U.S. patent application number 12/399638 was filed with the patent office on 2009-11-12 for anti-angiogenic peptides from the n-terminus of endostatin.
Invention is credited to Judah Folkman, Kashi Javaherian, Robert Tjin Tham Sjin.
Application Number | 20090280190 12/399638 |
Document ID | / |
Family ID | 37394278 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280190 |
Kind Code |
A1 |
Folkman; Judah ; et
al. |
November 12, 2009 |
ANTI-ANGIOGENIC PEPTIDES FROM THE N-TERMINUS OF ENDOSTATIN
Abstract
Provided herein are anti-angiogenic comprising the N-terminal
end of endostatin, nucleic acids encoding the same, pharmaceutical
preparations comprising an effective amount of the peptide and
nucleic acids and use of the pharmaceuticals in treating or
preventing diseases or conditions associated with undesirable
angiogenesis.
Inventors: |
Folkman; Judah; (Brookline,
MA) ; Javaherian; Kashi; (Lexington, MA) ;
Sjin; Robert Tjin Tham; (Framingham, MA) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
37394278 |
Appl. No.: |
12/399638 |
Filed: |
March 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11364855 |
Feb 28, 2006 |
7524811 |
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12399638 |
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PCT/US04/28143 |
Aug 30, 2004 |
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11364855 |
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60499264 |
Aug 29, 2003 |
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60539213 |
Jan 26, 2004 |
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Current U.S.
Class: |
424/641 ;
435/320.1; 514/1.1; 530/324; 530/325; 530/326; 530/327; 530/328;
536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/78 20130101 |
Class at
Publication: |
424/641 ;
530/324; 530/325; 530/326; 530/328; 530/327; 536/23.1; 435/320.1;
514/12; 514/13; 514/14; 514/15 |
International
Class: |
A61K 33/30 20060101
A61K033/30; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; C07K 14/00 20060101 C07K014/00; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; A61K 38/08 20060101
A61K038/08; A61K 38/10 20060101 A61K038/10; A61K 38/16 20060101
A61K038/16 |
Goverment Interests
STATEMENT OF RIGHTS
[0002] This invention was made with government support under Grant
R01 CA064481 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. An isolated peptide comprising about 10 to about 35 amino acids
of the N-terminal region of an endostatin protein including the
first histidine of the endostatin protein, or a variant thereof,
wherein the isolated peptide or variant thereof has anti-tumor
activity.
2. The isolated peptide of claim 1, comprising from about 20 to
about 30 amino acids.
3. The isolated peptide of claim 1, which is selected from the
group consisting of SEQ ID NOs: 2, 4, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 96, 98, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, 125, 126, 128, 129, 130, and
131.
4. The isolated peptide of claim 1, comprising SEQ ID NO: 2.
5. The isolated peptide of claim 1, comprising SEQ ID NO: 4.
6. The isolated peptide of claim 1, comprising about 12 amino acids
of SEQ ID NOs: 2 or 4.
7. A nucleic acid encoding an isolated peptide or variant thereof
of claim 1.
8. A nucleic acid which is selected from the group consisting of
SEQ ID NOs: 1, 3, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,
119, 121, and 123.
9. A vector comprising a nucleic acid of claim 7.
10. A pharmaceutical composition comprising an isolated peptide or
variant thereof of claim 1.
11. A pharmaceutical composition of claim 10, further comprising a
second peptide.
12. A pharmaceutical composition of claim 10, further comprising an
effective amount of zinc.
13. A device for introducing a composition into a subject
comprising the pharmaceutical composition of claim 10.
14. The device of claim 13 that is a stent or a syringe.
15. A method of preventing angiogenesis in a tissue, comprising
contacting a tissue with a peptide of claim 1 to thereby prevent
angiogenesis.
16. A method of preventing tumor growth in a tissue, comprising
contacting the tissue with a peptide of claim 1 to thereby prevent
tumor growth.
17. A method for preventing angiogenesis in a subject, comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide of claim 1, to thereby prevent
angiogenesis in the subject.
18. A method for preventing tumor growth in a subject, comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide of claim 1, to thereby prevent tumor
growth in the subject.
19. A method for treating or preventing an angiogenic associated
disease in a subject comprising administering to the subject a
pharmaceutical composition of claim 11.
20. The method of claim 21, wherein the angiogenic associated
disease is selected from the group consisting of: atherosclerosis,
hemangioma, cancer, solid tumors, leukemia, metastasis,
telangiectasia psoriasis scleroderma, pyogenic granuloma,
myocardial angiogenesis, plaque neovascularization, coronary
collaterals, cerebral collaterals, arteriovenous malformations,
ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular
glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis,
diabetic neovascularization, macular degeneration, wound healing,
peptic ulcer, fractures, keloids, phemphigoid, trachoma,
vasculogenesis and hematopoiesis.
21. A method of treating cancer in a subject, comprising
administering to a subject in need thereof a therapeutically
effective amount of a peptide of claim 1, to thereby treat cancer
in the subject.
22. The method of claim 1, wherein the cancer is selected from the
group consisting of: endometrial, breast, prostate, colon, lung,
liver, lymph node, kidney, pancreas, prostate, ovary, spleen, small
intestine, stomach, skin, testes, head and neck, esophagus, brain
(glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma,
ependymomas), blood cells, and bone marrow.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of
PCT/US04/028143, which was filed on Aug. 30, 2004, which claims the
benefit of U.S. Provisional Applications 60/499,264, filed on Aug.
29, 2003 and 60/539,213, filed on Jan. 26, 2004; the contents of
each application is hereby incorporated by reference in its
entirety.
BACKGROUND
[0003] Endostatin, a 183 amino acid proteolytic cleavage fragment
corresponding to the C-terminus of collagen 18, has been the
subject of investigation by a number of laboratories because of its
anti-tumor activity with no toxic side effects (O'Reilly et al.
(1997) Cell, 88: 277-285; Kisker et al. (2001) Cancer Res,
61:7669-7674; Dhanabal et al. (1999) Cancer Res, 59: 189-197; Yoon
et al. (1999) Cancer Res, 59: 6251-6256; Folkman and Kalluri,
(2003) Cancer Medicine, 6th edition, pp. 161-194. Hamilton: B.C.
Decker Inc.). A number of anti-angiogenic activities have been
reported for this protein, such as inhibition of endothelial cell
proliferation, migration, and tube formation. Endostatin also
suppresses vascular endothelial growth factor (VEGF)-induced
vascular permeability (Takahashi et al. (2003) Faseb J, 17:
896-898). However, the mechanism of action of endostatin remains
unknown. Endostatin inhibits endothelial cell migration by
inhibiting phosphorylation of focal adhesion kinase via binding to
.alpha.5.beta.1 integrin (Wickstrom et al. (2002) Cancer Res, 62:
5580-5589). It also has been shown that cell surface glypicans are
low-affinity endostatin receptors (Karumanchi et al. (2001) Mol
Cell, 7: 811-822). Endostatin has been implicated in several
signaling pathways, such as downregulation of c-myc (Shichiri and
Hirata (2001) Faseb J, 15: 1044-1053), cyclin-D1 (Hanai et al.
(2002) J Biol Chem, 277: 16464-16469) and RhoA activity (Wickstrom
et al. (2003) J Biol Chem, 278: 37895-37901), blockage of VEGF
signaling (Hajitou et al. (2002) Faseb J, 16: 1802-1804; Kim et al.
(2002) J Biol Chem, 277: 27872-27879), and inhibition of the
wnt-signaling pathway (Hanai et al. (2002) J Cell Biol, 158:
529-539). Furthermore, endostatin has been shown to bind and
inactivate metalloproteinases (Kim et al. (2000) Cancer Res, 60:
5410-5413; Nyberg et al. (2003) J Biol Chem, 278: 22404-22411; Lee
et al. (2002) FEBS Lett, 519: 147-152) and to regulate a spectrum
of genes which suppress angiogenesis (Abdollahi et al. (2004) Mol
Cell, 13: 649-663).
[0004] The crystal structures of both murine and human endostatin
have been elucidated (Hohenester et al. (1998) Embo J, 17:
1656-1664; Ding et al. (1998) Proc Natl Acad Sci USA, 95:
10443-10448) and show a noncovalently held dimer at high
concentration required for crystallization (Ding et al. (1998) Proc
Natl Acad Sci USA, 95: 10443-10448). The presence of two disulfide
bonds results in a highly folded structure. Endostatin binds one
atom of zinc per monomer via the three histidines in the N-terminus
of the molecule (histidines 1, 3, and 11) and asparatic 76. The
heparin binding property of endostatin is mediated by noncontiguous
arginines clustered over the three dimensional globular surface of
the molecule (Sasaki et al. (1999) Embo J, 18: 6240-6248).
[0005] Oligomeric endostatin (NC1 and dimer) have been shown to be
primarily associated with laminin in the basement membrane
(Javaherian et al. (2002) J Biol Chem, 277: 45211-45218). This
association may be important for some of the biological functions
displayed by endostatin. On the other hand, the heparin binding
properties of endostatin manifest themselves in its interaction
with the cell surface. It is likely that endostatin has a number of
biological functions mediated by different regions of the
protein.
SUMMARY
[0006] The instant disclosure is based on the surprising finding
that the N-terminal region of endostatin is responsible for its
anti-angiogenic activity. Based on these findings, the disclosure
features anti-angiogenic peptides comprising at least about 12
amino acids of SEQ ID Nos. 2 or 4. Other anti-angiogenic peptides
comprise at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24 or 25 amino acids of SEQ ID Nos. 2 or 4. Exemplary
anti-angiogenic peptides are selected from the group consisting of
SEQ ID Nos. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124-131.
[0007] Also featured are pharmaceutical compositions comprising a
pharmaceutically acceptable carrier and an effective amount of an
anti-angiogenic peptide comprising at least about 12 amino acids of
SEQ ID Nos. 2 or 4. Certain pharmaceutical compositions are
comprised of an anti-angiogenic protein as disclosed herein and a
second peptide. Other pharmaceutical compositions additionally
comprise an effective amount of zinc. Devices, such as syringes and
stents which comprise a peptide disclosed herein, are also
described.
[0008] Further disclosed are nucleic acids encoding anti-angiogenic
peptides, which comprise at least about 12 amino acids of SEQ ID
Nos. 2 or 4, as well as pharmaceutical compositions comprising a
disclosed nucleic acid in a suitable vector for expression of an
effective amount of anti-angiogenic peptide to a subject. Preferred
nucleic acids comprise at least about 36, 54 or 60 nucleotides of
SEQ ID Nos.: 1, 3 or 5. Other preferred nucleic acids are selected
from the group consisting of SEQ ID Nos.: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121 and 123.
[0009] Also provided are methods for using the disclosed peptides
for treating or preventing a disease or condition that results from
angiogenesis (an angiogenic associated disease), such as a cancer
or tumor growth.
[0010] Other features and advantages of the disclosed
anti-angiogenic peptides will become apparent based on an
understanding of the following detailed description and claims.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a graph showing treatment of human pancreatic
carcinoma (BxPC3) with human endostatin peptides.
[0012] FIG. 2A-C are graphs showing that the N-terminal domain of
endostatin is responsible for its anti-tumor properties. FIG. 2A is
a graph showing treatment of LLC with murine Fc-endostatin and
murine peptides P1, P2, P5, and P6 (mP1, mP2, mP5 and mP6,
respectively); FIG. 2B are images of LLC sections showing CD31
staining; FIG. 2C is a graph showing the determination of vessel
density (*p<0.015 vs. PBS (control)); and FIG. 2D is a schematic
diagram showing the crystal structure of endostatin.
[0013] FIG. 3A-E are graphs showing that the zinc binding site of
endostatin is important for anti-tumor activity. FIG. 3A is a
schematic diagram of mP1 and mP1-H1/3A; FIG. 3B is a graph showing
zinc binding to mP1 and mP1-H1/3A; FIG. 3C is a graph showing
treatment of LLC with mP1 and mP1-H1/3A; FIG. 3D shows images of
LLC tumor sections stained with CD31; and FIG. 3E is a graph
showing the determination of vessel density.
[0014] FIG. 4 is a graph showing the tumor volume of mice having
LLC treated twice daily with mP1 or mP1-H with or without zinc on
days 4, 7, 10 and 14.
[0015] FIG. 5 is a graph showing inhibition of endothelial cell
migration by endostatin peptides.
[0016] FIGS. 6A and B show inhibition of VEGF-induced permeability
by endostatin peptides. FIG. 6A is a graph showing quantification
of Evan's blue dye extracted from the skin by incubation with
formamide for 5 days at room temperature as measured at 620 nm; and
FIG. 6B shows representative pictures of a Miles assay (V is VEGF,
P is PBS).
[0017] FIG. 7 is a graph showing the tumor volume in mice to which
mP1 endostatin peptides mP1, mP1-15, mP1-20 or PBS was administered
as a function of days following the beginning of the treatment.
[0018] FIG. 8 shows the nucleic acid and amino acid sequence of
human and murine endostatin proteins.
DETAILED DESCRIPTION
Definitions
[0019] As used herein, the following terms and phrases shall have
the meanings set forth below. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art.
[0020] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.
[0021] The term "bioavailable" when referring to a compound is
art-recognized and refers to a form of a compound that allows for
it, or a portion of the amount of compound administered, to be
absorbed by, incorporated to, or otherwise physiologically
available to a subject or patient to whom it is administered.
[0022] As used herein, the term "composition" is intended to
encompass a product comprising specified ingredients in specific
amounts, as well as any product which results, directly or
indirectly, from combination of the specific ingredients in the
specified amounts.
[0023] "Conservative substitutions" are changes between amino acids
of broadly similar molecular properties. For example, interchanges
within the aliphatic group alanine, valine, leucine and isoleucine
can be considered as conservative. Sometimes substitution of
glycine for one of these can also be considered conservative. Other
conservative interchanges include those within the aliphatic group
aspartate and glutamate; within the amide group asparagine and
glutamine; within the hydroxyl group serine and threonine; within
the aromatic group phenylalanine, tyrosine and tryptophan; within
the basic group lysine, arginine and histidine; and within the
sulfur-containing group methionine and cysteine. Sometimes
substitution within the group methionine and leucine can also be
considered conservative. Preferred conservative substitution groups
are aspartate-glutamate; asparagine-glutamine;
valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine;
and lysine-arginine.
[0024] The terms "parenteral administration" and "administered
parenterally" are art-recognized and refer to modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intra-articular, subcapsular, subarachnoid, intraspinal, and
intrasternal injection and infusion.
