a Therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, receptor-mediated
endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
262:4429-4432). Methods of introduction include, but are not
limited to, topical, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, ophthalmic, and
oral routes. The compounds may be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. It is preferred
that administration is localized, but it may be systemic. In
addition, it may be desirable to introduce the pharmaceutical
compositions of the invention into the central nervous system by
any suitable route, including intraventricular and intrathecal
injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration can also be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent.
[0096] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0097] For topical application, the purified troponin subunit is
combined with a carrier so that an effective dosage is delivered,
based on the desired activity (i.e., ranging from an effective
dosage, for example, of 1.0 .mu.M to 1.0 mM to prevent localized
angiogenesis, endothelial cell migration, and/or inhibition of
capillary endothelial cell proliferation. In one embodiment, a
topical troponin subunit, fragment or homolog is applied to the
skin for treatment of diseases such as psoriasis. The carrier may
in the form of, for example, and not by way of limitation, an
ointment, cream, gel, paste, foam, aerosol, suppository, pad or
gelled stick.
[0098] A topical Therapeutic for treatment of some of the eye
disorders discussed infra consists of an effective amount of
troponin subunit, fragment, or homolog, in a ophthalmologically
acceptable excipient such as buffered saline, mineral oil,
vegetable oils such as corn or arachis oil, petroleum jelly,
Miglyol 182, alcohol solutions, or liposomes or liposome-like
products. Any of these compositions may also include preservatives,
antioxidants, antibiotics, immunosuppressants, and other
biologically or pharmaceutically effective agents which do not
exert a detrimental effect on the troponin subunit.
[0099] For directed internal topical applications, for example for
treatment of ulcers or hemorrhoids, the troponin subunit, fragment,
or homolog composition may be in the form of tablets or capsules,
which can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; or a
glidant such as colloidal silicon dioxide. When the dosage unit
form is a capsule, it can contain, in addition to material of the
above type, a liquid carrier such as a fatty oil. In addition,
dosage unit forms can contain various other materials which modify
the physical form of the dosage unit, for example, coatings of
sugar, shellac, or other enteric agents.
[0100] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0101] In another embodiment, the Therapeutic can be delivered in a
vesicle, in particular a liposome. See, Langer et al., 1990,
Science 249:1527-1533; Treat et al., 1989, in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, N.Y., pp. 353-365; Lopez-Berestein, ibid., pp.
317-327.
[0102] In yet another embodiment, the Therapeutic can be delivered
in a controlled release system. In one embodiment, an infusion pump
may be used to administer troponin subunit, such as for example,
that used for delivering insulin or chemotherapy to specific organs
or tumors (see Langer, supra; Sefton, CRC Crit. Ref. Biomed., 1987,
Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al.,
1989, N. Engl. J. Med. 321:574.
[0103] In a preferred form, the troponin subunit, fragment, or
homolog is administered in combination with a biodegradable,
biocompatible polymeric implant which releases the troponin
subunit, fragment, or homolog over a controlled period of time at a
selected site. Examples of preferred polymeric materials include
polyanhydrides, polyorthoesters, polyglycolic acid, polylactic
acid, polyethylene vinyl acetate, and copolymers and blends
thereof. See, Medical Applications of Controlled Release, Langer
and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.; Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), 1984, Wiley, New York; Ranger and Peppas, 1983, J.
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 71:105. In yet another
embodiment, a controlled release system can be placed in proximity
of the therapeutic target, i.e., the brain, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, 1989, supra, vol. 2, pp.
115-138).
[0104] Other controlled release systems are discussed in the review
by Langer (1990, Science 249:1527-1533).
[0105] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a Therapeutic, and a pharmaceutically
acceptable carrier.
[0106] The pharmaceutical compositions of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0107] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents. Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents such as acetates, citrates or
phosphates. Antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid; and
agents for the adjustment of tonicity such as sodium chloride or
dextrose are also envisioned. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0108] These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides, microcrystalline cellulose, gum tragacanth
or gelatin. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciencesn by E. W. Martin. Such
compositions will contain a therapeutically effective amount of the
Therapeutic, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0109] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0110] The amount of the Therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro
assays such as those discussed in section 5.2 may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. However, suitable dosage ranges for
intravenous administration of full-length troponin subunits are
generally about 20-500 micrograms of active compound per kilogram
body weight. Suitable dosage ranges for intranasal administration
of full-length troponin subunits are generally about 0.01 pg/kg
body weight to 1 mg/kg body weight. Suitable dosage ranges for
intravenous administration of troponin fragments are generally
about 10 micrograms to 1 milligram of active compound per kilogram
body weight, preferably about 1-50 milligrams per administration,
more preferably about 1-20 milligrams per human. Effective doses
may be extrapolated from dose-response curves derived from in vitro
or animal model test bioassays or systems.
[0111] Administration of the doses recited above can be repeated.
In a preferred embodiment, the doses recited above are administered
2 to 7 times per week. The duration of treatment depends upon the
patient's clinical progress and responsiveness to therapy.
[0112] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0113] Modifications and variations of the compositions of the
present invention, and methods for use, will be obvious to those
skilled in the art from the foregoing detailed description. Such
modifications and variations are intended to fall within the scope
of the appended claims.
[0114] The following non-limiting examples demonstrate the
discovery of troponin subunit inhibition of angiogenic stimulus
induced endothelial cell proliferation, and means for determining
the effective dosage of troponin subunit, fragment, or homolog to
inhibit angiogenesis, as well as for identifying troponin subunit
fragments and homologs (i.e., those fragments or homologs of
troponin subunit capable of inhibiting angiogenesis. The troponin
subunit used in the examples is purified as described infra.
6. EXAMPLES
Example 1
Purification of Troponin Subunit Components
Cardiac Troponin Isolation from Tissue
[0115] The procedures of Ebashi et al., 1968, J. Biochem.
64:465-477; Yasui et al., 1968, J. Biol. Chem. 243:735-742;
Hartshorne et al., 1969, Biochim. Biophys. Acta, 175:30; Schaub et
al., 1969, Biochem. J. 115:993-1004; Greater et al., 1971, J. Biol.
Chem. 246:4226-4233; and Greater et al., 1973, J. Biol. Chem.
248:2125-2133 for purifying troponin can be used. Rabbit back and
leg muscles are removed, cleaned of fat and connective tissue, and
ground. The ground muscle (1 kg) is stirred for 5 min. in 2 liters
of a solution containing 20 mM KCl, 1 mM KHCO.sub.3, 0.1 mM
CaCl.sub.2, and 0.1 mM DTT..sup.1 The suspension is filtered
through cheesecloth, and the washing of the residue is repeated
four times. Two liters of 95% ethanol are then added to the washed
residue and the solution filtered after 10 min. The ethanol
extraction is repeated twice. The residue is then washed 3 times
with 2 liters of diethyl ether for 10 min. Finally the residue is
allowed to dry at room temperature for 2 to 3 hours. .sup.1 The
abbreviations used are: DDT, dithiothreitol; EGTA, ethylene glycol
bis(.beta.-aminoethyl ether)-N,N'-tetraacetate; SDS, sodium dodecyl
sulfate; SE-, sulfoethyl.
[0116] The dried powder (from 1 kg of muscle) is extracted
overnight at 22.degree. with 2 liters of a solution containing 1 M
KCl, 25 mM Tris (pH 8.0), 0.1 mM CaCl.sub.2, and 1 mM DTT. After
filtration through cheesecloth, the residue is once more extracted
with 1 liter of 1 M KCl.
[0117] The extracts are combined and cooled to 4.degree. C. Solid
ammonium sulfate is added to produce approximately 40% saturation
(230 g per liter). After 30 min. the solution is centrifuged and
125 g of ammonium sulfate is then added per liter of supernatant
(60% saturation). After centrifugation the precipitate is dissolved
in 500 ml of a solution containing 5 mM Tris (pH 7.5), 0.1 mM
CaCl.sub.2, and 0.1 mM DTT and dialyzed against 15 liters of the
same solution for 6 hours and against a fresh solution
overnight.
[0118] Solid KCl is added to a final concentration of 1 M and 1 M
KCl solution is added to bring the volume to 1 liter. The pH is
then adjusted to 4.6 by addition of HCl, and the tropomyosin
precipitate is removed by centrifugation. The pH of the supernatant
is adjusted to 7.0 with KOH, and 450 g of ammonium sulfate are
added per liter (70% saturation). The precipitate is dissolved in a
solution containing 5 mM Tris (pH 7.5, 0.1 mM CaCl.sub.2, and 0.1
mM DTT, and dialyzed overnight against the same solution. Solid KCl
is added to bring its concentration to 1 M, the pH adjusted to 4.6,
and the precipitate which forms is removed by centrifugation. The
neutralized supernatant is dialyzed against 2 mM Tris (pH 7.5)
until the Nessler reaction is negative. The final yield of troponin
is usually 2.5 to 3.0 g per kg of fresh muscle.