[0025] A "patient," "subject" or "host" to be treated by the
subject method may mean either a human or non-human animal.
[0026] The term "percent identical" refers to sequence identity
between two amino acid sequences or between two nucleotide
sequences. Identity can each be determined by comparing a position
in each sequence which may be aligned for purposes of comparison.
When an equivalent position in the compared sequences is occupied
by the same base or amino acid, then the molecules are identical at
that position; when the equivalent site occupied by the same or a
similar amino acid residue (e.g., similar in steric and/or
electronic nature), then the molecules can be referred to as
homologous (similar) at that position. Expression as a percentage
of homology, similarity, or identity refers to a function of the
number of identical or similar amino acids at positions shared by
the compared sequences. Various alignment algorithms and/or
programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and
BLAST are available as a part of the GCG sequence analysis package
(University of Wisconsin, Madison, Wis.), and can be used with,
e.g., default settings. ENTREZ is available through the National
Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, Md. In one embodiment, the
percent identity of two sequences can be determined by the GCG
program with a gap weight of 1, e.g., each amino acid gap is
weighted as if it were a single amino acid or nucleotide mismatch
between the two sequences. Other techniques for alignment are
described in Methods in Enzymology, vol. 266: Computer Methods for
Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic
Press, Inc., a division of Harcourt Brace & Co., San Diego,
Calif., USA. Preferably, an alignment program that permits gaps in
the sequence is utilized to align the sequences. The Smith-Waterman
is one type of algorithm that permits gaps in sequence alignments.
See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program
using the Needleman and Wunsch alignment method can be utilized to
align sequences. An alternative search strategy uses MPSRCH
software, which runs on a MASPAR computer. MPSRCH uses a
Smith-Waterman algorithm to score sequences on a massively parallel
computer. This approach improves ability to pick up distantly
related matches, and is especially tolerant of small gaps and
nucleotide sequence errors. Nucleic acid-encoded amino acid
sequences can be used to search both protein and DNA databases.
Databases with individual sequences are described in Methods in
Enzymology, ed. Doolittle, supra. Databases include Genbank, EMBL,
and DNA Database of Japan (DDBJ).
[0027] The term "pharmaceutically acceptable carrier" is
art-recognized and refers to a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting any subject composition or component
thereof from one organ, or portion of the body, to another organ,
or portion of the body. Each carrier must be "acceptable" in the
sense of being compatible with the subject composition and its
components and not injurious to the patient. Some examples of
materials which may serve as pharmaceutically acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer
solutions; and (21) other non-toxic compatible substances employed
in pharmaceutical formulations.
[0028] The terms "polynucleotide", and "nucleic acid" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. The following are non-limiting examples of
polynucleotides: coding or non-coding regions of a gene or gene
fragment, loci (locus) defined from linkage analysis, exons,
introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,
ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. The term "recombinant" polynucleotide means a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin
which either does not occur in nature or is linked to another
polynucleotide in a nonnatural arrangement. The term
"oligonucleotide" may be used to refer to a single stranded
polynucleotide having less than about 100 nucleotides, less than
about, e.g, 75, 50, 25, or 10 nucleotides.
[0029] The terms "polypeptide", "peptide" and "protein" (if single
chain) are used interchangeably herein to refer to polymers of
amino acids. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" refers to either natural and/or
unnatural or synthetic amino acids, including glycine and both the
D or L optical isomers, and amino acid analogs and
peptidomimetics.
[0030] The term "prophylactic" or "therapeutic" treatment is
art-recognized and refers to administration of a drug to a host. If
it is administered prior to clinical manifestation of the unwanted
condition (e.g., disease or other unwanted state of the host
animal) then the treatment is prophylactic, i.e., it protects the
host against developing the unwanted condition, whereas if
administered after manifestation of the unwanted condition, the
treatment is therapeutic (i.e., it is intended to diminish,
ameliorate or maintain the existing unwanted condition or side
effects therefrom).
[0031] The term "synthetic" is art-recognized and refers to
production by in vitro chemical or enzymatic synthesis.
[0032] The terms "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" are art-recognized and refer to the administration of
a subject composition, therapeutic or other material other than
directly into the central nervous system, such that it enters the
patient's system and, thus, is subject to metabolism and other like
processes.
[0033] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. The term thus means any substance
intended for use in the diagnosis, cure, mitigation, treatment or
prevention of disease or in the enhancement of desirable physical
or mental development and/or conditions in an animal or human.
[0034] The term "therapeutic effect" is art-recognized and refers
to a local or systemic effect in animals, particularly mammals, and
more particularly humans caused by a pharmacologically active
substance. The phrase "therapeutically-effective amount" means that
amount of such a substance that produces some desired local or
systemic effect at a reasonable benefit/risk ratio applicable to
any treatment. The therapeutically effective amount of such
substance will vary depending upon the subject and disease
condition being treated, the weight and age of the subject, the
severity of the disease condition, the manner of administration and
the like, which can readily be determined by one of ordinary skill
in the art. For example, certain compositions described herein may
be administered in a sufficient amount to produce a at a reasonable
benefit/risk ratio applicable to such treatment.
[0035] The term "treating" is art-recognized and refers to curing
as well as ameliorating at least one symptom of any condition or
disease or preventing a condition or disease from worsening.
[0036] The term "vector" refers to a nucleic acid capable of
transporting another nucleic acid to which it has been linked. One
type of vector which may be used in accord with the invention is an
episome, i.e., a nucleic acid capable of extra-chromosomal
replication. Other vectors include those capable of autonomous
replication and expression of nucleic acids to which they are
linked. Vectors capable of directing the expression of genes to
which they are operatively linked are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of "plasmids"
which refer to circular double stranded DNA molecules which, in
their vector form are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression
vectors which serve equivalent functions and which become known in
the art subsequently hereto.
Exemplary Compositions
[0037] Peptides
[0038] Provided herein are peptides or polypeptides and variants
thereof that inhibit, e.g., angiogenesis and thereby inhibit tumor
growth and/or formation. Peptides may comprise an N-terminal amino
acid sequence of an endostatin protein. The endostatin protein may
be a mammalian protein, such as from a human, a non-human primate,
a canine, a feline, an equine, a bovine, an ovine, a sheep, or a
rodent (e.g., mouse or rat). A nucleotide sequence encoding human
endostatin is set forth in SEQ ID NO: 151 and the protein encoded
thereby is set forth as SEQ ID NO: 152 and is identical to GenBank
Accession number AAF01310 except that it is lacking the initiator
methionine of AAF01310. A nucleotide sequence encoding mouse
endostatin is set forth in SEQ ID NO: 153 and the protein encoded
thereby is set forth as SEQ ID NO: 154 and is identical to GenBank
Accession number AAF69009. The nucleotide and amino acid sequences
of other species are also publicly available.
[0039] In one embodiment, the peptide comprises about 5 to about 40
amino acids of the N-terminal region of an endostatin protein. The
peptide may comprise from about 10 to about 30 or 35 amino acids or
from about 20 to about 30 or 35 amino acids of the N-terminal
region of an endostatin protein. For example, a peptide may
comprise about amino acids 1 to about amino acids 35, 30, 25, 20,
15, or 10 of an endostatin protein, such as a protein having SEQ ID
NO: 2 or 4. The amino acid sequences of these human and mouse
peptides are HSHRDFQPVLHLVALNSPLSGGMRGIR (SEQ ID NO: 2) and
HTHQDFQPVLHLVALNTPLSGGMRGIR (mP1; SEQ ID NO: 4), respectively. SEQ
ID NO:2 is encoded by the following nucleic acid sequence:
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacagccccctgtcaggcggcatgcggg-
gcatccgc (SEQ ID NO: 1). SEQ ID NO: 4 is encoded by the following
nucleic acid sequence:
catactcatcaggactttcagccagtgctccacctggtggcactgaacaccccctgtctggaggcatgcgtgg-
tatccgt (SEQ ID NO: 3).
[0040] Peptides, which are slightly different from SEQ ID NO: 1
have been shown to retain anti-angiogenic activity. One such
peptide is HSHRDFQPVLHLVALNSPLSGGMRG (hP1; SEQ ID NO: 6), which is
encoded by the nucleic acid sequence:
catactcatcaggactttcagccagtgctccacctggtggcactgaacaccccctgtctggaggcatgcgtgg-
t (SEQ ID NO: 5). SEQ ID NO: 6 does not contain the two most
C-terminal amino acid residues in SEQ ID NO: 2. Further
anti-angiogenic peptides may lack one or more amino acids at the N-
or C-terminus of SEQ ID Nos. 2 or 4. Exemplary anti-angiogenic
peptides are as follows:
TABLE-US-00001 (SEQ ID NO: 8) SHRDFQPVLHLVALNSPLSGGMRGIR; (SEQ ID
NO: 10) HRDFQPVLHLVALNSPLSGGMRGIR; (SEQ ID NO: 12)
RDFQPVLHLVALNSPLSGGMRGIR; (SEQ ID NO: 14) DFQPVLHLVALNSPLSGGMRGIR;
(SEQ ID NO: 16) FQPVLHLVALNSPLSGGMRGIR; (SEQ ID NO: 18)
QPVLHLVALNSPLSGGMRGIR; (SEQ ID NO: 20) PVLHLVALNSPLSGGMRGIR; (SEQ
ID NO: 22) VLHLVALNSPLSGGMRGIR; (SEQ ID NO: 24) LHLVALNSPLSGGMRGIR;
(SEQ ID NO: 26) HLVALNSPLSGGMRGIR; (SEQ ID NO: 28)
LVALNSPLSGGMRGIR; (SEQ ID NO: 30) VALNSPLSGGMRGIR; (SEQ ID NO: 32)
ALNSPLSGGMRGIR; (SEQ ID NO: 34) LNSPLSGGMRGIR; (SEQ ID NO: 36)
NSPLSGGMRGIR; (SEQ ID NO: 38) HSHRDFQPVLHLVALNSPLSGGMRGI; (SEQ ID
NO: 40) HSHRDFQPVLHLVALNSPLSGGMR; (SEQ ID NO: 42)
HSHRDFQPVLHLVALNSPLSGGM; (SEQ ID NO: 44) HSHRDFQPVLHLVALNSPLSGG;
(SEQ ID NO: 46) HSHRDFQPVLHLVALNSPLSG; (SEQ ID NO: 48)
HSHRDFQPVLHLVALNSPLS; (SEQ ID NO: 50) HSHRDFQPVLHLVALNSPL; (SEQ ID
NO: 52) HSHRDFQPVLHLVALNSP; (SEQ ID NO: 54) HSHRDFQPVLHLVALNS; (SEQ
ID NO: 56) HSHRDFQPVLHLVALN; (SEQ ID NO: 58) HSHRDFQPVLHLVAL; (SEQ
ID NO: 60) HSHRDFQPVLHLVA; (SEQ ID NO: 62) HSHRDFQPVLHLV; (SEQ ID
NO: 64) HSHRDFQPVLHL; (SEQ ID NO: 66) THQDFQPVLHLVALNTPLSGGMRGIR;
(SEQ ID NO: 68) HQDFQPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 70)
QDFQPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 72) DFQPVLHLVALNTPLSGGMRGIR;
(SEQ ID NO: 74) FQPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 76)
QPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 78) PVLHLVALNTPLSGGMRGIR; (SEQ
ID NO: 80) VLHLVALNTPLSGGMRGIR; (SEQ ID NO: 82) LHLVALNTPLSGGMRGIR;
(SEQ ID NO: 84) HLVALNTPLSGGMRGIR; (SEQ ID NO: 86)
LVALNTPLSGGMRGIR; (SEQ ID NO: 88) VALNTPLSGGMRGIR; (SEQ ID NO: 90)
ALNTPLSGGMRGIR; (SEQ ID NO: 92) LNTPLSGGMRGIR; (SEQ ID NO: 94)
NTPLSGGMRGIR; (SEQ ID NO: 96) HTHQDFQPVLHLVALNTPLSGGMRGI; (SEQ ID
NO: 98) HTHQDFQPVLHLVALNTPLSGGMRG; (SEQ ID NO: 100)
HTHQDFQPVLHLVALNTPLSGGMR; (SEQ ID NO: 102) HTHQDFQPVLHLVALNTPLSGGM;
(SEQ ID NO: 104) HTHQDFQPVLHLVALNTPLSGG; (SEQ ID NO: 106)
HTHQDFQPVLHLVALNTPLSG; (mP1-20; SEQ ID NO: 108)
HTHQDFQPVLHLVALNTPLS; (SEQ ID NO: 110) HTHQDFQPVLHLVALNTPL; (SEQ ID
NO: 112) HTHQDFQPVLHLVALNTP; (SEQ ID NO: 114) HTHQDFQPVLHLVALNT;
(SEQ ID NO: 116) HTHQDFQPVLHLVALN; (mP1-15; SEQ ID NO: 118)
HTHQDFQPVLHLVAL; (SEQ ID NO: 120) HTHQDFQPVLHLVA; (SEQ ID NO: 122)
HTHQDFQPVLHLV; (SEQ ID NO: 124) HTHQDFQPVLHL; (SEQ ID NO: 126)
HSHRDFVALNSPLSGGMRGIR; (SEQ ID NO: 128) HSHRDFQPVLHLLSGGMRGIR; (SEQ
ID NO: 130) QPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 132)
HTHQDFVALNTPLSGGMRGIR; and (SEQ ID NO: 134)
HTHQDFQPVLHLLSGGMRGIR.
[0041] Other anti-angiogenic peptides are based on the following
consensus sequences: HXaaHXaaDFQPVLHLVALNXaaPLSGGMRGIR (SEQ ID NO:
130) or HXaaHXaaDFQPVLHLVALNXaaPLSG (SEQ ID NO: 131), wherein Xaa
is any amino acid.
[0042] Other peptides having anti-angiogenic activity may comprise,
consist of or consist essentially of any of the amino acid
sequences described above. Yet other peptides may comprise, consist
of or consist essentially of an amino acid sequence that has at
least about 70%, 80%, 90%, 95%, 98% or 99% identity or homology
with an N-terminal endostatin peptide. For example, peptides that
differ from a sequence in a naturally occurring endostatin protein
in about 1, 2, 3, 4, 5 or more amino acids would be expected to
retain anti-angiogenic activity. Peptides that are similar to the
sequences described above may contain substitutions, e.g.,
conservative substitutions, deletions or additions. The differences
are preferably in regions that are not significantly conserved
among different species. Such regions can be identified by aligning
the amino acid sequences of endostatin proteins from various animal
species. For example, amino acids 2, 4, and 17 of SEQ ID NO: 2, 4,
or 6, and the highlighted amino acids in e.g. SEQ ID NOs: 8, 10,
12, 30, 32, or 34 may be substituted without negatively impacting
the anti-angiogenic activity, since these amino acids differ in the
human and mouse sequences. These amino acids can be substituted,
e.g., with those found in another species. Amino acid 9 can also be
substituted, since that one is different in the Gallus gallus
species. Other amino acids that may be substituted, inserted or
deleted at these or other locations can be identified by
mutagenesis studies coupled with biological assays.
[0043] Also encompassed herein are endostatin peptides that are
fused to a heterologous peptide, such as a peptide that can be used
for detecting; purifying; stabilizing; or solubilizing the
endostatin peptide.
[0044] A peptide may by linked to an immunoglobulin (Ig) constant
heavy or light chain domain or portion thereof. For example, a
peptide may be linked to a CH1, CH2 and/or CH3 domain of a heavy
chain. If the constant region is from a light chain, it may be from
a kappa or lambda light chain. If the constant region is from a
heavy chain, it may be from an antibody of any one of the following
classes of antibodies: IgG, IgA, IgE, IgD, and IgM. IgG may be
IgG1, IgG2, IgG3 or IgG4. The constant domain may be an Fc
fragment. The constant domain may be from a mammalian antibody,
e.g., a human antibody. Soluble receptor-IgG fusion proteins are
common immunological reagents and methods for their construction
are known in the art (see e.g., U.S. Pat. Nos. 5,225,538,
5,726,044; 5,707,632; 750,375, 5,925,351, 6,406,697 and Bergers et
al. Science 1999 284: 808-12). Preferred as immunoglobulin is the
constant part of the heavy chain of human IgG, particularly IgG1,
where dimerization between two heavy chains takes place at the
hinge region. It is recognized that inclusion of the CH2 and CH3
domains of the Fc region as part of the fusion polypeptide
increases the in vivo circulation half-life of the polypeptide
comprising the Fc region, and that of the oligomer or dimer
comprising the polypeptide.
[0045] An Fc portion of human IgG1 which includes the hinge region,
and domains CH2 and CH3 has the nucleotide sequence 5' gag ccc aaa
tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg
ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg
atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa
gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat
gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc
agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag
tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc
aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc
cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc
ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag
aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc
ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag
agc ctc tcc ctg tct ccg ggt aaa tga 3' (SEQ ID NO: 132), which
encodes a peptide having the amino acid sequence: Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys (SEQ ID NO: 133).
[0046] Constant Ig domains may also contain one or more mutations
that reduce or eliminate one or more effector function, e.g.,
binding to Fc receptors and complement activation (see, e.g., S.
Morrison, Annu. Rev. Immunol., 10, pp. 239-65 (1992); Duncan and
Winter (1988) Nature 332: 738-740; and Xu et al. (1994) J. Biol.
Chem. 269: 3469-3474). For example, mutations of amino acids
corresponding to Leu 235 and Pro 331 of human IgGI to Glu and Ser
respectively, are provided. Such constructs are further described
in U.S. Pat. No. 6,656,728.
[0047] The constant Ig domain may be linked to the N-terminus or
C-terminus of a peptide.
[0048] The peptide may also be linked to a linker sequence with a
thrombin cleavage site, such as between the peptide and an
immunoglobulin domain. An exemplary nucleotide sequence encoding
such a site has the following nucleotide sequence: 5' tct aga ggt
ggt cta gtg ccg cgc ggc agc ggt tcc ccc ggg ttg cag 3' (SEQ ID NO:
134), which encodes a peptide having the amino acid sequence: Ser
Arg Gly Gly Leu Val Pro Arg Gly Ser Gly Ser Pro Gly Leu Gln (SEQ ID
NO: 135).
[0049] A peptide may also be fused to a signal sequence. For
example, when prepared recombinantly, a nucleic acid encoding the
peptide may be linked at its 5' end to a signal sequence, such that
the peptide is secreted from the cell.
[0050] Peptides may be used as a substantially pure preparation,
e.g., wherein at least about 90% of the peptides in the preparation
are the desired peptide. Compositions comprising at least about
50%, 60%, 70%, or 80% of the desired peptide may also be used.
[0051] Peptides may be denatured or non-denatured and may be
aggregated or non-aggregated as a result thereof. Peptides can be
denatured according to methods known in the art.
[0052] Peptides may be conjugated to zinc. Thus, peptides may be in
a composition comprising Zn.sup.2+, e.g., in sufficient quantities
that most of the peptides are conjugated to one or more Zn.sup.2+
molecule. Binding of Zn.sup.2+ to a peptide can be demonstrated by
the following assay. Zinc and peptide solutions are mixed,
optionally incubated together, and then dialyzed to remove the zinc
that is not bound to the peptides. Detection of zinc in the peptide
solution can then be performed by atomic absorption.
[0053] Yet other peptides that are encompassed herein are those
that comprise modified amino acids. Exemplary peptides are
derivative peptides that may be one modified by glycosylation,
pegylation, phosphorylation or any similar process that retains at
least one biological function of the peptide from which it was
derived.
[0054] Peptides may also comprise one or more non-naturally
occurring amino acids. For example, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into peptides. Non-classical amino acids include, but are
not limited to, the D-isomers of the common amino acids,
2,4-diaminobutyric acid, alpha-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu,
epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino
acids such as beta-methyl amino acids, Calpha-methyl amino acids,
Nalpha-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0055] In other specific embodiments, branched versions of the
peptides listed herein are provided, e.g., by substituting one or
more amino acids within the sequence with an amino acid or amino
acid analog with a free side chain capable of forming a peptide
bond with one or more amino acids (and thus capable of forming a
"branch"). Cyclical peptides are also contemplated.
[0056] Also included are peptide derivatives which are
differentially modified during or after synthesis, e.g., by
benzylation, glycosylation, acetylation, phosphorylation,
amidation, pegylation, derivatization by known protecting/blocking
groups, proteolytic cleavage, linkage to an antibody molecule or
other cellular ligand, etc. In specific embodiments, the peptides
are acetylated at the N-terminus and/or amidated at the
C-terminus.
[0057] Also provided are derivatives of endostatin peptides, such
as chemically modified peptides and peptidomimetics.
Peptidomimetics are compounds based on, or derived from, peptides
and proteins. Peptidomimetics can be obtained by structural
modification of known peptide sequences using unnatural amino
acids, conformational restraints, isosteric replacement, and the
like. The subject peptidomimetics constitute the continuum of
structural space between peptides and non-peptide synthetic
structures; peptidomimetics may be useful, therefore, in
delineating pharmacophores and in helping to translate peptides
into nonpeptide compounds with the activity of the parent
peptides.
[0058] Moreover, mimetopes of the subject peptides can be provided.
Such peptidomimetics can have such attributes as being
non-hydrolyzable (e.g., increased stability against proteases or
other physiological conditions which degrade the corresponding
peptide), increased specificity and/or potency for stimulating cell
differentiation. For illustrative purposes, peptide analogs can be
generated using, for example, benzodiazepines (e.g., see Freidinger
et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,
ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma
lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.
R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p
123), C-7 mimics (Huffman et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,
1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. (1986)
J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and
Function (Proceedings of the 9th American Peptide Symposium) Pierce
Chemical Co. Rockland, Ill., 1985), .beta.-turn dipeptide cores
(Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al.
(1986) J Chem Soc Perkin Trans 1: 1231), .beta.-aminoalcohols
(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann
et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones
(Natarajan et al. (1984) Biochem Biophys Res Commun 124:141), and
methyleneamino-modified (Roark et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,
1988, p 134). Also, see generally, Session III: Analytic and
synthetic methods, in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988).
[0059] In addition to a variety of sidechain replacements which can
be carried out to generate peptidomimetics, the description
specifically contemplates the use of conformationally restrained
mimics of peptide secondary structure. Numerous surrogates have
been developed for the amide bond of peptides. Frequently exploited
surrogates for the amide bond include the following groups (i)
trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv)
phosphonamides, and (v) sulfonamides.
##STR00001##
[0060] Additionally, peptidomimietics based on more substantial
modifications of the backbone of a peptide can be used.
Peptidomimetics which fall in this category include (i)
retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called
peptoids).
##STR00002##
[0061] Furthermore, the methods of combinatorial chemistry are
being brought to bear, on the development of new peptidomimetics.
For example, one embodiment of a so-called "peptide morphing"
strategy focuses on the random generation of a library of peptide
analogs that comprise a wide range of peptide bond substitutes.
##STR00003##
[0062] In an exemplary embodiment, the peptidomimetic can be
derived as a retro-inverso analog of the peptide. Such
retro-inverso analogs can be made according to the methods known in
the art, such as that described by the Sisto et al. U.S. Pat. No.
4,522,752. A retro-inverso analog can be generated as described,
e.g., in WO 00/01720. It will be understood that a mixed peptide,
e.g. including some normal peptide linkages, may be generated. As a
general guide, sites which are most susceptible to proteolysis are
typically altered, with less susceptible amide linkages being
optional for mimetic switching. The final product, or intermediates
thereof, can be purified by HPLC.
[0063] Peptides may comprise at least one amino acid or every amino
acid that is a D stereoisomer. Other peptides may comprise at least
one amino acid that is reversed. The amino acid that is reversed
may be a D stereoisomer. Every amino acid of a peptide may be
reversed and/or every amino acid may be a D stereoisomer.
[0064] In another illustrative embodiment, a peptidomimetic can be
derived as a retro-enantio analog of a peptide. Retro-enantio
analogs such as this can be synthesized with commercially available
D-amino acids (or analogs thereof) and standard solid- or
solution-phase peptide-synthesis techniques, as described, e.g., in
WO 00/01720. The final product may be purified by HPLC to yield the
pure retro-enantio analog.
[0065] In still another illustrative embodiment, trans-olefin
derivatives can be made for the subject peptide. Trans-olefin
analogs can be synthesized according to the method of Y. K. Shue et
al. (1987) Tetrahedron Letters 28:3225 and as described in WO
00/01720. It is further possible to couple pseudodipeptides
synthesized by the above method to other pseudodipeptides, to make
peptide analogs with several olefinic functionalities in place of
amide functionalities.
[0066] Still another class of peptidomimetic derivatives include
the phosphonate derivatives. The synthesis of such phosphonate
derivatives can be adapted from known synthesis schemes. See, for
example, Loots et al. in Peptides: Chemistry and Biology, (Escom
Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in
Peptides: Structure and Function (Proceedings of the 9th American
Peptide Symposium, Pierce Chemical Co. Rockland, Ill., 1985).
[0067] Many other peptidomimetic structures are known in the art
and can be readily adapted for use in the subject peptidomimetics.
To illustrate, a peptidomimetic may incorporate the
1-azabicyclo[4.3.0]nonane surrogate (see Kim et al. (1997) J. Org.
Chem. 62:2847), or an N-acyl piperazic acid (see Xi et al. (1998)
J. Am. Chem. Soc. 120:80), or a 2-substituted piperazine moiety as
a constrained amino acid analogue (see Williams et al. (1996) J.
Med. Chem. 39:1345-1348). In still other embodiments, certain amino
acid residues can be replaced with aryl and bi-aryl moieties, e.g.,
monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a
biaromatic, aromatic-heteroaromatic, or biheteroaromatic
nucleus.
[0068] The subject peptidomimetics can be optimized by, e.g.,
combinatorial synthesis techniques combined with high throughput
screening.
[0069] Moreover, other examples of mimetopes include, but are not
limited to, protein-based compounds, carbohydrate-based compounds,
lipid-based compounds, nucleic acid-based compounds, natural
organic compounds, synthetically derived organic compounds,
anti-idiotypic antibodies and/or catalytic antibodies, or fragments
thereof. A mimetope can be obtained by, for example, screening
libraries of natural and synthetic compounds for compounds capable
of inhibiting angiogenesis and/or tumor growth. A mimetope can also
be obtained, for example, from libraries of natural and synthetic
compounds, in particular, chemical or combinatorial libraries
(i.e., libraries of compounds that differ in sequence or size but
that have the same building blocks). A mimetope can also be
obtained by, for example, rational drug design. In a rational drug
design procedure, the three-dimensional structure of a compound of
the present invention can be analyzed by, for example, nuclear
magnetic resonance (NMR) or x-ray crystallography. The
three-dimensional structure can then be used to predict structures
of potential mimetopes by, for example, computer modelling. The
predicted mimetope structures can then be produced by, for example,
chemical synthesis, recombinant DNA technology, or by isolating a
mimetope from a natural source (e.g., plants, animals, bacteria and
fungi).
[0070] "Peptides, variants and derivatives thereof" or "peptides
and analogs thereof" are included in "peptide therapeutics" and is
intended to include any of the peptides or modified forms thereof,
e.g., peptidomimetics, described herein. Preferred peptide
therapeutics have anti-angiogenic activity. For example, they may
reduce or inhibit angiogenesis by a factor of at least about 50%, 2
fold, 5 fold, 10 fold, 30 fold or 100 fold, as determined, e.g., in
an assay described herein.
[0071] Assays for testing candidate peptides for anti-angiogenesis
and inhibition of tumor growth or formation are known in the art
and exemplary ones are further described herein.