Cardiac Troponin Isolation from Tissue
[0119] Bovine hearts are obtained approximately 30 min. after death
and immediately cut open, rinsed of blood, and immersed in ice. The
left ventricle is removed, trimmed of excess fat and connective
tissue, and ground. All subsequent extraction and preparation steps
are performed at 0-3.degree. except where noted. The ground muscle
(500 g) is homogenized in a Waring Blender for 1 min. in 2.5 liters
of solution containing 0.09 M KH.sub.2PO.sub.4, 0.06 M
K.sub.2HPO.sub.4, 0.3 M KCl, 5 mM 2-mercaptoethanol, pH 6.8. The
homogenized muscle suspension is then stirred for 30 min. and
centrifuged at 1000.times.g for 20 min. The precipitate is
re-extracted for 30 min. and centrifuged. The residue is then
washed with 2.5 liters of 5 mM 2-mercaptoethanol and centrifuged at
1000.times.g for 10 min., followed by two successive washings and
centrifugations with 1.5 liters of 50 mM KCl, 5 mM Tris-HCl (pH
8.1), and 5 mM 2-mercaptoethanol. The residue is then washed and
centrifuged twice with 1.5 liters of 50 mM Tris-HCl (pH 8.1), and 5
mM 2-mercaptoethanol. The volume of the residue is measured, and
the residue is mixed with 0.5 volume of 3 M KCl, 50 mM Tris-HCl (pH
8.1), and 5 mM 2-mercaptoethanol. After a 16- to 20-hour extraction
at 0.degree., the suspension is centrifuged at 15,000.times.g for
10 min. The sediment is discarded, and the supernatant is adjusted
to pH 7.6 with 0.05 N HCl. The filamentous precipitate which forms
upon pH adjustment is removed by filtering the extract through
nylon gauze. The protein that precipitates between 30 and 50%
ammonium sulfate saturation is collected, dissolved in a solution
containing 1 M KCl, and 1 mM potassium phosphate (pH 6.8), and 5 mM
2-mercaptoethanol, and dialyzed against the same solution for 4
hours and against a fresh solution overnight. The protein solution
is clarified by centrifugation at 105,000.times.g for 30 min. The
troponin is then purified by chromatography on a hydroxylapatite
column with the protein being eluted between 0.08 and 0.10 M
phosphate. Greater et al., 1972 Cold Spring Harbor Symp. Quant.
Biol. 37:235-244. Rabbit cardiac troponin is prepared in a similar
manner using a pooled batch of hearts which has been stored at
-20.degree. C. prior to extraction.
[0120] The troponin subunits are separated by DEAE-Sephadex
chromatography in 6 M urea. Bovine cardiac tropomyosin is prepared
from the 50% ammonium sulfate saturation supernatant from the
troponin extraction scheme (see above). Ammonium sulfate is added
to 65% saturation, and the precipitate is dissolved in and dialyzed
versus 1 M KCl, 1 mM potassium phosphate (pH 7.0), and 5 mM
2-mercaptoethanol. The protein is then purified by hydroxylapatite
chromatography.
Protein Determination
[0121] Protein concentrations are determined by the biuret method
of Gornall et al. using bovine serum albumin as a standard. Gornall
et al., 1949, J. Biol. Chem., 177:751-766.
Separation of Components
[0122] A sequence of SP-Sephadex and DEAE-Sephadex chromatography
gives complete separation of the three cardiac troponin
components.
Recombinant Troponin Isolation and Reconstitution Protocols
Troponin I and T
[0123] DNA encoding various troponin subunits and isoforms are
known in the art. See, e.g., Wu et al., 1994, DNA Cell. Biol.
13:217-233; Schreier et al., 1990, Biol. Chem. 265:21247-21253; and
Gahlmann et al., 1990, J. Biol. Chem. 265:12520-12528.
[0124] To express a troponin subunit, DNA encoding the subunit is
subcloned into a high copy number expression plasmid, such as
KP3998, using recombinant techniques known in the art.
[0125] To express the cloned cDNA, E. coli transformed with the
insert-containing pKP1500 vector is grown overnight at 37.degree.
C., then inoculated into 4 liters of Luria-Bertani broth (LB)
medium and grown at 42.degree. C. until mid-log phase.
Isopropyl-1-thio-.beta.-D-galactopyranoside is then added to 0.5
mM, and the culture is allowed to grow at 42.degree. C. overnight.
Purification of expressed troponin subunit, fragment, or homolog
may be adapted from published procedures (Reinach et al., 1988, J.
Biol. Chem. 250:4628-4633 and Xu et al., 1988, J. Biol. Chem.
263:13962-13969). The cells are harvested by centrifugation and
suspended in 20 ml of 20 mM Tris, 20% sucrose, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 1 mg/ml lysozyme, pH 7.5. After
incubation on ice for 30 min., 80 ml of 20 mM Tris, 1 mM EDTA, 0.2
mM phenylmethylsulfonyl fluoride, 0.5 mM DTT is added and the cells
broken in a French press (SLM Instruments). The cell debris is
pelleted; the supernatant is made 35% in saturated
(NH.sub.4).sub.2SO.sub.4 and stirred on ice for 30 min. After
sedimentation, the supernatant is made 50 mM in NaCl, 5 mM in
CaCl.sub.2, 1 mM in MgCl.sub.2, and 1 mM in DTT and then loaded
onto a 1.5.times.25-cm phenyl-Sepharose (Pharmacia LKB
Biotechnology Inc.) column. The column is washed first with 50 mM
Tris, 50 mM NaCl, 5 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM DTT, pH
7.5, then with 50 mM Tris, 1 mM NaCl, 0.1 mM CaCl.sub.2, 1 mM DTT,
pH 7.5, until no more protein is eluted. The crude troponin subunit
is then eluted with 50 mM Tris, 1 mM EDTA, 1 mM DTT, pH 7.5.
Fractions that contain troponin subunit, fragment, or homolog are
pooled, dialyzed against 25 mM Tris, 6 M urea (United States
Biochemical Corp.), 1 mM MgCl.sub.2, 1 mM DTT, pH 8.0, and loaded
onto a 1.5.times.25-cm DES2 (Whatman) column. The column is eluted
with a 0-0.6 M NaCl linear gradient. Troponin subunit, fragment, or
homolog which elutes from the column is dialyzed against 0.1 mM
NH.sub.4HCO.sub.3, 1 mM .beta.-mercaptoethanol, lyophilized, and
stored. Purity is assessed by SDS-polyacrylamide gel
electrophoresis and UV spectrophotometry. Typical yields of 6 mg of
purified recombinant troponin subunit, fragment, or homolog/liter
of bacterial culture are expected.
[0126] The lyophilized recombinant protein is resuspended in a take
up buffer consisting of 6M urea, 20 mM Hepes (pH 7.5), 0.5M NaCl, 2
mM EDTA, and 5 mM DTT. The mixture is nutated at room temperature
for 1 hour. The solution is then dialyzed at 4.degree. C. for six
hours with 1 exchange against a dialysis buffer consisting of 0.5M
NaCl, 20 mM Hepes (pH 7.5), and 0.5mM DTT.
[0127] Protein concentration is determined for each subunit at
280.lamda.. The extension coefficient of Troponin I is 0.40 and
Troponin T is 0.50.
Troponin C
[0128] The lyophilized recombinant protein is resuspended in a take
up buffer consisting of 0.1 M NaCl, 20 mM Hepes (pH 7.5), 2mM EDTA,
and 5 mM DTT. This solution is dialyzed for 6 hours at 4.degree. C.
with one exchange against a dialysis buffer of 0.1 M NaCl, 20 mM
Hepes (pH 7.5), and 0.5 mM DTT.
[0129] Protein concentration is determined by measuring absorbance
at 280.lamda.. The extension coefficient for troponin C is
0.18.
Reconstitution of Combined Subunits
[0130] Protein concentrations having the same reconstitution molar
ratios of troponin subunits C, I, and T are maintained for all
various combinations. These concentrations of the respective
proteins are combined in a reconstitution buffer consisting of 0.1
M NaCl, 0.1 M CaCl2, 5 mM DTT, 5 mM Hepes (pH 7.5). Dialysis is for
20-24 hours at 4.degree. C. with three exchanges over a dialysis
buffer consisting of 0.1 M NaCl, 0.1 m CaCl.sub.2, 0.5 mM DTT, and
5 mM Hepes (pH 7.5).