[0072] Nucleic Acids
[0073] Also disclosed are nucleic acids encoding anti-angiogenic
peptides. Preferred nucleic acids are as follows:
TABLE-US-00002 (SEQ ID NO: 1)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtcaggcggcatgcggggcatccgc. (SEQ ID NO: 3)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatgcgtggtatccgt. (SEQ ID NO: 5)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatgcgtggt. (SEQ ID NO: 7)
agccaccgcgacttccagccggtgctccacctggttgcgctcaacagccc
cctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 9)
caccgcgacttccagccggtgctccacctggttgcgctcaacagccccct
gtcaggcggcatgcggggcatccgc; (SEQ ID NO: 11)
cgcgacttccagccggtgctccacctggttgcgctcaacagccccctgtc
aggcggcatgcggggcatccgc; (SEQ ID NO: 13)
gacttccagccggtgctccacctggttgcgctcaacagccccctgtcagg
cggcatgcggggcatccgc; (SEQ ID NO: 15)
ttccagccggtgctccacctggttgcgctcaacagccccctgtcaggcgg
catgcggggcatccgc; (SEQ ID NO: 17)
cagccggtgctccacctggttgcgctcaacagccccctgtcaggcggcat gcggggcatccgc;
(SEQ ID NO: 19) ccggtgctccacctggttgcgctcaacagccccctgtcaggcggcatgcg
gggcatccgc; (SEQ ID NO: 21)
gtgctccacctggttgcgctcaacagccccctgtcaggcggcatgcgggg catccgc; (SEQ ID
NO: 23) ctccacctggttgcgctcaacagccccctgtcaggcggcatgcggggcat ccgc;
(SEQ ID NO: 25) cacctggttgcgctcaacagccccctgtcaggcggcatgcggggcatccg
c; (SEQ ID NO: 27)
ctggttgcgctcaacagccccctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 29)
gttgcgctcaacagccccctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 31)
gcgctcaacagccccctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 33)
ctcaacagccccctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 35)
aacagccccctgtcaggcggcatgcggggcatccgc; (SEQ ID NO: 37)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtcaggcggcatgcggggcatc; (SEQ ID NO: 39)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtcaggcggcatgcgg; (SEQ ID NO: 41)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtcaggcggcatg; (SEQ ID NO: 43)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtcaggcggc; (SEQ ID NO: 45)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag ccccctgtcaggc;
(SEQ ID NO: 47) cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
ccccctgtca; (SEQ ID NO: 49)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag ccccctg; (SEQ ID
NO: 51) cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag cccc;
(SEQ ID NO: 53) cacagccaccgcgacttccagccggtgctccacctggttgcgctcaacag
c; (SEQ ID NO: 55)
cacagccaccgcgacttccagccggtgctccacctggttgcgctcaac; (SEQ ID NO: 57)
cacagccaccgcgacttccagccggtgctccacctggttgcgctc; (SEQ ID NO: 59)
cacagccaccgcgacttccagccggtgctccacctggttgcg; (SEQ ID NO: 61)
cacagccaccgcgacttccagccggtgctccacctggtt; (SEQ ID NO: 63)
cacagccaccgcgacttccagccggtgctccacctg; (SEQ ID NO: 65)
actcatcaggactttcagccagtgctccacctggtggcactgaacacccc
cctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 67)
catcaggactttcagccagtgctccacctggtggcactgaacacccccct
gtctggaggcatgcgtggtatccgt; (SEQ ID NO: 69)
caggactttcagccagtgctccacctggtggcactgaacacccccctgtc
tggaggcatgcgtggtatccgt; (SEQ ID NO: 71)
gactttcagccagtgctccacctggtggcactgaacacccccctgtctgg
aggcatgcgtggtatccgt; (SEQ ID NO: 73)
atttcagccagtgctccacctggtggcactgaacacccccctgtctggag
gcatgcgtggtatccgt; (SEQ ID NO: 75)
cagccagtgctccacctggtggcactgaacacccccctgtctggaggcat gcgtggtatccgt;
(SEQ ID NO: 77) ccagtgctccacctggtggcactgaacacccccctgtctggaggcatgcg
tggtatccgt; (SEQ ID NO: 79)
gtgctccacctggtggcactgaacacccccctgtctggaggcatgcgtgg tatccgt; (SEQ ID
NO: 81) ctccacctggtggcactgaacacccccctgtctggaggcatgcgtggtat ccgt;
(SEQ ID NO: 83) cacctggtggcactgaacacccccctgtctggaggcatgcgtggtatccg
t; (SEQ ID NO: 85)
ctggtggcactgaacacccccctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 87)
gtggcactgaacacccccctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 89)
gcactgaacacccccctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 91)
ctgaacacccccctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 93)
aacacccccctgtctggaggcatgcgtggtatccgt; (SEQ ID NO: 95)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatgcgtggtatc; (SEQ ID NO: 97)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatgcgtggt; (SEQ ID NO: 99)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatgcgt; (SEQ ID NO: 101)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggcatg; (SEQ ID NO: 103)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtctggaggc; (SEQ ID NO: 105)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac ccccctgtctgga;
(SEQ ID NO: 107) catactcatcaggactttcagccagtgctccacctggtggcactgaacac
ccccctgtct; (SEQ ID NO: 109)
catactcatcaggactttcagccagtgctccacctggtggcactgaacac ccccctg; (SEQ ID
NO: 111) catactcatcaggactttcagccagtgctccacctggtggcactgaacac
cccc;
(SEQ ID NO: 113) catactcatcaggactttcagccagtgctccacctggtggcactgaacac
c; (SEQ ID NO: 115)
catactcatcaggactttcagccagtgctccacctggtggcactgaac; (SEQ ID NO: 117)
catactcatcaggactttcagccagtgctccacctggtggcactg; (SEQ ID NO: 119)
catactcatcaggactttcagccagtgctccacctggtggca; (SEQ ID NO: 121)
catactcatcaggactttcagccagtgctccacctggtg; and (SEQ ID NO: 123)
catactcatcaggactttcagccagtgctccacctg.
[0074] Nucleic acids include vectors, such as expression vectors
for producing a peptide, e.g., viral vectors. Also encompassed
herein are cells comprising a nucleic acid encoding a peptide
described herein and methods for producing peptides comprising
culturing these cells to produce a peptide. These methods can be
used of producing recombinant peptides or for expression of a
peptide in a cell, e.g., in a cell of a subject.
[0075] Appropriate vectors may be introduced into host cells using
well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0076] The vector may contain a selectable marker for propagation
in a host. Generally, a plasmid vector is introduced in a
precipitate, such as a calcium phosphate precipitate, or in a
complex with a charged lipid. If the vector is a virus, it may be
packaged in vitro using an appropriate packaging cell line and then
transduced into host cells.
[0077] Preferred vectors comprise cis-acting control regions to the
polynucleotide of interest. Appropriate trans-acting factors may be
supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0078] In certain embodiments, the vectors provide for specific
expression, which may be inducible and/or cell type-specific.
Particularly preferred among such vectors are those inducible by
environmental factors that are easy to manipulate, such as
temperature and nutrient additives.
[0079] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0080] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating site at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0081] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline, kanamycin, or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Sf9 cells; animal cells such as CHO, COS and Bowes melanoma
cells; and plant cells. Appropriate culture mediums and conditions
for the above-described host cells are known in the art.
[0082] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE9, pQE10 available from Qiagen; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A available from Stratagene; pET series of vectors available
from Novagen; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
available from Pharmacia. Among preferred eukaryotic vectors are
pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and
pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable
vectors will be readily apparent to the skilled artisan.
[0083] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3, T5 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters, the trp promoter and the xyI/tet chimeric promoter.
Suitable eukaryotic promoters include the CMV immediate early
promoter, the HSV thymidine kinase promoter, the early and late
SV40 promoters, the promoters of retroviral LTRs, such as those of
the Rous sarcoma virus (RSV), and metallothionein promoters, such
as the mouse metallothionein-I promoter.
[0084] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals (for
example, Davis, et al., Basic Methods In Molecular Biology
(1986)).
[0085] Transcription of DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300
nucleotides that act to increase transcriptional activity of a
promoter in a given host cell-type. Examples of enhancers include
the SV40 enhancer, which is located on the late side of the
replication origin at nucleotides 100 to 270, the cytomegalovirus
early promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers.
[0086] A recombinant soluble form of a polypeptide of the present
invention may also be produced, e.g., by deleting at least a
portion of the transmembrane domain, such that the protein is not
capable to localize itself to a cell membrane. Also within the
scope of the invention are nucleic acids encoding splice variants
or nucleic acids representing transcripts synthesized from an
alternative transcriptional initiation site, such as those whose
transcription was initiated from a site in an intron. Such
homologues can be cloned by hybridization or PCR using standard
methods known in the art.
[0087] The polynucleotide sequence may also encode for a leader
sequence, e.g., the natural leader sequence or a heterologous
leader sequence. Alternatively, the nucleic acid can be engineered
such that the natural leader sequence is deleted and a heterologous
leader sequence inserted in its place. The term "leader sequence"
is used interchangeably herein with the term "signal peptide". For
example, the desired DNA sequence may be fused in the same reading
frame to a DNA sequence which aids in expression and secretion of
the polypeptide from the host cell, for example, a leader sequence
which functions as a secretory sequence for controlling transport
of the polypeptide from the cell. The protein having a leader
sequence is a preprotein and may have the leader sequence cleaved
by the host cell to form the mature form of the protein.
[0088] For secretion of the translated polypeptide into the lumen
of the endoplasmic reticulum, into the periplasmic space or into
the extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide, for example, the amino
acid sequence KDEL. The signals may be endogenous to the
polypeptide or they may be heterologous signals.
[0089] The polypeptides may be expressed in a modified form, such
as a fusion protein, and may include not only secretion signals,
but also additional heterologous functional regions. For instance,
a region of additional amino acids, particularly charged amino
acids, may be added to the N-terminus or C-terminus of the
polypeptide to improve stability and persistence in the host cell,
during purification, or during subsequent handling and storage.
Also, peptide moieties may be added to the polypeptide to
facilitate purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art. An example of such a
fusion protein may comprise a heterologous region from
immunoglobulin that is useful to solubilize proteins.
Exemplary Methods
[0090] Anti-angiogenic peptides or an anti-angiogenic peptide
encoding nucleic acid vector may be administered to a subject in
need thereof to prevent or reduce angiogenesis and/or cell
proliferation, e.g., tumor growth, or any disease or disorder
associated therewith. The subject may be a human or animal, such as
a mammal.
[0091] Unregulated angiogenesis occurs in a multiplicity of disease
states, tumor metastasis and abnormal growth by endothelial cells
and supports the pathological damage seen in these conditions. The
diverse pathological states created due to unregulated angiogenesis
have been grouped together as angiogenic dependent or angiogenic
associated diseases. Therapies directed at control of the
angiogenic processes could lead to the abrogation or mitigation of
these diseases. Thus, angiogenesis is associated with any new
tissue growth, whether normal or disease associated. Accordingly,
the therapeutics described herein can be used to inhibit
angiogenesis associated with any new tissue growth, whether normal
or disease associated.
[0092] Therapeutics may be contacted with a tissue to prevent
angiogenesis and/or cell proliferation, e.g., tumor growth, or any
disease or disorder associated therewith. Diseases and processes
that are mediated by angiogenesis include hemangioma, solid tumors,
leukemia, metastasis, telangiectasia psoriasis scleroderma,
pyogenic granuloma, myocardial angiogenesis, plaque
neovascularization, coronary collaterals, cerebral collaterals,
arteriovenous malformations, ischemic limb angiogenesis, corneal
diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,
retrolental fibroplasia, arthritis, diabetic neovascularization,
macular degeneration, wound healing, peptic ulcer, fractures,
keloids, phemphigoid, trachoma, vasculogenesis, hematopoiesis,
ovulation, menstruation, and placentation. The therapeutics
described herein may also be used for treating or repressing the
growth of a cancer or reducing tumor mass.
[0093] Angiogenesis is prominent in solid tumor formation and
metastasis. Angiogenic factors have been found associated with
several solid tumors such as rhabdomyosarcomas, retinoblastoma,
Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor cannot
expand without a blood supply to provide nutrients and remove
cellular wastes. Tumors in which angiogenesis is important include
solid tumors, and benign tumors such as acoustic neuroma,
neurofibroma, trachoma and pyogenic granulomas. Prevention of
angiogenesis could halt the growth of these tumors and the
resultant damage to the animal due to the presence of the
tumor.
[0094] Angiogenesis is important in two stages of tumor metastasis.
The first stage where angiogenesis stimulation is important is in
the vascularization of the tumor which allows tumor cells to enter
the blood stream and to circulate throughout the body. After the
tumor cells have left the primary site, and have settled into the
secondary, metastasis site, angiogenesis must occur before the new
tumor can grow and expand. Therefore, prevention of angiogenesis
could lead to the prevention of metastasis of tumors and possibly
contain the neoplastic growth at the primary site.
[0095] It should be noted that angiogenesis has been associated
with blood-born tumors such as leukemias, any of various acute or
chronic neoplastic diseases of the bone marrow in which
unrestrained proliferation of white blood cells occurs, usually
accompanied by anemia, impaired blood clotting, and enlargement of
the lymph nodes, liver, and spleen. It is believed that
angiogenesis plays a role in the abnormalities in the bone marrow
that give rise to leukemia-like tumors.
[0096] The compositions described herein may be used for treating
atherosclerosis. As such, compositions comprising the peptides
described herein may be used to prevent or regress atherosclerosis
growth or plaque formation.
[0097] Generally, the compositions described herein may be used for
treating inflammatory-disorders, such as immune and non-immune
inflammation, chronic articular rheumatism, psoriasis disorders
associated with inappropriate or inopportune invasion of vessels,
such as diabetic retinopathy, neovascular glaucoma, restenosis,
capillary proliferation in atherosclerotic plaques and
osteoporosis. Cancer-associated disorders that can be treated
include solid tumors, solid tumor metastasis, angiofibromas,
retrolental fibroplasia, hemangiomas, and Kaposi sarcomas.
[0098] One example of a disease mediated by angiogenesis is ocular
neovascular disease. This disease is characterized by invasion of
new blood vessels into the structures of the eye such as the retina
or cornea. It is the most common cause of blindness and is involved
in approximately twenty eye diseases. In age-related macular
degeneration, the associated visual problems are caused by an
ingrowth of chorioidal capillaries through defects in Bruch's
membrane with proliferation of fibrovascular tissue beneath the
retinal pigment epithelium. Angiogenic damage is also associated
with diabetic retinopathy, retinopathy of prematurity, corneal
graft rejection, neovascular glaucoma and retrolental
fibroplasia.
[0099] Diseases associated with corneal neovascularization that can
be treated as described herein include but are not limited to,
diabetic retinopathy, retinopathy of prematurity, corneal graft
rejection, neovascular glaucoma and retrolental fibroplasia,
epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens
overwear, atopic keratitis, superior limbic keratitis, pterygium
keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,
Mycobacteria infections, lipid degeneration, chemical burns,
bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes
zoster infections, protozoan infections, Kaposi sarcoma, Mooren
ulcer, Terrien's marginal degeneration, mariginal keratolysis,
trauma, rheumatoid arthritis, systemic lupus, polyarteritis,
Wegeners sarcoidosis, Scieritis, Steven's Johnson disease, and
periphigoid radial keratotomy.