[0131] Protein concentration is approximated by measuring
absorption at 278.lamda.. The troponin trimer has an extension
coefficient of 0.45 at 278.lamda..
Example 2
Inhibition of Endothelial Cell Proliferation Measured by DNA
Synthesis
[0132] The inhibitory effect of troponin subunit, fragment, or
homolog on the proliferation of bFGF-stimulated EC can be measured
according to the following procedure.
Endothelial Cell DNA Synthesis
[0133] On day one, 5,000 bovine capillary endothelial cells in
DMEM/10% CS/1% GPS are plated onto each well of a 96-well
pregelatinized tissue culture plate. On day two, the cell media is
changed to DMEM, 2% CS, 1% GPS, 0.5% BSA (complete medium),
supplemented with 10 .mu.l of 1 mg/ml "cold" thymidine per 50 ml of
medium. On day three, test samples in complete medium are added in
duplicate. Additionally, beta Fibroblast Growth Factor (bFGF) is
added to each well except for the appropriate controls, to a final
concentration of 0.2 ng/well. On day four, 5 .mu.l of 1:13 diluted
.sup.3H-Thymidine stock is added to each well and the plate is
incubated for 5-6 hours. Following incubation, the medium is
aspirated, and the remainder is rinsed once with PBS, then twice
for 5 minutes each with methanol followed by two rinses each for 10
minutes with 5% TCA. The cells are then rinsed with water three
times, dried to the plate, and 100 .mu.l of 0.3 N NaOH is added to
each well. The contents of the well are then transfered to the
scintillation counter vials and 3 mls of Ecolume added to each
vial. Samples are then counted on the scintillation counter.
3T3 Cell DNA Synthesis
[0134] DNA synthesis in bFGF-stimulated 3T3 cells provides a
control with which to evaluate results obtained for bFGF stimulated
endothelial cell proliferation. DNA synthesis in the 3T3 cells can
be determined according to the following method.
[0135] BALB/c 3T3 cells are trypsinized and resuspended at a
concentration of 5.times.10.sup.4 cells/ml. Aliquots of 200 .mu.l
are plated into 0.3 cm.sup.2 microtiter wells (Microtest II tissue
Culture Plates, Falcon). After reaching confluence, in a period of
2 to 3 days, the cells are further incubated for a minimum of 5
days in order to deplete the media of growth promoting factors.
These growth conditions yield confluent monolayers of non-dividing
BALB/c 3T3 cells. Test samples are dissolved in 50 .mu.l of 0.15 M
NaCl and added to microtiter wells, along with [.sup.3H]TdR. After
an incubation of at least 24 hours, the media is removed and the
cells are washed in PBS. Fixation of the cells and removal of
unincorporated [.sup.3H]TdR is accomplished by the following
successive steps; addition of methanol twice for periods of 5
minutes, 4 washes with H.sub.2O, addition of cold 5% TCA twice for
periods of 10 minutes, and 4 washes with H.sub.2O. DNA synthesis is
measured either by liquid scintillation counting or by
autoradiography using a modification of the method described by
Haudenschild et al., 1976, M. Exp. Cell Res. 98:175. For
scintillation counting, cells are lysed in 150 .mu.l of 0.3 N NaOH
and counted in 5 ml of Insta-Gel liquid scintillation cocktail
(Packard) using a Packard Tri-Carb liquid scintillation counter.
Alternatively, autoradiography may be used to quantitate DNA
synthesis by punching out the bottoms of the microtiter wells and
mounting them on glass slides with silastic glue. The slides are
dipped in a 1 g/ml solution of NTB2 nuclear track emulsion (Kodak)
and exposed for 3-4 days. The emulsion is developed with Microdol-X
solution (Kodak) for 10 minutes, rinsed with distilled H.sub.2O,
and fixed with Rapid Fixer (Kodak) for three minutes. The
autoradiographs are stained with a modified Giemsa stain. At least
1000 nuclei are counted in each well and DNA synthesis, expressed
as the percentage of nuclei labeled. Cell division is measured by
counting the number of cells in microtiter wells with the aid of a
grid after 40-48 hour incubations with test samples.
Example 3
Inhibition of Endothelial Cell Proliferation Measured by
Colorimetric Determination of Cellular Acid Phosphatase Activity
and Electronic Cell Counting
[0136] A quick and sensitive screen for inhibition of EC
proliferation in response to treatment with a troponin subunit,
homolog, or derivative of the invention involves incubating the
cells in the presence of varying concentrations of the inhibitor
and determining the number of endothelial cells in culture based on
the colorimetric determination of cellular acid phosphatase
activity, described by Connolly, et al., 1986, J. Anal. Biochem.
152:136-140.
[0137] The effect of troponin on the proliferation of capillary
endothelial cells (EC) was measured in an assay which measures the
ability of this protein to interfere with stimulation of
endothelial cell proliferation by a known angiogenesis factor
(bFGF).
[0138] Capillary endothelial cells and Balb/c 3T3 cells were
separately plated (2.times.10.sup.3/0.2 ml) onto gelatin-coated
96-well tissue culture dishes on day 1. On day 2, cells were refed
with Dulbecco's modified Eagle's medium (Gibco) with 5% calf serum
(Hyclone) (DMEM/5) and bFGF (10 ng/ml) (FGF Co.) and increasing
concentrations of one or more troponin subunits. These substances
were added simultaneously in volumes that did not exceed 10% of the
final volume. Wells containing phosphate buffered saline (PBS)
(Gibco) alone and PBS+bFGF were included as controls. On day 5,
media was removed and cells were washed with PBS and lysed in 10
.mu.l of buffer containing 0.1 M sodium acetate (pH 5.5), 0.1%
Triton X-100.TM. and 100 mM p-nitrophenyl phosphate (Sigma 104
phosphatase substrate). After incubation for 2 hours at 37.degree.
C., the reaction was stopped with the addition of 10 .mu.l of 1 N
NAOH. Color development was determined at 405 nm using a rapid
microplate reader (Bio-Tek).
[0139] Percent inhibition was determined by comparing the cell
number of wells exposed to stimulus with those exposed to stimulus
and troponin subunits.
[0140] All three troponin subunits were found to inhibit
bFGF-stimulated EC proliferation, as measured by the colorimetric
assay.
[0141] Troponin C inhibited bFGF-stimulated endothelial cell
proliferation in a dose-dependent manner in all concentrations
tested (FIG. 1). Percent inhibition of bovine endothelial cell
proliferation ("BCE") was 54%, 86%, 83%, and 100% at concentrations
of 280 nM, 1.4 .mu.M, 2.8 .mu.M and 5.6 .mu.M, respectively. An
inhibition of 100% was observed at a concentration of 20 .mu.g/well
(5.6 .mu.M). IC.sub.50 represents the concentration at which 50%
inhibition of bFGF growth factor-induced stimulation was observed.
The IC.sub.50 of troponin C was determined to be 278 nM.
[0142] Troponin I inhibited bFGF-stimulated BCE proliferation at
concentrations of 1 and 5 .mu.g/well, but inhibition was not
observed in the sample tested at 10 .mu.g/well (FIG. 2). The
percent inhibition of BCE was 33% and 46% at concentrations of 240
nM and 1.2 .mu.M, respectively. The IC.sub.50 of troponin I was
determined to be 1.14 .mu.M.
[0143] Troponin T inhibited bFGF-stimulated EC proliferation at
concentrations of 10 and 20 .mu.g/well, but not at concentrations
of 1 and 5 .mu.g/well (FIG. 3). BCE proliferation was inhibited 23%
and 62% at 1.6 .mu.M and 3.3 .mu.M, respectively. The IC.sub.50 of
troponin T was determined to be 2.14 .mu.M.
[0144] The combination of troponin subunits C and I inhibited EC at
all concentrations tested (FIG. 4). The percent inhibition of
proliferation of BCE was 52%, 54% 73% and 47% at 130 nM, 645 nM,
1.3 .mu.M and 2.6 .mu.M, respectively. The IC.sub.50 of this
combination was determined to be 110 nM.
[0145] The combination of troponin subunits C, I and T was observed
to inhibit bFGF-stimulated BCE proliferation by 16% at a
concentration of 360 nM (5 .mu.g/well, FIG. 5).
[0146] The troponin samples tested had no detectable inhibitory
effect on the growth of Balb/c 3T3 cells, a non-endothelial cell
type.
Example 4
Inhibition of Capillary Endothelial Cell Miaration by Troponin
[0147] Determination of the ability of the troponin subunit,
fragment, or homolog to inhibit the angiogenic process of capillary
EC migration in response to an angiogenic stimulus, can be
determined using a modification of the Boyden chamber technique is
used to study the effect of troponin subunit, fragment, or homolog
on capillary EC migration. Falk et al., 1980, J. immunol.