[0100] Diseases associated with retinal/choroidal
neovascularization that can be treated as described herein include,
but are not limited to, diabetic retinopathy, macular degeneration,
sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum,
Pagets disease, vein occlusion, artery occlusion, carotid
obstructive disease, chronic uveitis/vitritis, mycobacterial
infections, Lyme's disease, systemic lupus erythematosis,
retinopathy of prematurity, Eales disease, Bechets disease,
infections causing a retinitis or choroiditis, presumed ocular
histoplasmosis, Bests disease, myopia, optic pits, Stargarts
disease, pars planitis, chronic retinal detachment, hyperviscosity
syndromes, toxoplasmosis, trauma and post-laser complications.
Other diseases include, but are not limited to, diseases associated
with rubeosis (neovasculariation of the angle) and diseases caused
by the abnormal proliferation of fibrovascular or fibrous tissue
including all forms of proliferative vitreoretinopathy, whether or
not associated with diabetes.
[0101] Another disease in which angiogenesis is believed to be
involved is rheumatoid arthritis. The blood vessels in the synovial
lining of the joints undergo angiogenesis. In addition to forming
new vascular networks, the endothelial cells release factors and
reactive oxygen species that lead to pannus growth and cartilage
destruction. The factors involved in angiogenesis may actively
contribute to, and help maintain, the chronically inflamed state of
rheumatoid arthritis.
[0102] Factors associated with angiogenesis may also have a role in
osteoarthritis. The activation of the chondrocytes by
angiogenic-related factors contributes to the destruction of the
joint. At a later stage, the angiogenic factors would promote new
bone formation. Therapeutic intervention that prevents the bone
destruction could halt the progress of the disease and provide
relief for persons suffering with arthritis.
[0103] Chronic inflammation may also involve pathological
angiogenesis. Such disease states as ulcerative colitis and Crohn's
disease show histological changes with the ingrowth of new blood
vessels into the inflamed tissues. Bartonellosis, a bacterial
infection found in South America, can result in a chronic stage
that is characterized by proliferation of vascular endothelial
cells. Another pathological role associated with angiogenesis is
found in atherosclerosis. The plaques formed within the lumen of
blood vessels have been shown to have angiogenic stimulatory
activity.
[0104] One of the most frequent angiogenic diseases of childhood is
the hemangioma. In most cases, the tumors are benign and regress
without intervention. In more severe cases, the tumors progress to
large cavernous and infiltrative forms and create clinical
complications. Systemic forms of hemangiomas, the hemangiomatoses,
have a high mortality rate. Therapy-resistant hemangiomas exist
that cannot be treated with therapeutics currently in use.
[0105] Angiogenesis is also responsible for damage found in
hereditary diseases such as Osler-Weber-Rendu disease, or
hereditary hemorrhagic telangiectasia. This is an inherited disease
characterized by multiple small angiomas, tumors of blood or lymph
vessels. The angiomas are found in the skin and mucous membranes,
often accompanied by epistaxis (nosebleeds) or gastrointestinal
bleeding and sometimes with pulmonary or hepatic arteriovenous
fistula.
[0106] Another disease that can be treated according to the present
invention is acquired immune deficiency syndrome.
[0107] The therapeutic peptides may also be used for decreasing
overproliferation of normal, vascularized tissues, such as adipose
tissue, benign polyps, hypertrophied cardiac tissue, hypertrophied
renal tissue, hypertrophied prostatic tissue, tissue containing
amyloid deposits, and uterine fibroids. The therapeutics may be
administered in an amount effective to reduce the vascular supply
to the tissue, or to decrease the size or growth of the
vascularized tissue, such as adipose tissue, polyps (e.g.,
intestinal or nose polyps), and muscle (including cardiac) tissue.
Accordingly, subject that may be treated include a subject having
polypsis, and enlarged prostate, cardiac or renal hypertrophy or is
obese or overweight. The peptide therapeutics may be administered
in an amount and time period which results in blood levels
regulating the size and/or growth of the vascularized tissue to be
treated.
[0108] The therapies for losing weight are applicable to both
normal overweight individuals and individuals with genetic defects.
The method should also be useful in most cases involving weight
gains due to hormonal or metabolic defects or drug side effects. In
addition to promoting loss of body fat while maintaining lean body
mass and being able to sustain weight loss during chronic
administration, other benefits of the treatments include
normalization of blood glucose levels in obesity related
diabetes.
[0109] In addition, any disease or secondary condition developing
as a result from a disease associated with angiogenesis, which can
be treated according to the methods described herein, may also be
treated. For example, any condition resulting from being overweight
or obese may be treated or prevented as described herein. Exemplary
diseases include hyperlipidemia, dyslipogenesis,
hypercholesterolemia, impaired glucose tolerance, high blood
glucose sugar level, syndrome X, hypertension, atherosclerosis and
lipodystrophy, hypertension, high blood cholesterol, dyslipidemia,
type 2 diabetes, insulin resistance, glucose intolerance,
hyperinsulinemia, coronary heart disease, angina pectoris,
congestive heart failure, stroke, gallstones, cholescystitis and
cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and
respiratory problems, some types of cancer (such as endometrial,
breast, prostate, colon, lung, liver, lymph node, kidney, pancreas,
ovary, spleen, small intestine, stomach, skin, testes, head and
neck, esophagus, brain (glioblastomas, medulloblastoma,
astrocytoma, oligodendroglioma, ependymomas), blood cells, bone
marrow or other tissue), complications of pregnancy, poor female
reproductive health (such as menstrual irregularities, infertility,
irregular ovulation), bladder control problems (such as stress
incontinence); uric acid nephrolithiasis; and psychological
disorders (such as depression, eating disorders, distorted body
image, and low self esteem).
[0110] Angiogenesis is also involved in normal physiological
processes such as reproduction and wound healing. Angiogenesis is
an important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis could be
used to induce amenorrhea, to block ovulation or to prevent
implantation by the blastula.
[0111] In wound healing, excessive repair or fibroplasia can be a
detrimental side effect of surgical procedures and may be caused or
exacerbated by angiogenesis. Adhesions are a frequent complication
of surgery and lead to problems such as small bowel obstruction.
Therefore in this and in other situations, it may be desirable to
prevent wound healing with the therapeutic peptides described
herein.
[0112] Peptides may be provided in pharmaceutical compositions and
administered to a subject in need thereof. Peptides may also be
contacted with a tissue or cells in vitro.
[0113] Therapeutic methods generally comprise administering to a
subject, e.g., a subject in need thereof, a therapeutically
effective amount of a therapeutic, e.g., a peptide or variant or
derivative thereof or nucleic acid encoding such, as, e.g.,
described herein. Subjects may be mammals, such as humans,
non-human primates, canines, felines, equines, porcines, ovines,
bovines, sheep, mice and rat. In particular, methods described
herein can be used for veterinary purposes. The method may comprise
first diagnosing a subject with a disease or disorder in which
administration of a peptide described herein may be beneficial,
such as a malignant or benign tumor growth. The method may also
comprise determining the effect of the therapeutic a certain time
after its administration. For example, the size of the tumor growth
may be evaluated about one week, one month or two months after
starting of the treatment. The evaluation may also comprising
obtaining a sample of tissue, e.g., a tumor, and determining the
level of angiogenesis.
[0114] The therapeutics can be administered in a "growth inhibitory
amount," i.e., an amount of the peptide that is therapeutically
effective to inhibit or decrease proliferation of cells or tissues.
The therapeutics may also be administered in an "antiangiogenic
amount," i.e., an amount of a therapeutic that is therapeutically
effective to inhibit or decrease angiogenesis. The therapeutics may
be administered to mammals, preferably humans, either alone or, in
combination with pharmaceutically acceptable carriers, excipients
or diluents, in a pharmaceutical composition, according to standard
pharmaceutical practice. Therapeutics may be administered directly
into the tissue in which one desires to inhibit angiogenesis or
tumor growth. The therapeutics may also be administered orally or
parenterally, including intravenously, intramuscularly,
intraperitoneally, subcutaneously, rectally and topically. One or
more therapeutics can be injected directly into a tumor of the
subject to be treated.
[0115] The peptide or variant or derivative thereof may be
co-administered with zinc. For example a therapeutic composition
may be administered in a therapeutically effective dose (see below)
together with a therapeutically effective dose of Zn.sup.2+ (see
below). The peptide or variant or derivative thereof may be
combined with the zinc solution prior to administration or at the
time of administration, such as to allow zinc to interact with the
peptide. Alternatively, the peptide or variant or derivative
thereof is administered separately from the zinc, either before or
after administration of the zinc, provided that both are present in
the circulation at least during a common period. For example, a
solution of zinc may be administered about a few minutes to a few
hours before or after administration of the peptide. In yet other
embodiments, no zinc is administered to a subject to whom a peptide
is administered. Peptides that are administered may, however, still
bind zinc when the subject has zinc in their blood circulation.
[0116] Toxicity and therapeutic efficacy of the therapeutics can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Reagents
which exhibit large therapeutic indices are preferred. While
reagents that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such reagents to
the site of affected tissue in order to, e.g., minimize potential
damage to normal cells and, thereby, reduce side effects.
[0117] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such therapeutics lies preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any therapeutic used, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test therapeutic which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
[0118] The dosage of the therapeutic will depend on the disease
state or condition being treated and other clinical factors such as
weight and condition of the human or animal and the route of
administration of the compound. For treating humans or animals,
between approximately 0.5 mg/kilogram to 500 mg/kilogram of the
therapeutic can be administered. A more preferable range is about 1
mg/kilogram to about 100 mg/kilogram or from about 2 mg/kilogram to
about 50 mg/kilogram with the most preferable range being from
about 2 mg/kilogram to about 10 mg/kilogram. Depending upon the
half-life of the therapeutic in the particular animal or human, the
therapeutic can be administered between several times per day to
once a week. It is to be understood that the methods have
application for both human and veterinary use. The methods of the
present invention contemplate single as well as multiple
administrations, given either simultaneously or over an extended
period of time.
[0119] When the peptide or analog or derivative thereof is
administered with zinc, zinc may be included in the therapeutic
composition comprising the peptide or analog or derivative thereof
in a concentration from about 0.1 to about 100 mg/kg/day; about 1
to about 10 mg/kg/day; or about 2-5 mg/kg/day. Zinc may be
administered in the form of Zn.sup.2+ or a salt thereof. The amount
of zinc may vary depending on the amount of zinc in a subject's
circulation (e.g., blood) and on the amount of peptide administered
to the subject. The amount of zinc that may be necessary can be
determined, e.g., by taking a blood sample of a subject having
received a particular dose of peptide alone or with zinc and
determining the amount of peptide to which zinc is complexed, e.g.,
as described above.
[0120] Pharmaceutical compositions containing a therapeutic may be
in a form suitable for oral use, for example, as tablets, troches,
lozenges, aqueous or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any
method known to the art for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets may
contain the active ingredient (i.e., therapeutic) in admixture with
non-toxic pharmaceutically acceptable excipients which are suitable
for the manufacture of tablets. These excipients may be for
example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example,
microcrystalline cellulose, sodium crosscarmellose, corn starch, or
alginic acid; binding agents, for example starch, gelatin,
polyvinyl-pyrrolidone or acacia, and lubricating agents, for
example, magnesium stearate, stearic acid or talc. The tablets may
be uncoated or they may be coated by known techniques to mask the
unpleasant taste of the drug or delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a water soluble taste
masking material such as hydroxypropylmethyl-cellulose or
hydroxypropylcellulose, or a time delay material such as ethyl
cellulose, cellulose acetate buryrate may be employed.
[0121] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water soluble carrier such as
polyethyleneglycol or an oil medium, for example peanut oil, liquid
paraffin, or olive oil.
[0122] Aqueous suspensions may contain the active material in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
[0123] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as butylated
hydroxyanisol or alpha-tocopherol.
[0124] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
These compositions may be preserved by the addition of an
anti-oxidant such as ascorbic acid.
[0125] Pharmaceutical compositions may also be in the form of an
oil-in-water emulsions. The oily phase may be a vegetable oil, for
example olive oil or arachis oil, or a mineral oil, for example
liquid paraffin or mixtures of these. Suitable emulsifying agents
may be naturally-occurring phosphatides, for example soy bean
lecithin, and esters or partial esters derived from fatty acids and
hexitol anhydrides, for example sorbitan monooleate, and
condensation products of the said partial esters with ethylene
oxide, for example polyoxyethylene sorbitan monooleate. The
emulsions may also contain sweetening, flavouring agents,
preservatives and antioxidants.
[0126] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative,
flavoring and coloring agents and antioxidant.
[0127] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous solution. Among the acceptable vehicles
and solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution.
[0128] The sterile injectable preparation may also be a sterile
injectable oil-in-water microemulsion where the active ingredient
is dissolved in the oily phase. For example, the active ingredient
may be first dissolved in a mixture of soybean oil and lecithin.
The oil solution then introduced into a water and glycerol mixture
and processed to form a microemulation.
[0129] The injectable solutions or microemulsions may be introduced
into a patient's blood-stream by local bolus injection.
Alternatively, it may be advantageous to administer the solution or
microemulsion in such a way as to maintain a constant circulating
concentration of the instant compound. In order to maintain such a
constant concentration, a continuous intravenous delivery device
may be utilized. An example of such a device is the Deltec
CADD-PLUS.TM. model 5400 intravenous pump. For example, one could
generate blood levels of therapeutic peptides in the range of about
100 to 500 ng/ml, or about 200 to 400 ng/ml or about 250-300
ng/ml.
[0130] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous to or oleagenous suspension for
intramuscular and subcutaneous administration. This suspension may
be formulated according to the known art using those suitable
dispersing or wetting agents and suspending agents which have been
mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane-diol. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0131] In certain embodiments, it may be preferable to administer a
therapeutic locally, such as by local injection. For example, a
therapeutic may be injected directly into the tissue showing
excessive proliferation in which one desired to inhibit
angiogenesis. In one embodiment, a therapeutic is administered
locally into a tumor bed.
[0132] In one embodiment, a therapeutic peptide is incorporated
into a topical formulation containing, e.g., a topical carrier that
is generally suited to topical drug administration and comprising
any such material known in the art. The topical carrier may be
selected so as to provide the composition in the desired form,
e.g., as an ointment, lotion, cream, microemulsion, gel, oil,
solution, or the like, and may be comprised of a material of either
naturally occurring or synthetic origin. It is preferable that the
selected carrier not adversely affect the active agent or other
components of the topical formulation. Examples of suitable topical
carriers for use herein include water, alcohols and other nontoxic
organic solvents, glycerin, mineral oil, silicone, petroleum jelly,
lanolin, fatty acids, vegetable oils, parabens, waxes, and the
like. Formulations may be colorless, odorless ointments, lotions,
creams, microemulsions and gels.