118:239-247 (1980). A blind-well Boyden chamber, consists of two
wells (upper and lower) separated by a porous membrane. J. Exp.
Med. 115:453-456 (1962). A known concentration of growth factor is
placed in the lower wells and a predetermined number of cells and
troponin subunit, fragment, or homolog is placed in the upper
wells. Cells attach to the upper surface of the membrane, migrate
through and attach to the lower membrane surface. The membrane can
then be fixed and stained for counting, using the method of Glaser
et al., 1980, Nature 288:483-484.
[0148] Migration is measured using blind well chambers (Neuroprobe,
no. 025-187) and polycarbonate membranes with 8 micron pores
(Nucleopore) precoated with fibronectin (6.67 .mu.g/ml in PBS)
(human, Cooper). Basic FGF (Takeda Co.) diluted in DMEM with 1%
calf serum (DMEM/1) is added to the lower well at a concentration
of 10 ng/ml. The upper wells receive 5.times.10.sup.5 capillary
EC/ml and increasing concentrations of purified troponin subunit,
fragment or homolog is used within 24 hours of purification.
Control wells receive DMEM/1, either with or without bFGF. The
migration chambers are incubated at 37.degree. C. in 10% C0.sub.2
for 4 hours. The cells on the upper surface of the membrane are
then wiped off by drawing the membrane over a wiper blade
(Neuroprobe). The cells which have migrated through the membrane
onto the lower surface are fixed in 2% glutaraldehyde followed by
methanol (4.degree. C.) and stained with hematoxylin. Migration is
quantified by counting the number of cells on the lower surface in
16 oil immersion fields and comparing this number with that
obtained for the control.
Example 5
Inhibition in vivo of Neovascularization by Troponin as Determined
by the Chick Chorioallantoic Membrane Assay
[0149] The chick chorioallantoic membrane assay (CAM), may be used
to determine whether troponin subunit, fragment or homolog is
capable of inhibiting neovascularization in vivo. Taylor and
Folkman, 1982, Nature (London) 297:307-312. The effect of troponin
subunit, fragment or homolog on growing embryonic vessels is
studied using chick embryos in which capillaries appear in the yolk
sac at 48 h and grow rapidly over the next 6-8 days.
[0150] Three day post fertilization chick embryos are removed from
their shells and placed in plastic petri dishes (1005, Falcon). The
specimens are maintained in humidified 5% CO.sub.2 at 37.degree. C.
On day 6 of development, samples of purified troponin subunit,
fragment or homolog are mixed in methylcellulose disks and applied
to the surfaces of the growing CAMs above the dense subectodermal
plexus. Control specimens in which CAMS are implanted with empty
methylcellulose disks are also prepared. The CAMS are injected
intravascularly with India ink/Liposyn to more clearly delineate
CAM vascularity. Taylor et al., 1982, Nature 297:307-312.
[0151] Following a 48 hour exposure of the CAMs to the troponin
subunit, fragment, or homolog, the area around the implant is
observed and evaluated. Test specimens having avascular zones
completely free of India-ink filled capillaries surrounding the
test implant indicate the presence of an inhibitor of embryonic
neovascularization. In contrast, the control specimens show
neovascularization in close proximity or in contact with the
methylcellulose disks.
[0152] Histological mesodermal studies are performed on the CAMs of
test and control specimens. The specimens are embedded in JB-4
plastic (Polysciences) at 4.degree. C. and 3 .mu.m sections are cut
using a Reichert 2050 microtome. Sections are stained with
toluidine blue and micrographs are taken on a Zeiss photomicroscope
using Kodak TM .times.100 and a green filter.
Example 6
Inhibition in vivo of Neovascularization by Troponin as Determined
by the Rabbit Corneal Pocket Assay
[0153] Male NZW rabbits weighing 4-5 lbs. are anesthetized with
intravenous pentobarbital (25 mg/kg) and 2% xylocaine solution is
applied to the cornea. The eye is proptosed and rinsed
intermittently with Ringer's solution to prevent drying. The adult
rabbit cornea has a diameter of approximately 12 mm. An
intracorneal pocket is made by an incision approximately 0.15 mm
deep and 1.5 mm long in the center of the cornea with a No. 11
scalpel blade, using aseptic technique. A 5 mm-long pocket is
formed within the corneal stroma by inserting a 1.5 mm wide,
malleable iris spatula. In the majority of animals, the end of the
corneal pocket is extended to within 1 mm of the corneal-scleral
junction. In a smaller series of 22 rabbits implanted with tumor
alone, pockets are placed at greater distances--2-6 mm from the
corneal-scleral junction by starting the incision away from the
center.
[0154] In the first assay, polymer pellets of ethylene vinyl
acetate (EVAc) copolymer are impregnated with test substance and
surgically implanted in a pocket in the rabbit cornea approximately
1 mm from the limbus. When this assay system is being used to test
for angiogenesis inhibitors, either a piece of V2 carcinoma or some
other angiogenic stimulant is implanted distal to the polymer, 2 mm
from the limbus. On the opposite eye of each rabbit, control
polymer pellets that are empty are implanted next to an angiogenic
stimulant in the same way. In these control corneas, capillary
blood vessels start growing towards the tumor implant in 5-6 days,
eventually sweeping over the blank polymer. In test corneas, the
directional growth of new capillaries from the limbal blood vessels
towards the tumor occurs at a reduced rate and is often inhibited
such that an avascular region around the polymer is observed (FIG.
1). This assay is quantitated by measurement of the maximum vessel
lengths with a stereoscopic microscope.
Example 7
Isolation of Troponin I from Cartilaae
Purification of Troponin I from Cartilage
[0155] Troponin I was purified from bovine veal scapulae using a
modification of a protocol previously described by us (Moses, et
al., 1990, Science 2488, 1408-1410). Briefly, veal scapulae were
vacuum frozen immediately after slaughter and stored at -20.degree.
C. until used. Cartilage was scraped first with a periosteal
elevator (Arista) and then with a scalpel blade (No. 10,
Bard-Parker) until clean of all muscle and connective tissue.
Cartilage slices were extracted in 2 M NaCl, precipitated with HCl
and ammonium sulfate (25-20%, and fractionated using a series of
chromatography steps: gel filtration on A-1.5 m Sepharose (Bio-Rad)
in the presence of 4M guanidine-HCl, ion exchange on a Bio-Rex 70
(Bio-Rad) cation exchange column, gel filtration on a Sephadex G-75
(superfine) (Pharmacia) column, reversed-phase high-performance
liquid chromatography (HPLC) on a Hi-Pore 304 column (Bio-Rad) and
gel filtration on a Progel-TSK G3000SWXL column (3.0 cm.times.7.8
mm) (Supelco). Fractions obtained from each column step were tested
for their ability to inhibit capillary endothelial cell (EC)
proliferation which was stimulated by basic Fibroblast Growth
Factor (bFGF) as described below. Fractions containing inhibitory
activity were pooled and concentrated in a Savant Speed Vac
concentrator for amino acid and sequence analysis. Unless otherwise
stated, all reagents were obtained from Sigma.
Trypsin Digestion, HPLC Separation and Microsequencing
[0156] Proteins were each reduced, S-carboxyamidomethylated and
subjected to digestion with trypsin. The resulting peptide mixtures
were fractionated by narrow-bore high performance liquid
chromatography using a Zorbax C18 1.0 mm by 150 mm reverse-phase
column on a Hewlett-Packard 1090 HPLC with a 1040 diode array
detector. Optimum fractions were chosen based on differential UV
absorbance at 205, 277 nm and 292 nm, peak symmetry and resolution
(Lane, et al., 1991, J. Prot; Chem. 10, 151-160). These fractions
were then further screened for length and homogeneity by
matrix-assisted laser desorption time-of-flight mass spectrometry
(MALDI-TOF/MS) on a Thermo Bioanalysis Lasermat 2000 (Hemel,
England). Tryptic peptide sequences were determined by electrospray
ionization/tandem mass spectrometry on a Finnigan TSQ7000 (San
Jose, Calif.) triple quadrupole mass spectrometer as described in
Nash et al. (Nash, et al., 1996, Curr; Biol. 6, 968-980).
Alternatively, peptides were submitted to automated Edman
degradation on a PE/ABD 477A (Foster City, Calif.) protein
sequencer.
Cloning and Expression of Human Troponin I
[0157] Human intercostal cartilage tissue was obtained according to
bioethical guidelines pertaining to discarded clinical material.
The cDNA encoding a fragment of human fast-twitch skeletal muscle
troponin I was amplified by standard reverse transcriptase
polymerase chain reaction (RT-PCR) from the total RNA isolated from
a core sample of human cartilage using primers based on the
nucleotide sequence of human fast-twitch skeletal muscle TnI (Zhu,
et al., 1994, Biochim. Biophys. Acta 1217, 338-340): forward primer
5'-GCTCTGCAAACAGCTGCACGCCAAG-3' (SEQ ID NO:4) and reverse primer
5,-GCCCAGCAGGGCCTTGAGCATGGCA-3' (SEQ ID NO:5) which was cloned into
PCR2.1 (Invitrogen) and sequenced in both directions. The cDNA
encoding the full-length open reading frame (ORF) of human
fast-twitch skeletal muscle troponin I was cloned from human
skeletal muscle mRNA with Pfu polymerase (Stratagene) under
standard PCR conditions, using forward primer
(5'-CTCACCATGGGAGATGAGGAGAAGC-3') (SEQ ID NO:6) and the reverse
primer (5 -GCCTCGAGTGGCCTAGGACTCGGAC-3') (SEQ ID NO:7). The PCR
product was cloned into the expression vector Pet24d (Novagen)
using 5'-Ncol and 3'-Xhol sites and sequenced as above.
[0158] Tissue expression of TnI was analyzed by RT-PCR as described
above. Total RNA (400 ng/sample) was isolated from rat skeletal
muscle, liver (Clontech), xyphoid and Swarm rat chondrosarcoma. The
design of the forward (5'-GAACACTGCCCGCCTCTGCACATC-3') (SEQ ID
NO:8) and reverse (5'-GAGCCCAGCAGCGCCTTCAGCATG-3') (SEQ ID NO:9)
primers was based on the nucleotide sequence of rat fast-twitch
skeletal muscle TnI.
[0159] Recombinant (r) human TnI was expressed according to
standard protocols (Sambrook, et al., 1989, Molecular Cloning: A
laboratory manual. (Cold Spring Harbor Press, New York, N.Y.)).
After 5 hrs of expression, bacteria were harvested by
centrifugation. Following centrifugation at 12,000.times.g for 15
min, the pellet was resuspended in 1.0 ml of Buffer A (15 mM
Tris-HCl, 0.1 mM EDTA, pH 7.0). The cells were disrupted by
sonication. The inclusion bodies were isolated by centrifugation at
12,000.times.g once for 15 min in Buffer A, followed by
centrifugation once at 11,000.times.g once for 15 min in Buffer
A.
Purification of Recombinant Troponin I
[0160] The washed pellet was dissolved in 6 M urea, 0.5 M NaCl, 5
mM HEPES, 2 mM EDTA, 5 mM DTT (pH 7.5), and nutated in the above
buffer for 6-8 hours at 4.degree. C. The sample was then dialyzed
against 0.5 M NaCl, 5 mM HEPES, 5 mM DTT (pH 7.5) and concentrated
using an Amicon concentrator (YM-10, MWCO 10,000 Da) prior to
application to a Progel-TSK G3000SWXL column (30 cm.times.7.8 mm).
The sample was eluted using the above buffer (0.5 M NaCl, 5 mM
HEPES, 5 mM DTT, pH 7.5). Some of the inhibitory preparations were
further fractionated on a Q-Sepharose HP column (Pharmacia Biotech)
and tested as described below with no difference in biological
activity. Purified rTnI was dialyzed against phosphate buffered
saline (PBS) containing 0.5 mM DTT prior to testing. Protein
concentration was determined by scanning densitometric comparison
(IS-1000 Digital Imaging System, Version 2.00, Alpha Innotech
Corp.) with known protein standards (Novex) coelectrophoresed on
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) followed by staining with Coomassie Blue.
Western Blot Analysis
[0161] Immunoblotting was conducted on samples of native TnI
(purified from cartilage as described above), recombinant TnI
(purified as described above) and bovine chondrocyte lysates
prepared as described below according to standard protocols.
Cultures of primary bovine scapular chondrocytes were established
and maintained as previously described by us (Moses, et al., 1990,
J. Cell. Biol. 119, 474-481). Cells were rinsed with PBS and to
each 10 cm culture dish was added 1 ml of boiling
2.times.-concentrated electrophoresis sample buffer (250 mM
Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue and
2% B-mercaptoethanol). Cells were scraped from the dishes using a
disposable cell scraper (Costar), transferred to a microcentrifuge
tube and boiled for an additional 5 min. Following several passages
though a 26 gauge needle (Becton Dickinson), the sample was
clarified by centrifugation (2000.times.g), diluted to 0.1% SDS,
and the protein concentration determined using a DO Protein Assay
(BioRad). All samples were separated by polyacrylamide gel
electrophoresis on a 4/12% acrylamide mini-gel according to Laemmli
(Laemmli, 1970, Nature 227, 680-685). Proteins were then
transferred to nitrocellulose (Hybond-ECL, Amersham) using a
Transblot apparatus (Biorad), incubated with a monoclonal antibody
to rabbit skeletal muscle TnI (Advanced Immunochemical Inc.) and
developed using the ECL western blotting system according to the
manufacturer's protocol (Amersham).
Results
[0162] An in vitro assay which measures the inhibition of basic
fibroblast growth factor (bFGF)-stimulated proliferation of
capillary endothelial cells (EC) was used to monitor purification
(Moses, et al., 1990, Science 2488, 1408-1410; Moses, et al., 1990,
J. Cell. Biol. 119, 474-481; Connolly, et al., 1986, Anal. Biochem.
152, 136-140). All cartilage-derived fractions obtained from a
series of chromatography steps described below were screened for
this inhibitory bioactivity. Inhibitory activity eluted at an
approximate molecular weight of 25,000 Da from the A-1.5 m size
exclusion column, at approximately 0.2M NaCl from the Biorex 70
cation exchange column, at approximately 23,000 Da from the
Sephadex G-75 gel filtration column, at an acetonitrile
concentration of approximately 38.5%, and at an approximate Mr of
22,000 Da from the Progel-TSK G3000SWXL column. Inhibitory
fractions obtained from the final chromatography step were
subjected to tryptic digestion and the resultant peptides were
sequenced by microcapillary LC-ESI tandem mass spectrometry or
automated Edman degradation. The sequences of three peptide
fragments were obtained and were identified as fragments of
troponin I (FIG. 6).
[0163] Since there had been no previous reports in the literature
that cartilage cells, the chondrocytes, contain TnI, the cDNA
encoding human cartilage TnI was cloned using a standard PCR
strategy (Wu and Moses, 1996, Gene 18, 243-246) (FIG. 7A).
Sequencing of the PCR product revealed its identity to human fast
skeletal muscle TnI (FIG. 7B) (SEQ ID NO:16). TnI expression levels
of rat xiphoid cartilage, Swarm rat chondrosarcoma and liver, were
also determined by RT-PCR and were significantly lower than that of
rat skeletal muscle, with the expression level in liver appearing
to be slightly lower than that of cartilage or chondrosarcoma (FIG.
7C).
[0164] In order to obtain sufficient amounts of TnI to investigate
its potential as an antiangiogenic factor, a cDNA encoding full
length human fast skeletal muscle troponin I was cloned into
expression vector pET-24d and transformed into E. coli BL21(DE3) p
LysS strain. The expression level of recombinant human skeletal
muscle troponin I was approximately 30-40% of total cellular
protein. Following purification, recombinant TnI migrated as a
single band, at approximately 21 kDa on SDS-PAGE (FIG. 8).
Example 8
Capillagy Endothelial Cell (EC) Proliferation
Cell Culture
[0165] Capillary EC, isolated from bovine adrenal cortex (Folkman,
et al., 1979, Proc. Natl. Acad. Sci. USA 76, 5217-5221) were
obtained from Children's Hospital. (Boston, Mass.). These cells
were demonstrated to be endothelial by staining with antisera to
von Willebrand factor and by their uptake of fluoresceinated,
acetylated low density lipoprotein. Cells were maintained in
culture in DME (Dulbecco's Modified Eagle's Medium, Gibco
Laboratories) with 10% calf serum (Hyclone) (DME/10) supplemented
with 3 ng/ml bFGF or Vascular Endothelial Growth Factor (VEGF) in
preparation for these assays.
[0166] BALB/c mouse 3T3 cells were maintained in DME/10,
L-glutamine (292 .mu.g/ml) as previously described (Klagsbrun, et
al., 1977, Exp. Cell Res. 105, 99-108). Bovine aortic smooth muscle
cells (SMC), isolated by explant from the medial layer of bovine
aortas, were obtained from Children's Hospital (Boston, Mass.).