[0133] Various additives, known to those skilled in the art, may be
included in formulations, e.g., topical formulations. Examples of
additives include, but are not limited to, solubilizers, skin
permeation enhancers, opacifiers, preservatives (e.g.,
anti-oxidants), gelling agents, buffering agents, surfactants
(particularly nonionic and amphoteric surfactants), emulsifiers,
emollients, thickening agents, stabilizers, humectants, colorants,
fragrance, and the like. Inclusion of solubilizers and/or skin
permeation enhancers is particularly preferred, along with
emulsifiers, emollients and preservatives. An optimum topical
formulation comprises approximately: 2 wt. % to 60 wt. %,
preferably 2 wt. % to 50 wt. %, solubilizer and/or skin permeation
enhancer; 2 wt. % to 50 wt. %, preferably 2 wt. % to 20 wt. %,
emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. %
preservative, with the active agent and carrier (e.g., water)
making of the remainder of the formulation.
[0134] Other active agents may also be included in formulations,
e.g., other anti-inflammatory agents, analgesics, antimicrobial
agents, antifungal agents, antibiotics, vitamins, antioxidants, and
sunblock agents commonly found in sunscreen formulations including,
but not limited to, anthranilates, benzophenones (particularly
benzophenone-3), camphor derivatives, cinnamates (e.g., octyl
methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl
methane), p-aminobenzoic acid (PABA) and derivatives thereof, and
salicylates (e.g., octyl salicylate).
[0135] Topical formulations may also be used as preventive, e.g.,
chemopreventive, compositions. When used in a chemopreventive
method, susceptible skin is treated prior to any visible condition
in a particular individual.
[0136] Therapeutics may also be administered in the form of a
suppository for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter,
glycerinated gelatin, hydrogenated vegetable oils, mixtures of
polyethylene glycols of various molecular weights and fatty acid
esters of polyethylene glycol.
[0137] For topical use, creams, ointments, jellies, solutions or
suspensions, etc., containing the therapeutics may be employed. For
purposes of this application, topical application shall include
mouth washes and gargles.
[0138] Therapeutics may be administered in intranasal form via
topical use of suitable intranasal vehicles and delivery devices,
or via transdermal routes, using those forms of transdermal skin
patches well known to those of ordinary skill in the art. To be
administered in the form of a transdermal delivery system, the
dosage administration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
[0139] The therapeutic peptides may be administered on a
dose-schedule that maintains a constant concentration in the
circulation. They may also be administered on a dose-schedule in
which therapy is periodically discontinued, e.g., a once daily
bolus injection.
[0140] Single therapeutic peptides or variants or derivatives
thereof may be administered. Alternatively, two or more different
peptides may be co-administered. For example, a therapeutic peptide
may be administered on its own or it may be co-adminstered with
other angiogenic inhibitors, such as TNP-470, described by U.S.
Pat. No. 5,290,807; Angiostatin, described by U.S. Pat. No.
5,639,725; Endostatin, and Thalidomide. Other angiogenic inhibitors
are described in Genetic Engineering News, Oct. 1, 1998 and in U.S.
Pat. No. 6,306,819.
[0141] In other embodiments, the therapeutics are co-administered
with other well known therapeutic agents that are selected for
their particular usefulness against the condition that is being
treated. For example, the instant therapeutics may be useful in
combination with known anti-cancer and cytotoxic agents. Similarly,
the instant therapeutics may be useful in combination with agents
that are effective in the treatment and prevention of benign or
malignant tumors. The therapeutic agents may be added to
chemotherapy, radiotherapy or used in combination with
immunotherapy or vaccine therapy.
[0142] Drugs that can be co-administered to a subject being treated
with a therapeutic described herein include antineoplastic agents
selected from vinca alkaloids, epipodophyllotoxins, anthracycline
antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D,
taxol, colchicine, cytochalasin B, emetine, maytansine, or
amsacrine.
[0143] Classes of compounds that can be used as the
chemotherapeutic agent (antineoplastic agent) include: alkylating
agents, antimetabolites, natural products and their derivatives,
hormones and steroids (including synthetic analogs), and
synthetics. Examples of compounds within these classes are given
below. Alkylating agents (including nitrogen mustards, ethylenimine
derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil
mustard, Chlormethine, Cyclophosphamide (Cytoxan.TM.), Ifosfamide,
Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine,
Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,
Streptozocin, Dacarbazine, and Temozolomide. Antimnetabolites
(including folic acid antagonists, pyrimidine analogs, purine
analogs and adenosine deaminase inhibitors): Methotrexate,
5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,
6-Thioguanine, Fludarabine phosphate, Pentostatine, and
Gemcitabine. Natural products and their derivatives (including
vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and
epipodophyllotoxins): Vinblastine, Vincristine, Vindesine,
Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin,
Idarubicin, paclitaxel (paclitaxel is commercially available as
Taxol.RTM. and is described in more detail below in the subsection
entitled "Microtubule Affecting Agents"), Mithramycin,
Deoxycoformycin, Mitomycin-C, L-Asparaginase, Interferons
(especially IFN-a), Etoposide, and Teniposide. Hormones and
steroids (including synthetic analogs): 17.alpha.-Ethinylestradiol,
Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,
Dromostanolone propionate, Testolactone, Megestrolacetate,
Tamoxifen, Methylprednisolone, Methyltestosterone, Prednisolone,
Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,
Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate,
Leuprolide, Flutamide, Toremifene, Zoladex. Synthetics (including
inorganic complexes such as platinum coordination complexes):
Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine,
Mitotane, Mitoxantrone, Levamisole, and Hexamethylmelamine.
[0144] Methods for the safe and effective administration of most of
these chemotherapeutic agents are known to those skilled in the
art. In addition, their administration is described in the standard
literature. For example, the administration of many of the
chemotherapeutic agents is described in the "Physicians' Desk
Reference" (PDR), e.g., 2004 edition (Thomson PDR, Montvale, N.J.
07645-1742, USA).
[0145] If formulated as a fixed dose, such combination products
employ the combinations described herein within the dosage range
described herein and the other pharmaceutically active agent(s)
within its approved dosage range. Combinations of the instant
invention may also be used sequentially with known pharmaceutically
acceptable agent(s) when a multiple combination formulation is
inappropriate.
[0146] Radiation therapy, including x-rays or gamma rays which are
delivered from either an externally applied beam or by implantation
of tiny radioactive sources, may also be used in combination with a
therapeutic described herein to treat cancer.
[0147] When a therapeutic is administered into a human subject, the
daily dosage will normally be determined by the prescribing
physician with the dosage generally varying according to the age,
weight, and response of the individual patient, as well as the
severity of the patient's symptoms.
[0148] Therapeutic peptides or analogs thereof described herein can
be labeled isotopically or with other molecules or proteins for use
in the detection and visualization of endostatin binding sites with
state of the art techniques, including, but not limited to,
positron emission tomography, autoradiography, flow cytometry,
radioreceptor binding assays, and immunohistochemistry. Labeled
peptides or analogs thereof may be used for detecting and
quantifying the presence of an antibody specific for an endostatin
in a body fluid.
[0149] A person of skill in the art will understand that nucleic
acids encoding endostatin peptides can be used instead of the
peptides in most embodiments described herein. For example,
angiogenesis can be inhibited in a tissue by introducing into the
tissue a nucleic acid encoding a peptide described herein. A
nucleic acid may be linked to or comprise a transcriptional control
element, such as a promoter or an enhancer. A nucleic acid may
further be included in a vector, such as an expression vector.
Exemplary expression vectors include viral vectors, such as an
adenovirus and an adeno associated virus (AAV) vector. When using a
non-viral vector, various methods can be used for facilitating
entry of the nucleic acid into a cell, such as liposomes.
Exemplary Kits
[0150] Also provided herein are kits, e.g., therapeutic kits. A kit
may comprise a therapeutic peptide described herein and optionally
a device for administration of the therapeutic peptide. A kit may
also comprise a therapeutic peptide in a lyophilized form and a
solution or buffer to solubilize the therapeutic peptide. A kit may
also comprise instructions for use.
[0151] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2.sup.nd Ed.,
ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
EXEMPLIFICATION
[0152] The invention now being more generally described, it will
now be more readily understood by reference to the following
examples, which are included merely for purposes of illustration of
certain aspects and embodiments of the present invention, and are
not intended to limit the invention.
Example 1
Identification of a 27 Amino Acid Endostatin Peptide that is
Responsible for Anti-Tumor Activity
[0153] Overlapping peptides with 24-27 amino acids derived from
both mouse endostatin and human endostatin were synthesized (Table
1).
TABLE-US-00003 TABLE 1 Overlapping mouse and human endostatin
peptides Name Sequence Mouse Peptides mP1:
HTHQDFQPVLHLVALNTPLSGGMRGIR; (SEQ ID NO: 4) mP2: MRGIRGA DFQAFQQARA
VGLSGTFR; (SEQ ID NO: 136) mP3: TFRAFLSSRLQDLYSIVRRADRGSV; (SEQ ID
NO: 137) mP4: GSVPIVNLKDEVLSPSWDSLFSGSQ; (SEQ ID NO: 138) mP5:
GSQGQVQPGARIFSFDGRDVLRHPA; (SEQ ID NO: 139) mP6:
HPAWPQKSVWHGSDPSGRRLMESY; (SEQ ID NO: 140) mP7:
ETWRTETTGATGQASSLLSGRLLEQ; (SEQ ID NO: 141) mP8:
KAASAHNSYIVLAIENSFMTSFSKKK. (SEQ ID NO: 142) Human Peptides hP1 1
HSHRDFQPVLHLVALNSPLSGGMRG 25 (SEQ ID NO: 6) hP2 23
MRGIRGADFQAFQQARAVGLAGTFR 47 (SEQ ID NO: 143) hP3 45
TFRAFLSSRLQDLYSIVRRADRAAV 69 (SEQ ID NO: 144) hP4 67
AAVPIVNLKDELLFPSWEALFSGSE 91 (SEQ ID NO: 145) hP5 89
GSEGPLKPGARIFSFDGKDVLRHPT 113 (SEQ ID NO: 146) hP6 111
HPTWPQKSVWHGSDPNGRRLTESY 134 (SEQ ID NO: 147) hP7 136
ETWRTEAPSATGQASSLLGGRLLGQ 160 (SEQ ID NO: 148) hP8 158
LGQSAASAHHAYIVLAIENSFMTASKKK 183 (SEQ ID NO: 149)
[0154] Peptides were approximately 1/7th-1/8th the size of full
length endostatin. Three cysteines, 33,165,173, were replaced by
alanines (underlined in Table 1), and cysteine 135 was omitted, to
prevent the formation of disulfide bonds. Two additional lysines
were added at the C-terminal of hP8 to increase its solubility
(double underlined). Most peptides were water soluble, except for
hP2 at high concentrations (>2.5 mg/ml). Also, all peptides were
approximately 70% pure. However, no difference in tumor inhibition
was observed when peptides of more than 95% purity were used.
[0155] These peptides were initially tested for anti-tumor activity
employing the human pancreatic tumor cells BxPC-3 cells, which were
implanted in the s.c. dorsa of SCID mice. For systemic treatment,
human endostatin peptides, P1-P8, were administered subcutaneously
(s.c.) twice a day at 7 mg/kg/d due to the high clearance rate from
the mouse circulation. Full-length Fc-Endostatin, hFcES was
administered subcutaneously only once per day at a dose of 20
mg/kg/d. PBS was used as a control. Tumors were measured every
three days and the final measurements at 28 days are shown in FIG.
1 (T/C is indicated in each bar and the group sizes had an n equal
to 3.
[0156] The N-terminal hP1 peptide of endostatin inhibited BxPC-3 by
39% (p=0.077) and full-length endostatin by 44% (p=0.0057) (FIG.
1). Two other peptides, hP2 and hP5, also showed some small
anti-tumor activity, where hP2 inhibited BxPC-3 by 19% (p=0.48) and
hP5 by 29% (p=0.15). Other peptides had no effect (FIG. 1). Thus,
most of the anti-tumor activity was associated with the N-terminal
hP1 peptide compared to full-length endostatin. Although tumor
inhibition by hP1, hP2 and hP5 was not statistically significant
due to the small number of mice per group (n=3), the trends
suggesting tumor inhibition by these peptides prompted further
study of such peptides on tumor inhibition in the murine LLC model.
Moreover, peptides and full-length endostatin were not at equimolar
concentrations. However, this data suggests that the anti-tumor
property of endostatin may be located within its N-terminal
domain.
The N-Terminal 27 Amino Acid Peptide of Endostatin is Responsible
for its Anti-Tumor Property
[0157] The murine LLC tumor model (O'Reilly et al. (1994) Cell, 79:
315-328) was used to further characterize these peptides because
this tumor grows more rapidly than BxPC-3 cells, allowing for
shorter treatment periods. Murine analogs of the endostatin
peptides were synthesized. The only difference between the human
and murine peptides was that murine P1 peptide contained 27 amino
acids instead of 25 amino acids for human P1. We tested only those
peptides that showed any anti-tumor activity in the previous
experiment (see FIG. 1).
[0158] Unlike the BxPC-3 treatment, LLC tumors were treated at
equimolar concentrations of murine endostatin and murine peptides
(mP1, mP2, mP5, and mP6). mP6 was used as a representative control
peptide, because it showed no anti-tumor activity (see FIG. 1).
Control mice were treated with PBS. In these experiments, LLC cells
were implanted into the s.c. dorsa of C57B1/6J mice, and
systemically treated. Peptides (mP1, mP2, mP5, and mP6) were
injected (s.c.) twice a day at a dose of 2.8 mg/kg/d, whereas
endostatin and PBS were administered once a day at a dose of 20
mg/kg/d. The N-terminal mP1 endostatin peptide inhibited LLC by 44%
(p<0.035), which is comparable to the inhibition by full-length
endostatin (53%, p<0.01) (FIG. 2A; T/C is indicated in the
figure). No or insignificant activity was detected employing mP2,
mP5 and mP6 at the same concentration as the mP1 peptide (FIG. 2A).
Thus, this result suggests that the 27 amino acids mP1 peptide
contains all of the anti-tumor activity associated with
endostatin.