These cells were cultured in DME/10 on uncoated tissue culture
plastic as previously described (D'Amore and Smith, 1993, Growth
Factors 8, 61-75).
[0167] Briefly, capillary EC (2,000 cells per well) were plated on
gelatinized 96-Well culture plates in DMEM supplemented with 5%
(v/v) calf serum and incubated for 24 hours. On day 2, cells were
treated with bFGF (Scios Nova; 1 ng/ml) and challenged with the
test fractions and/or with purified TnI. For experiments in which
VEGF was used as the mitogen, 800 cells per well were plated and
allowed to incubate for 3 hours before VEGF (Biomedical
Technologies Incorporated; 30 ng/ml) and TnI was added. Control
wells contained cells alone and cells stimulated with bFGF or VEGF.
On day 5, growth medium was removed from the plates; cells were
lysed in buffer containing the detergent Triton x-100 and the
phosphatase substrate p-nitrophenyl phosphate. After incubation for
2 h at 37.degree. C., NaOH was added to terminate the reaction.
Color development was determined using a rapid multiwell plate
reader (Dynatech MR 5000) (Moses, et al., 1990, Science 2488,
1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481;
Connolly, et al., 1986, Anal. Biochem. 152, 136-140). EC inhibitory
activity was verified by electronic cell counting assays as
previously described by us (Moses, et al., 1990, Science 2488,
1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481).
Tritiated thymidine incorporation assays were conducted according
to the method of Shing (Shing, 1990, in Methods in Enzymolggy, eds.
Barnes, D., Mather, J. P. and Sato, G. H. (Academic Press, New
York), pp. 91-95).
Results
[0168] Purified rTnI was tested for its ability to inhibit bFGF and
VEGF-stimulated capillary EC and was found to inhibit EC
proliferation in a dose-dependent and saturable manner with an
IC.sub.50 (the inhibitory concentration at which one observes 50%
suppression of proliferation) of approximately 65 nM when bFGF was
used as the mitogen (FIG. 9A) and approximately 1.5 nM when VEGF
was used (FIG. 9B). Native TnI inhibited capillary EC proliferation
in an equipotent manner. Tritiated thymidine assays demonstrated
that recombinant TnI inhibited capillary EC DNA synthesis in a
dose-dependent and saturable manner with an IC.sub.50 of
approximately 240 nM. This suppression of proliferation appears to
be unique to endothelial cells given the fact that TnI did not
suppress the growth of any of the non-endothelial cells tested
including bovine aortic smooth muscle cells and Balb/c 3T3 cells
even when tested at doses which were over 5.times. higher than that
required to obtain an IC.sub.50 value for capillary EC.
Example 9
Cell Specificity
[0169] To determine whether the proliferation of bovine aortic SMC
and Balb c/3T3 cells was inhibited by TnI, the following assays
were conducted. SMC were plated into multiwell dishes (2.1
cm.sup.2/well) at a density of 10,000 cells/well. After allowing
the cells to attach overnight, fresh media was applied containing
either 3 ng/ml PDGF-BB alone or in combination with increasing
concentrations of purified TnI. Following incubation for 72 hrs at
37.degree. C. in 10% CO.sub.2, the cells were rinsed in PBS,
detached by trypsinization and counted electronically. The effect
of TnI on quiescent BALB/c mouse 3T3 cells was assessed by
measuring the incorporation of tritiated thymidine into DNA in
96-well plates as previously described (Shing, 1990, in Methods in
Enzymolqgy, eds. Barnes, D., Mather, J. P. and Sato, G. H.
(Academic Press, New York), pp. 91-95).
Example 10
Chick Chorioallantoic Membrane (CAM) Assay
[0170] All procedures were carried out in a laminar flow hood under
sterile conditions. The eggs were stored in a Favorite Egg
Incubator (Leahy) at 37.degree. C. and 65% relative humidity. On
day 3 of development, fertilized White Leghorn eggs (SPAFAS) were
cracked and the embryos removed from their shells and placed in
plastic petri dishes. On day 6, test substances including native
rabbit TnI (Greater and Gergely, 1971, J. Biol. Chem. 246,
4226-4233) and recombinant human TnI and appropriate buffer
controls were mixed in methylcellulose, disks and applied to the
surfaces of the growing CAMs above the dense subectodermal plexus.
Forty-eight hours following implantation of the plastic disc, the
eggs were examined for vascular reactions under a dissecting scope
(60.times.) and photographed (Moses, et al., 1990, Science 2488,
1408-1410; Moses, et al., 1990, J. Cell. Biol. 119, 474-481).
[0171] The CAM assay was used to determine whether rTnI was an
inhibitor of angiogenesis in vivo. The results shown in FIG. 10
demonstrate the significant inhibition of embryonic
neovascularization as evidenced by the large avascular zone caused
by 130 picomoles of rTnI. This effect was observed in 66% of the
eggs tested at this dose and 100% of the eggs tested at a dose of
approximately 380 picomoles. This observation was reproduced in
three separate sets of CAM assays using three different TnI
preparations. Over 125 CAMs were tested in this series of
experiments.
Example 11
Mouse Corneal Pocket Assay
[0172] Inhibition of angiogenesis in vivo was also demonstrated
using the mouse corneal pocket assay (Chen, et al., 1995, Cancer.
Res. 55, 4230-4233; O'Reilly, et al., 1996, Nat. Med. 2, 689-692).
Briefly, pellets composed of bFGF (40 ng/ml), sucrose octasulfate,
and Hydron were implanted into corneal micropockets of six C57BL/6
mice as previously described (U.S. Pat. No. 5,837,680 to Moses et
al.). Troponin I (50 mg/kg) was administered systemically every 12
hours by subcutaneous injection. On the sixth day of treatment,
corneal angiogenesis was evaluated using slit lamp microscopy and
photographed.
Results
[0173] In another in vivo assay, the mouse corneal pocket assay,
systemic administration of rTnI significantly inhibited bFG
F-induced angiogenesis (FIG. 11B) when compared to corneas of
control mice which received vehicle alone (FIG. 11A).
[0174] Taken together, the in vivo studies described in Section 6,
Examples 10 and 11 show rTnI to be a potent inhibitor of
neovascularization when compared to other inhibitors tested in
these same assays (Moses, et al., 1995, in International Review of
Cytology, 161, 1-48).
Example 12
B16-BL6 Melanoma Model
[0175] Murine melanoma B16-BL6 were cultured in RPMI 1640 (Gibco)
supplemented with 10% (v/v) fetal calf serum (Hyclone), L-glutamine
and NaHCO.sub.3. Cells were washed with EBSS (Gibco) and
trypsinized for 3 to 5 mm with 0.25% TRL/0.2% EDTA to which culture
buffer was added for washing. This preparation was then centrifuged
for 10 mm at 1000 rpm, the cell pellet resuspended in fresh culture
media, cell number determined using a coulter counter and cell
viability determined with trypan blue (100% viability). The cell
suspension was adjusted to 2.5.times.10.sup.5 cells/ml for
implantation. B16-BL6 cells (5.times.10.sup.5/0.2 ml) were injected
into the tail veins of C57BL/6 mice (approximately 6-7 weeks old).
One day following tumor cell inoculation, mice were treated with
rTnI systemically, twice per week, with a dose of either 1 mg/kg
(n=10) or 20 mg/kg (n=10) or vehicle (150 mM NaCl, 20 mM citrate,
pH3) over, a 28 day period. On day 30, animals were sacrificed, the
number of lung surface metastases counted and the lungs
weighed.
Results
[0176] Recombinant TnI was tested for its ability to inhibit lung
metastasis in vivo caused by a very aggressive variant of the B16
melanoma cell line, B16-BL6 (Saiki, et al., 1989 Cancer Res. 49,
3815-3822). Recombinant TnI, administered systemically, inhibited
lung metastases by 52% (p<0.04 one tailed t-test) at a dose of 1
mg/kg when given only twice weekly (n=10), and by 64% (p<0.02;
one tailed t-test) at a dose of 20 mg/kg twice weekly (n=10), [lung
metastasis control (68.6.+-.7.5 SEM) (n=10); 1 mg/kg (32.8.+-.1-4.8
SEM); 20mg/kg (25.0.+-.7.5 SEM)] with no observed toxicity (i.e.,
no weight or appetite loss, etc.). Lung weights were comparable in
control and treated groups.
[0177] As shown by the data, TnI inhibited lung metastasis.