[0159] To determine the effect on angiogenesis after mP1 treatment,
LLC tumors treated with mP1, mP2, and endostatin at equimolar
concentration were analyzed for vessel density (CD31). FIG. 2B
shows CD31 staining of LLC sections from mice treated with PBS
(control), Fc-Endostatin (20 mg/kg/d), mP1 (2.8 mg/kg/d), and mP2
(2.8 mg/kg/d). In each case, peptides were administered twice a day
s.c. and LLC tumor sections from day 13 were formalin-fixed
paraffin-embedded and then stained with CD31 (PECAM). FIG. 2C show
the determination of vessel density and the Y axis is expressed as
% CD31/high power field (hpf). Treatment of LLC with mP1 and
endostatin reduced vessel density significantly (approximately 65%,
p<0.015), while mP2 and PBS had no effect. Treatment with mP5
and mP6 showed similar results as mP2 treatment. These results
suggest that mP1 can inhibit LLC tumor growth by reducing vessel
density in similar manner to full length endostatin.
Histidines at Position 1 and 3 of Endostatin are Critical for Zinc
Binding
[0160] The crystal structure of endostatin reveals a highly folded
molecule (FIG. 2D). However, the N-terminal region resembles a
random coil structure consistent with our analysis that a synthetic
peptide corresponding to this domain can mimic the native molecule
(FIG. 2D).
[0161] Because the endostatin N-terminal domain is responsible for
its anti-tumor activity, we wanted to investigate the mP1 peptide
further. There is an atom of Zinc (Zn) associated with each
molecule of endostatin (Ding et al. (1998) Proc Natl Acad Sci USA,
95: 10443-10448). Based on our crystal structure analysis, three
histidines at positions 1, 3, and 11 plus, aspartic at position 76,
form the four coordinates for this Zn atom (Ding et al. (1998) Proc
Natl Acad Sci USA, 95: 10443-10448). mP1 contains the three
histidines mentioned above. This raises the possibility that this
peptide is capable of binding Zn, by having a molecule of water
occupying the fourth coordinate (FIG. 3A, left panel). It has been
shown previously that mutations of histidines 1 and 3 disrupt Zn
binding of endostatin (Boehm et al. (1998) Biochem Biophys Res
Commun, 252: 190-194). Therefore, a mutant of peptide mP1 was
synthesized where the histidines at position 1 and 3 were mutated
to alanines. This mutant peptide was called mP1-H1/3A (also
referred to mP1-H) and has the following amino acid sequence:
ATAQDFQPVLHLVALNTPLSGGMRGIR (SEQ ID NO: 150). The sequences of mP1
and mP1-H1/3A are also indicated in FIG. 3A.
[0162] To determine the Zn binding capacity of mP1 and mP1-H1/3A
flame atomic absorption was performed. Each peptide was dissolved
in 20 mM Tris, pH 8.0 at a concentration of 0.5 mg/ml, mixed with
excess Zn chloride (1 mM), and extensively dialyzed against the
above buffer for 72 hours with three changes in dialysis solution
(molecular weight cut off (MWCO)=1000 kDa; the molecular weight of
the peptides was taken to be 3000 kDa). Atomic absorption readings
of the final zinc concentrations (.mu.g/ml) were determined to be
9.63 and 1.05 for mP1 and mP1-H1/3A, respectively. These data
yielded zinc ratios of 0.1 per molecule of mP1-H1/3A and 0.9 for
mP1 (FIG. 3B). Therefore, mutating the histidines at position 1 and
3 to alanines abolished Zn binding (FIG. 3A, right panel).
The Zinc Binding Domain of Endostatin is Important for its
Anti-Tumor Activity
[0163] To determine if Zn binding is also important for the
anti-tumor property of endostatin, mP1 and mP1-H1/3A were tested
using the LLC tumor model. Peptides were administered twice a day
(s.c.) at a dose of 2.8 mg/kg/d. Peptide mP1 inhibited LLC by 42%
(p=0.031), whereas mP1-H1/3A had no effect (FIG. 3C). To determine
if there was a difference in angiogenesis, vessel density (CD31) of
LLC tumors was analyzed after mP1 and mP1-H1/3A treatment. There
was a considerable decrease in vessel density after mP1 treatment
(67% reduction, p<0.01), whereas mP1-H1/3A was similar to PBS
(FIGS. 3D and 3E). These data suggest that Zn binding is important
for the anti-tumor property of endostatin. The unpaired Student t
test was used for statistical analysis.
[0164] Additional treatments included subcutaneously injection the
mP1-H peptide together with 1 mM of Zn.sup.2+; subcutaneously
injecting the mP1 peptide alone or with 1 mM of Zn.sup.2+; or
subcutaneously injecting PBS (FIG. 4). The results, which are shown
in FIGS. 3 and 4, indicated that tumor volume was essentially not
affected by the administration of the mP1-H1/3A peptide, in which
histidines 1 and 3 are changed to alanines, since the tumor mass
was similar to that obtained when injecting PBS (the negative
control).
[0165] Thus, these results indicate that the histidines at
positions 1 and 3 in N-terminal endostatin peptides cannot be
replaced by alanines and that their substitution with another amino
acid would likely also reduce or eliminate the anti-tumor effect of
the peptides. The results further indicate that it may be
beneficial to include zinc in the composition comprising the
peptide, unless there is zinc already present in the
circulation.
The N-Terminal Fragment of Endostatin Retains its Capacity to
Inhibit Endothelial Cell Migration
[0166] Endostatin peptides were tested for anti-endothelial cell
migration activity. Inhibition of VEGF-induced migration of human
microvascular endothelial cells (HMVECs) was determined using
several doses of endostatin peptides (FIG. 5). Human peptides were
used because the cells were of human origin and the migration
response of the HMVECs was assayed using a modified Boyden chamber.
VEGF (5 ng/ml) was used a chemotactic agent and cells were
challenged with human endostatin (Entremed; EM-ES), human
Fc-Endostatin (nFcES), human P1 (hP1), human P2 (hP2), human P6
(hP6), and human P1-H1/3A (hP1-H1/3A). Total migration per membrane
was quantified from the capture images using Scion Image Software
(National Institutes of Health) and the unpaired Student T test was
used for statistical analysis.
[0167] Human recombinant endostatin (Entremed) inhibited EC
migration at 500 and 200 ng/ml (30%) but not at 100 ng/ml, whereas
human Fc-Endostatin inhibited equally well between 500 and 100
ng/ml (25-30%) (FIG. 5). Interestingly, slightly better inhibition
of EC migration was observed at 100 ng/ml for Fc-Endostatin and 200
ng/ml for endostatin (EntreMed) than higher concentrations, which
suggested a U-shaped endostatin response. The best inhibition for
hP1 was at 100, 62.5, and 25 ng/ml. No significant differences in
inhibition between these concentrations were observed. At higher
and lower concentrations less or no inhibition was observed. At 500
ng/ml of hP1 no inhibition was observed. Thus, similar to
full-length endostatin, there is a U-shaped response to hP1
inhibition of EC migration. Endostatin hP2 peptide had no effect at
all even at higher concentrations. However, hP6 inhibited EC
migration but at higher concentrations than hP1 and no inhibition
was observed for concentrations lower than 100 ng/ml.
[0168] To determine if the Zn binding site is important for
anti-endothelial cell migration activity, hP1-H1/3A was also
tested. This mutant peptide did show some small inhibition at 200
and 100 ng/ml. However, this inhibition was statistically not
significant. Thus, peptide hP1 could inhibit VEGF-induced EC
migration at equimolar concentrations (25 ng/ml) to full-length
endostatin or human endostatin (EntreMed) (200 ng/ml), whereas hP6
only inhibited at doses of 100 and 200 ng/ml. Interestingly, hP1
was more potent in inhibiting EC migration than full-length
endostatin. These results show that the N-terminal P1 peptide of
endostatin maintains the ability to inhibit VEGF-induced EC
migration and that the Zn binding site is critical for this
activity.
Anti-Permeability Activities of Endostatin Peptides
[0169] The ability of endostatin peptides to inhibit VEGF-induced
permeability was also tested using the Miles assay (Miles and Miles
(1952) J Physiol, 118: 228-257). Previously, endostatin has been
shown to inhibit VEGF-induced permeability using the Miles assay.
Immuno-compromised SCID mice were treated 5 days before performing
the Miles assay. Specifically, SCID mice were injected
subcutaneously (s.c.; twice a day) with human endostatin (EntreMed;
EM-ES) at a dose of 100 mg/kg/d; murine Fc-Endostatin at a dose of
20 mg/kg/d; murine endostatin peptides, mP1 and mP1-H1/3A, at a
dose of 2.8 mg/kg/d or 14 mg/kg/d; or with PBS (n=3) for 5 days. At
the high dose (14 mg/kg/d), both mP1 and mP1-H1/3A inhibited
VEGF-induced permeability as well as human endostatin (EntreMed)
and murine Fc-Endostatin (FIG. 6). However, similar results were
obtained when equimolar concentrations (2.8 mg/kg/d) were used
(FIG. 6). Because mP1-H1/3A showed the same inhibition as mP1, even
at equimolar concentration, this suggests that there is a
separation of activity between anti-tumor and
anti-permeability.
[0170] Smaller peptides derived from mP1 were also shown to inhibit
tumor growth. Two peptides, mP1-15 (SEQ ID NO: 118) and mP1-20 (SEQ
ID NO: 108), were tested for anti-tumor activity using the LLC
tumor model. Peptides were administered s.c. twice a day at dose of
2.8 mg/kg/day on days 4, 7, 10 and 14. PBS was used as a control.
FIG. 7 shows that both mP1-15 and mp1-20 inhibit tumor volume. (T/C
is indicated and the group size has an n equal to 5).
[0171] Thus, we have shown that a synthetic peptide, corresponding
to the N-terminus of endostatin, is responsible for its anti-tumor,
anti-migration, and anti-permeability activities. Zinc binding is
required for the anti-tumor and anti-migration activities, because
substitution of the two histidines at amino acid positions 1 and 3
in the peptide completely blocks its properties. However, Zn
binding was not required for anti-permeability property.
[0172] The zinc binding requirement of endostatin for inhibiting
tumor formation has been controversial, with conflicting results
reported from different groups (Boehm et al. (1998) Biochem Biophys
Res Commun, 252: 190-194; Yamaguchi et al. (1999) Embo J, 18:
4414-4423; Sim et al. (1999) Angiogenesis, 3: 41-51). Whereas, the
earliest report showed that the replacement of histidines 1 and 3
by alanines blocked the inhibitory effect of endostatin in LLC
(Boehm et al. (1998) Biochem Biophys Res Commun, 252: 190-194), two
later publications challenged this finding (Yamaguchi et al. (1999)
Embo J, 18: 4414-4423; Sim et al. (1999) Angiogenesis, 3: 41-51).
In one of these reports, a mutant endostatin was prepared by
deleting 5 amino acids in both C- and N-termini (Yamaguchi et al.
(1999) Embo J, 18: 4414-4423). This construct elicited anti-tumor
activity, similar to full-length endostatin. However, in the
employed renal Rc-9 carcinoma tumor model, the administration of
endostatin was initiated when the tumor size was 300 mm.sup.3, and
lasted for only 4 days, when the tumor size reached 500 mm.sup.3.
The injection sites were at the periphery of the tumor, and the
injection dosage was 10 .mu.g/kg/d. In contrast, in our experiments
we initiated treatment when LLC tumors reached a size of .about.100
mm.sup.3 and continued until tumors were .about.6000-7000 mm.sup.3.
Furthermore, we treated systemically and did not inject into the
periphery of the tumor.
[0173] Another publication which dealt with the relevance of zinc
binding to anti-tumor activity of endostatin, demonstrated that the
removal of 4 amino acids "HSHR" from N-terminus of human endostatin
did not affect its anti-tumor activity (Sim et al. (1999)
Angiogenesis, 3:41-51). Measurements of zinc binding revealed that
this mutant bound 2 atoms of zinc per molecule of endostatin,
whereas the wild type bound 10 atoms of zinc per endostatin
molecule. However, in our crystal structure studies of endostatin,
we have demonstrated that the endostatin employed for
crystallization studies, contains 1 atom of zinc/endostatin
molecule and the removal of the 4 amino acids "HSHR" from
N-terminus lacks zinc binding (Ding et al. (1998) Proc Natl Acad
Sci USA, 95: 10443-10448).
[0174] Endostatin is generated by proteolytic cleavage of collagen
18 (O'Reilly et al. (1997) Cell, 88: 277-285; Wen et al. (1999)
Cancer Res, 59: 6052-6056; Felbor et al. (2000) Embo J, 19:
1187-1194). The first amino acid at the N-terminus of endostatin is
a histidine. The presence of histidine is important for conferring
Zn binding to endostatin. Consequently, we are led to conclude that
the processing of collagen 18 to endostatin may be highly
regulated.
[0175] Several groups have shown that peptides derived from
endostatin have antiangiogenic effects (Wickstrom et al. (2004) J
Biol Chem, 279: 20178-20185; Cattaneo et al. (2003) Exp Cell Res,
283: 230-236; Chillemi et al. (2003) J Med Chem, 46: 4165-4172;
Morbidelli et al. (2003) Clin Cancer Res, 9: 5358-5369; Cho et al.
(2004) Oncol Rep, 11: 191-195). An N-terminal peptide comprising
amino acids 6-49 (lacking the zinc binding histidines) has
inhibited endothelial cell proliferation and migration (Cattaneo et
al. (2003) Exp Cell Res, 283: 230-236; Chillemi et al. (2003) J Med
Chem, 46: 4165-4172). A Matrigel assay, employing this peptide has
resulted in the inhibition of angiogenesis in vivo. However, no
antitumor data was presented. In another study, a C-terminal
peptide (amino acids 135-184) retaining the Cys135-Cys165 disulfide
bond, has demonstrated anti-tumor activity (Morbidelli et al.