Example 13
Inhibition of Endothelial Cell Proliferation Usina Fragments of
Troponin I
[0178] Recombinant peptides corresponding to fragments of rabbit
(rb) TnI (SEQ ID NO:10) (FIG. 12) were tested for ability to
inhibit bFGF-stimulated capillary EC as described above in Section
6, Examples 2 and 8. The rbTnI fragments (SEQ ID NOS:11-15) were
prepared according to Jha et al., 1996, Biochemistry
35(34):11026-11035. As shown in Table 2, various concentrations of
peptides corresponding to the amino-terminal (N') region (aa 1-94)
(SEQ ID NO:11); the N' and inhibitory (I') region (aa 1-120) (SEQ
ID NO:12); the I' region (aa 98-114) (SEQ ID NO:13); the carboxy
terminus (C') and I' region (C'+I') (aa 96-181) (SEQ ID NO:14); the
C' region (aa 122-181) (SEQ ID NO:15); and mixtures of the C'+I'
(SEQ ID NO:14) plus the N' (SEQ ID NO:11) fragments and the N'+I'
(SEQ ID NO:12) plus the C' (SEQ ID NO:15) fragments of TnI were
tested for inhibition of EC proliferation.
[0179] As shown in Table 2, the C'+I' fragment (SEQ ID NO:14)
significantly inhibited EC proliferation. The percent inhibition of
EC was 54% and 48% at concentrations of 0.1 .mu.g/well and 0.3
.mu.g/well, respectively. The IC.sub.50 was determined to be 0.1 to
0.2 .mu.g/well (0.05 .mu.M to 0.1 .mu.M). Furthermore, the N'+I'
(SEQ ID NO:12) fragment interfered with the inhibitory activity of
the C' (SEQ ID NO:15) fragment and the N' (SEQ ID NO:11) fragment
interfered with the inhibitory activity of the C'+I' (SEQ ID NO:14)
fragment.
[0180] As shown in Section 6, Example 3, supra, full-length TnI
inhibited EC proliferation approximately 46% at a concentration of
5 .mu.g/well (1.2 .mu.M). Thus, the C'+I' fragment had 25 to
50-fold EC inhibitory activity compared to the full-length TnI.
[0181] These results demonstrate that fragments of troponin
subunits, particularly the C'+I' fragment (SEQ ID NO:14), inhibited
EC proliferation in an assay that was developed to mimic the
process of neovascularization. Thus, troponin subunit fragments
inhibit angiogenesis. TABLE-US-00003 TABLE 2 Approx. SEQ ID Amino
Assay Assay Assay IC.sub.50 Approx. Fragment Region.sup.a NO: Acids
.mu.g/well .mu.g/ml MW nM % I.sup.b .mu.g/well IC.sub.50 .mu.M N'
1-94 11 94 0.01 0.05 10,906 5 -12 >0.3 >0.1 0.025 0.125 11 6
0.1 0.5 46 31 0.3 1.5 138 28 N' + I' 1-120 12 120 0.01 0.05 13,923
4 6 >>0.3 >>0.1 0.025 0.025 9 0 0.1 0.5 36 12 0.3 1.5
108 17 I' 98-114 13 17 4 20 1,972 10140 -12 >>40 >>100
10 50 25350 -25 20 100 50700 -6 40 200 101401 -34 C' + I' 96-181 14
86 0.01 0.05 9,978 5 25 0.1 to 0.05 0.025 0.125 13 28 0.2 to 0.1
0.5 50 54 0.1 0.3 1.5 150 48 C' 122-181 15 60 0.01 0.05 6,961 7 -1
>0.3 >0.2 0.025 0.125 18 -6 0.1 0.5 72 20 0.3 1.5 215 23 (C'
+ I') + 96-181 + 14, 11 180 0.01 0.05 20,884 7 17 >0.3 >0.2
N' 1-94 0.025 0.125 18 20 0.1 0.5 72 27 0.3 1.5 215 28 (N' + I') +
1-120 + 12, 15 180 0.01 0.05 20,884 7 -7 >>0.3 >>0.2 C'
122-181 0.025 0.125 18 -1 0.1 0.5 72 -6 0.3 1.5 215 -1 .sup.aRabbit
fast twitch skeletal muscle amino acid (aa) numbers Average MW/aa:
116.0 .sup.bPercent Inhibition
[0182]
Sequence CWU 1
1
20 1 160 PRT Homo sapiens 1 Met Thr Asp Gln Gln Ala Glu Ala Arg Ser
Tyr Leu Ser Glu Glu Met 1 5 10 15 Ile Ala Glu Phe Lys Ala Ala Phe
Asp Met Phe Asp Ala Asp Gly Gly 20 25 30 Gly Asp Ile Ser Val Lys
Glu Leu Gly Thr Val Met Arg Met Leu Gly 35 40 45 Gln Thr Pro Thr
Lys Glu Glu Leu Asp Ala Ile Ile Glu Glu Val Asp 50 55 60 Glu Asp
Gly Ser Gly Thr Ile Asp Phe Glu Glu Phe Leu Val Met Met 65 70 75 80
Val Arg Gln Met Lys Glu Asp Ala Lys Gly Lys Ser Glu Glu Glu Leu 85
90 95 Ala Glu Cys Phe Arg Ile Phe Asp Arg Asn Ala Asp Gly Tyr Ile
Asp 100 105 110 Pro Glu Glu Leu Ala Glu Ile Phe Arg Ala Ser Gly Glu
His Val Thr 115 120 125 Asp Glu Glu Ile Glu Ser Leu Met Lys Asp Gly
Asp Lys Asn Asn Asp 130 135 140 Gly Arg Ile Asp Phe Asp Glu Phe Leu
Lys Met Met Glu Gly Val Gln 145 150 155 160 2 182 PRT Homo sapiens
2 Met Gly Asp Glu Glu Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln 1
5 10 15 His Leu Lys Ser Val Met Leu Gln Ile Ala Ala Thr Glu Leu Glu
Lys 20 25 30 Glu Glu Ser Arg Arg Glu Ala Glu Lys Gln Asn Tyr Leu
Ala Glu His 35 40 45 Cys Pro Pro Leu His Ile Pro Gly Ser Met Ser
Glu Val Gln Glu Leu 50 55 60 Cys Lys Gln Leu His Ala Lys Ile Asp
Ala Ala Glu Glu Glu Lys Tyr 65 70 75 80 Asp Met Glu Val Arg Val Gln
Lys Thr Ser Lys Glu Leu Glu Asp Met 85 90 95 Asn Gln Lys Leu Phe
Asp Leu Arg Gly Lys Phe Lys Arg Pro Pro Leu 100 105 110 Arg Arg Val
Arg Met Ser Ala Asp Ala Met Leu Lys Ala Leu Leu Gly 115 120 125 Ser
Lys His Lys Val Cys Met Asp Leu Arg Ala Asn Leu Lys Gln Val 130 135
140 Lys Lys Glu Asp Thr Glu Lys Glu Arg Asp Leu Arg Asp Val Gly Asp
145 150 155 160 Trp Arg Lys Asn Ile Glu Glu Lys Ser Gly Met Glu Gly
Arg Lys Lys 165 170 175 Met Phe Glu Ser Glu Ser 180 3 258 PRT Homo
sapiens 3 Met Ser Asp Glu Glu Val Glu Gln Val Glu Glu Gln Tyr Glu
Glu Glu 1 5 10 15 Glu Glu Ala Gln Glu Glu Glu Glu Val Gln Glu Asp
Thr Ala Glu Glu 20 25 30 Asp Ala Glu Glu Glu Lys Pro Arg Pro Lys
Leu Thr Ala Pro Lys Ile 35 40 45 Pro Glu Gly Glu Lys Val Asp Phe
Asp Asp Ile Gln Lys Lys Arg Gln 50 55 60 Asn Lys Asp Leu Met Glu
Leu Gln Ala Leu Ile Asp Ser His Phe Glu 65 70 75 80 Ala Arg Lys Lys
Glu Glu Glu Glu Leu Val Ala Leu Lys Glu Arg Ile 85 90 95 Glu Lys
Arg Arg Ala Glu Arg Ala Glu Gln Gln Arg Ile Arg Ala Glu 100 105 110
Lys Glu Arg Glu Arg Gln Asn Arg Leu Ala Glu Glu Lys Ala Arg Arg 115
120 125 Glu Glu Glu Asp Ala Lys Arg Arg Ala Glu Asp Asp Leu Lys Lys
Lys 130 135 140 Lys Ala Leu Ser Ser Met Gly Ala Asn Tyr Ser Ser Tyr
Leu Ala Lys 145 150 155 160 Ala Asp Gln Lys Arg Gly Lys Lys Gln Thr
Ala Arg Glu Met Lys Lys 165 170 175 Lys Ile Leu Ala Glu Arg Arg Lys
Pro Leu Asn Ile Asp His Leu Gly 180 185 190 Glu Asp Lys Leu Arg Asp
Lys Ala Lys Glu Leu Trp Glu Thr Leu His 195 200 205 Gln Leu Glu Ile
Asp Lys Phe Glu Phe Gly Glu Lys Leu Lys Arg Gln 210 215 220 Lys Tyr
Asp Ile Thr Thr Leu Arg Ser Arg Ile Asp Gln Ala Gln Lys 225 230 235
240 His Ser Lys Lys Ala Gly Thr Pro Ala Lys Gly Lys Val Gly Gly Arg
245 250 255 Trp Lys 4 25 DNA Artificial Sequence Primer 4
gctctgcaaa cagctgcacg ccaag 25 5 25 DNA Artificial Sequence Primer
5 gcccagcagg gccttgagca tggca 25 6 25 DNA Artificial Sequence
Primer 6 ctcaccatgg gagatgagga gaagc 25 7 25 DNA Artificial
Sequence Primer 7 gcctcgagtg gcctaggact cggac 25 8 24 DNA
Artificial Sequence Primer 8 gaacactgcc cgcctctgca catc 24 9 24 DNA
Artificial Sequence Primer 9 gagcccagca gcgccttcag catg 24 10 181
PRT Oryctolagus cuniculus 10 Gly Asp Glu Glu Lys Arg Asn Arg Ala
Ile Thr Ala Arg Arg Gln His 1 5 10 15 Leu Lys Ser Val Met Leu Gln
Ile Ala Ala Thr Glu Leu Glu Lys Glu 20 25 30 Glu Gly Arg Arg Glu
Ala Glu Lys Gln Asn Tyr Leu Ala Glu His Cys 35 40 45 Pro Pro Leu
Ser Leu Pro Gly Ser Met Ala Glu Val Gln Glu Leu Cys 50 55 60 Lys
Gln Leu His Ala Lys Ile Asp Ala Ala Glu Glu Glu Lys Tyr Asp 65 70
75 80 Met Glu Ile Lys Val Gln Lys Ser Ser Lys Glu Leu Glu Asp Met
Asn 85 90 95 Gln Lys Leu Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro
Pro Leu Arg 100 105 110 Arg Val Arg Met Ser Ala Asp Ala Met Leu Lys
Ala Leu Leu Gly Ser 115 120 125 Lys His Lys Val Cys Met Asp Leu Arg
Ala Asn Leu Lys Gln Val Lys 130 135 140 Lys Glu Asp Thr Glu Lys Glu
Arg Asp Leu Arg Asp Val Gly Asp Trp 145 150 155 160 Arg Lys Asn Ile
Glu Glu Lys Ser Gly Met Glu Gly Arg Lys Lys Met 165 170 175 Phe Glu
Ser Glu Ser 180 11 94 PRT Oryctolagus cuniculus 11 Gly Asp Glu Glu
Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln His 1 5 10 15 Leu Lys
Ser Val Met Leu Gln Ile Ala Ala Thr Glu Leu Glu Lys Glu 20 25 30
Glu Gly Arg Arg Glu Ala Glu Lys Gln Asn Tyr Leu Ala Glu His Cys 35
40 45 Pro Pro Leu Ser Leu Pro Gly Ser Met Ala Glu Val Gln Glu Leu
Cys 50 55 60 Lys Gln Leu His Ala Lys Ile Asp Ala Ala Glu Glu Glu
Lys Tyr Asp 65 70 75 80 Met Glu Ile Lys Val Gln Lys Ser Ser Lys Glu
Leu Glu Asp 85 90 12 120 PRT Oryctolagus cuniculus 12 Gly Asp Glu
Glu Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln His 1 5 10 15 Leu
Lys Ser Val Met Leu Gln Ile Ala Ala Thr Glu Leu Glu Lys Glu 20 25
30 Glu Gly Arg Arg Glu Ala Glu Lys Gln Asn Tyr Leu Ala Glu His Cys
35 40 45 Pro Pro Leu Ser Leu Pro Gly Ser Met Ala Glu Val Gln Glu
Leu Cys 50 55 60 Lys Gln Leu His Ala Lys Ile Asp Ala Ala Glu Glu
Glu Lys Tyr Asp 65 70 75 80 Met Glu Ile Lys Val Gln Lys Ser Ser Lys
Glu Leu Glu Asp Met Asn 85 90 95 Gln Lys Leu Phe Asp Leu Arg Gly
Lys Phe Lys Arg Pro Pro Leu Arg 100 105 110 Arg Val Arg Met Ser Ala
Asp Ala 115 120 13 18 PRT Oryctolagus cuniculus 13 Lys Leu Phe Asp
Leu Arg Gly Lys Phe Lys Arg Pro Pro Leu Arg Arg 1 5 10 15 Val Arg
14 86 PRT Oryctolagus cuniculus 14 Asn Gln Lys Leu Phe Asp Leu Arg
Gly Lys Phe Lys Arg Pro Pro Leu 1 5 10 15 Arg Arg Val Arg Met Ser
Ala Asp Ala Met Leu Lys Ala Leu Leu Gly 20 25 30 Ser Lys His Lys
Val Cys Met Asp Leu Arg Ala Asn Leu Lys Gln Val 35 40 45 Lys Lys
Glu Asp Thr Glu Lys Glu Arg Asp Leu Arg Asp Val Gly Asp 50 55 60
Trp Arg Lys Asn Ile Glu Glu Lys Ser Gly Met Glu Gly Arg Lys Lys 65
70 75 80 Met Phe Glu Ser Glu Ser 85 15 60 PRT Oryctolagus cuniculus
15 Leu Lys Ala Leu Leu Gly Ser Lys His Lys Val Cys Met Asp Leu Arg
1 5 10 15 Ala Asn Leu Lys Gln Val Lys Lys Glu Asp Thr Glu Lys Glu
Arg Asp 20 25 30 Leu Arg Asp Val Gly Asp Trp Arg Lys Asn Ile Glu
Glu Lys Ser Gly 35 40 45 Met Glu Gly Arg Lys Lys Met Phe Glu Ser
Glu Ser 50 55 60 16 196 DNA Homo sapiens 16 gctctgcaaa cagctgcacg
ccaagatcga tgcggctgaa gaggagaagt acgacatgga 60 ggtgagggtg
cagaagacca gcaaggagct ggaggacatg aaccagaagc tatttgatct 120
gcgggccaag ttcaagcggc ccccactgcg gagggtgcgc atgtcggccg atgccatgct
180 caaggccctg ctgggc 196 17 182 PRT Homo sapiens 17 Met Gly Asp
Glu Glu Lys Arg Asn Arg Ala Ile Thr Ala Arg Arg Gln 1 5 10 15 His
Leu Lys Ser Val Met Leu Gln Ile Ala Ala Thr Glu Leu Glu Lys 20 25
30 Glu Glu Ser Arg Arg Glu Ala Glu Lys Gln Asn Tyr Leu Ala Glu His
35 40 45 Cys Pro Pro Leu His Ile Pro Gly Ser Met Ser Glu Val Gln
Glu Leu 50 55 60 Cys Lys Gln Leu His Ala Lys Ile Asp Ala Ala Glu
Glu Glu Lys Tyr 65 70 75 80 Asp Met Glu Val Arg Val Gln Lys Thr Ser
Lys Glu Leu Glu Asp Met 85 90 95 Asn Gln Lys Leu Phe Asp Leu Arg
Gly Lys Phe Lys Arg Pro Pro Leu 100 105 110 Arg Arg Val Arg Met Ser
Ala Asp Ala Met Leu Lys Ala Leu Leu Gly 115 120 125 Ser Lys His Lys
Val Cys Met Asp Leu Arg Ala Asn Leu Lys Gln Val 130 135 140 Lys Lys
Glu Asp Thr Glu Lys Glu Arg Asp Leu Arg Asp Val Gly Asp 145 150 155
160 Trp Arg Lys Asn Ile Glu Glu Lys Ser Gly Met Glu Gly Arg Lys Lys
165 170 175 Met Phe Glu Ser Glu Ser 180 18 10 PRT Artificial
Sequence Description of Artificial Sequence tryptic peptide 18 eu
Gln Ile Ala Ala Thr Glu Leu Glu Lys 1 5 10 19 14 PRT Artificial
Sequence Description of Artificial Sequence tryptic peptide 19 le
Asp Val Ala Glu Glu Glu Lys Tyr Asp Met Glu Val Lys 1 5 10 20 5 PRT
Artificial Sequence Description of Artificial Sequence tryptic
peptide 20 eu Phe Asp Leu Arg 1 5
* * * * *