(2003) Clin Cancer Res, 9: 5358-5369). However, the peptide was
administered at the tumor periphery and not systemically. Cho et
al. have shown that N-terminus, which includes the Zn binding site,
and the C-terminus of endostatin are not required for anti-tumor
activity (Cho et al. (2004) Oncol Rep, 11: 191-195). However, this
peptide and full-length endostatin were not tested at equimolar
concentrations. Our results differ from these groups in that the P1
peptide could inhibit tumor formation, migration, and permeability
at equimolar concentrations to full length endostatin. Furthermore,
at higher concentrations (14 mg/kg/d) mP2 could inhibit LLC tumor
formation as well as mP1 at 2.8 mg/kg/d. However, mP1 at 14 mg/kg/d
inhibited LLC tumor formation less than at 2.8 mg/kg/d. Thus, a
U-shaped curve seems to be associated with anti-tumor activity of
endostatin as a function of the protein concentration. Similar
results were observed for full-length endostatin using the
pancreatic BxPC-3 and ASPC-1 tumor models. Therefore, determination
of optimum endostatin concentration may be an important factor. In
vitro assays have demonstrated a similar biphasic characteristic by
endostatin such as seen in migration assays (see FIG. 5).
[0176] The fact that full-length endostatin is not required for its
anti-tumor activity explains the initial inconsistencies of
endostatin activity. Endostatin has two disulfide bonds.
Aggregation of endostatin in E. coli preparations is caused by
random inter-molecular disulfides after PBS dialysis. Whereas,
endostatin demonstrates a single protein molecule under reducing
conditions, most of the protein in an identical sample does not
enter the polyacrylamide gel under nonreducing condition. It is
probably the degree of nonspecific aggregation that is responsible
for the lack of activity in some of the preparations. Endostatin is
most likely released from the aggregate in animals over a period of
time, resulting in a denatured protein or partial fragments, which
are capable of demonstrating anti-tumor properties due to their
N-terminal peptide. Presumably, some of the preparations yield
larger aggregates, which make such a release inefficient and give
rise to a product that is incapable of eliciting antiangiogenic
response in mice.
[0177] What is the basis of endostatin's anti-tumor activity? A
large number of mechanisms have been proposed. One which has been
studied in more detail is the endostatin binding to integrin
.alpha.5.beta.1 (Wickstrom et al. (2002) Cancer Res, 62:
5580-5589). Based on the findings of these authors, an assembly of
several cell surface proteins and components including
.alpha.5.beta.1, are responsible for interactions between
endostatin and this integrin (Wickstrom et al. (2003) J Biol Chem,
278: 37895-37901). However, no anti-tumor data were presented to
confirm the above mechanism. More recently, the same authors have
shown that an 11 amino acid peptide derived from endostatin
containing arginines and showing heparin binding, is responsible
for antiangiogenic activity of endostatin (Wickstrom et al. (2004)
J Biol Chem, 279: 20178-20185). We speculate that the phenomena
observed by these investigators, reflects some of the properties
associated with heparin binding characteristic of endostatin, and
not its anti-tumor activity. Previously, we reported that
disruption of heparin binding of endostatin (accomplished by the
mutation of two discontinuous arginines on the protein surface)
blocked cell motility (Kuo et al. (2001) J Cell Biol, 152:
1233-1246). Furthermore, our endostatin hP3 peptide (see Table 1),
which contains the peptide reported by the authors, failed to
inhibit tumor growth.
Materials and Methods:
Cell Culture and Reagents
[0178] Human BxPC-3 pancreatic adenocarcinoma and Lewis lung
carcinoma (LLC) cells were grown and maintained as described
earlier (Kisker et al. (2001) Cancer Res, 61:7669-7674; O'Reilly et
al. (1994) Cell, 79: 315-328). For BxPC-3 tumor cell injection,
cells were grown in 900-cm2 roller bottles. Human microvascular
endothelial cells (HMVEC-d; Clonetics, Walkersville, Md.) were
cultured in microvascular endothelial cell growth medium (EGM-2 MV;
Clonetics) and maintained at 5% CO2 in a 37.degree. C. humidified
incubator. Recombinant human endostatin was a generous gift from
EntreMed Corporation (Rockville, Md.) and recombinant human and
murine FcEndostatin were prepared as described earlier (Bergers et
al. (1999) Science, 284: 808-812). Human and murine endostatin
peptides were synthesized by SynPep Corporation (Dublin, Calif.).
Peptides were resuspended in PBS or 50 mM Tris, 150 mM NaCl, pH
7.5. PECAM, purified rat anti-mouse CD31, was obtained from BD
Pharmingen (San Diego, Calif.) and human recombinant VEGF was
obtained from the NIH (Bethesda, Md.).
Animal Studies
[0179] All animal procedures were performed in compliance with
Boston Children's Hospital guidelines, and protocols were approved
by the Institutional Animal Care and Use Committee. Male (2427 g)
immunocompetent C57B1/6J mice (Jackson Laboratories, Bar Harbor,
Me.) and immunocompromised SCID mice (Massachusetts General
Hospital, Boston, Mass.) were used. Mice were 7-9 weeks of age.
They were acclimated, caged in groups of five in a barrier care
facility, and fed with animal chow and water ad libitum. Animals
were anesthetized via inhalation of isoflurane (Baxter, Deerfield,
Ill.) before all surgical procedures and observed until fully
recovered. Animals were euthanized by a lethal dose of CO2
asphyxiation.
Tumor Models
[0180] BxPC-3 and LLC cells were grown in cell culture as described
above. The cell concentration was adjusted to 50.times.10.sup.6
cells/ml. Mice were shaved and the dorsal skin was cleaned with
ethanol before tumor cell injection. A suspension of
5.times.10.sup.6 tumor cells in 0.1 ml RPMI-1640 (for BxPC-3) or
DMEM (for LLC) was injected subcutaneously (s.c.) into the dorsa of
mice at the proximal midline. BxPC-3 cells were implanted in SCID
mice and LLC in C57B1/6J mice.
[0181] Animals with Lewis Lung carcinoma (600-800 mm.sup.3 tumors)
were euthanized, and the skin overlying the tumor was cleaned with
Betadine and ethanol. Tumor tissue was excised under aseptic
conditions. A suspension of tumor cells in 0.9% normal saline was
made by passage of viable tumor tissue through a sieve and a series
of sequentially smaller hypodermic needles of diameter 22- to
30-gauge. The final concentration was adjusted to 1.times.10.sup.7
cells/ml, and the suspension was placed on ice. The injection of
tumor cells was performed as described above.
[0182] The mice were weighed and tumors were measured every 3-5
days in two diameters with a dial-caliper. Volumes were determined
using the formula a.sup.2.times.b.times.0.52 (where a is the
shortest and b is the longest diameter). Data is represented as
volume of treated tumor over control (T/C). At the completion of
each experiment, the mice were euthanized with CO2 asphyxiation.
Tumors were fixed in 10% buffered Formalin (Fisher Scientific, Fair
Lawn, N.J.) and paraffin-embedded.
[0183] For treatment of tumor-bearing mice, tumor volumes were
allowed to grow to approximately 100 mm.sup.3, and mice were
randomized. Treatment was performed by single bolus s.c.
injections. Peptides were administered twice a day (every 12 hrs).
Doses indicated for peptides were corrected for the purity of
peptides (approximately 70%). For example, mice injected with 4
mg/kg/d peptide, were actually injected with 2.8 mg/kg/d after
correction. The unpaired Student t-test was used for statistical
analysis.
Immunohistochemistry
[0184] Tumors were fixed in 10% buffered Formalin overnight at
4.degree. C. The next day, tumors were washed three times in PBS
and paraffin-embedded. Sections (5 .mu.m) were permeabilized with
40 .mu.g/ml proteinase K (Roche Diagnostics Corp.) in 0.2 M
Tris-HCl buffer (pH 7.6) for 25 minutes at 37.degree. C. and washed
with PBS. PECAM (1:250) was incubated at 4.degree. C. overnight.
Stainings were amplified using tyramide signal amplification direct
and indirect kits (NEN Life Science Products Inc., Boston, Mass.).
Sections were photographed at 400.times. magnification using a
NIKON TE300 microscope. Vessel density (average of three fields)
was determined by IPLab software. The unpaired Student t-test was
used for statistical analysis.
Cell Migration Assay
[0185] The motility response of HMVEC-d cells was assayed using a
modified Boyden chamber. Cells were plated in T75-cm2 flasks at
0.5.times.10.sup.6 cells per flask and allowed to grow for 48 h
(.about.70% confluent) prior to the migration assay. To facilitate
cell adhesion, the upper membrane of a transwell (8 mm pore;
Costar) was coated with fibronectin (10 mg/ml; Becton Dickinson,
Bedford, Mass.) for 1 h at 37.degree. C. Coated membranes were
rinsed with PBS and allowed to air dry immediately before use.
Cells were detached by trypsinization, treated with trypsinization
neutralization solution (Clonetics), and resuspended at a final
concentration of 1.times.10.sup.6 cells/ml in serum-free
endothelial basal medium (EBM; Clonetics) containing 0.1% BSA.
Cells (200,000 in 0.2 ml) were then treated with 0.2 ml of EBM/BSA
containing endostatin or peptides at the indicated concentrations.
Cells were incubated for 20 min. at 37.degree. C. with occasional
shaking. Cells (50,000 in 100 .mu.l) were added to the upper
chamber of the transwell. EBM or EBM supplemented with VEGF (5
ng/ml) was added to the lower chamber and cells were allowed to
migrate toward the bottom chamber for 4 h in a humidified incubator
containing 5% CO2. Transwell filters were rinsed once with PBS and
fixed and stained using a Diff-Quik staining kit (Baxter) following
the manufacturer's protocol. Non-migrated cells were removed from
the upper chamber with a cotton swab. Stained filters were cut out
of the chamber and mounted onto slides using Permount (Fisher). The
number of migrated cells was measured using microscopy (three
fields from each membrane were captured using a 40.times.
objective), and images were captured with a CCD camera using SPOT
software. Total migration per membrane was quantified from the
captured images using Scion Image software (National Institutes of
Health). All experiments were run in triplicate. The unpaired
Student t-test was used for statistical analysis.
Miles Vascular Permeability Assay (the Miles Assay)
[0186] SCID mice were injected subcutaneously (s.c.) with human
endostatin (EntreMed; 100 mg/kg/day), murine Fc-Endostatin (20
mg/kg/d), peptides (either 14 mg/kg/d or 2.8 mg/kg/d), and with
saline (200 .mu.l) (n=12) for 5 days prior to performing the Miles
assay (25). Briefly, Evan's blue dye (100 .mu.l of a 1% solution in
PBS) was injected intravenously (i.v.) into mice. After 10 minutes,
50 .mu.l of human recombinant VEGF (1 ng/.mu.l) or PBS were
injected intradermally into the pre-shaved back skin. After 20
minutes, the animals were euthanized and an area of skin that
included the blue spot resulting from leakage of the dye was
removed. Evan's blue dye was extracted from the skin by incubation
with formamide for 5 days at room temperature, and the absorbance
of extracted dye was measured at 620 nm using a spectrophotometer.
The unpaired Student t-test was used for statistical analysis.
Statistical Methods
[0187] Data are expressed as mean+S.D. Statistical significance was
assessed using the Student t-test. P<0.05 was considered
statistically significant.
INCORPORATION BY REFERENCE
[0188] The contents of all cited references (including literature
references, GenBank Accession numbers, issued patents, published
patent applications as cited throughout this application) are
hereby expressly incorporated by reference.
EQUIVALENTS
[0189] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
154181DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 1cacagccacc gcgacttcca
gccggtgctc cacctggttg cgctcaacag ccccctgtca 60ggcggcatgc ggggcatccg
c 81227PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 2His Ser His Arg Asp Phe Gln Pro
Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu Ser Gly Gly
Met Arg Gly Ile Arg 20 25381DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
3catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac ccccctgtct
60ggaggcatgc gtggtatccg t 81427PRTArtificial SequenceDescription of
Artificial Sequence Synthesized anti-angiogenic mouse peptide 4His
Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
15Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 20 25575DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 5catactcatc aggactttca gccagtgctc
cacctggtgg cactgaacac ccccctgtct 60ggaggcatgc gtggt
75625PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic human peptide 6His Ser His Arg Asp Phe
Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu Ser
Gly Gly Met Arg Gly 20 25778DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
7agccaccgcg acttccagcc ggtgctccac ctggttgcgc tcaacagccc cctgtcaggc
60ggcatgcggg gcatccgc 78826PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 8Ser His Arg
Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Ser 1 5 10 15Pro
Leu Ser Gly Gly Met Arg Gly Ile Arg 20 25975DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 9caccgcgact tccagccggt gctccacctg
gttgcgctca acagccccct gtcaggcggc 60atgcggggca tccgc
751025PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 10His Arg Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn Ser Pro 1 5 10 15Leu Ser Gly Gly Met Arg
Gly Ile Arg 20 251172DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
11cgcgacttcc agccggtgct ccacctggtt gcgctcaaca gccccctgtc aggcggcatg
60cggggcatcc gc 721224PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 12Arg Asp Phe
Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu 1 5 10 15Ser
Gly Gly Met Arg Gly Ile Arg 201369DNAArtificial SequenceDescription
of Artificial Sequence DNA encoding exemplary anti-angiogenic
peptide 13gacttccagc cggtgctcca cctggttgcg ctcaacagcc ccctgtcagg
cggcatgcgg 60ggcatccgc 691423PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 14Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser 1 5 10 15Gly
Gly Met Arg Gly Ile Arg 201566DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
15ttccagccgg tgctccacct ggttgcgctc aacagccccc tgtcaggcgg catgcggggc
60atccgc 661622PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 16Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly 1 5 10 15Gly Met Arg
Gly Ile Arg 201763DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
17cagccggtgc tccacctggt tgcgctcaac agccccctgt caggcggcat gcggggcatc
60cgc 631821PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 18Gln Pro Val Leu His
Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly 1 5 10 15Met Arg Gly
Ile Arg 201960DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
19ccggtgctcc acctggttgc gctcaacagc cccctgtcag gcggcatgcg gggcatccgc
602020PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 20Pro Val Leu His Leu Val Ala Leu
Asn Ser Pro Leu Ser Gly Gly Met 1 5 10 15Arg Gly Ile Arg
202157DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 21gtgctccacc tggttgcgct
caacagcccc ctgtcaggcg gcatgcgggg catccgc 572219PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 22Val Leu His Leu Val Ala Leu Asn Ser Pro
Leu Ser Gly Gly Met Arg 1 5 10 15Gly Ile Arg2354DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 23ctccacctgg ttgcgctcaa cagccccctg
tcaggcggca tgcggggcat ccgc 542418PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 24Leu His
Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly 1 5 10
15Ile Arg2551DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
25cacctggttg cgctcaacag ccccctgtca ggcggcatgc ggggcatccg c
512617PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 26His Leu Val Ala Leu Asn Ser Pro
Leu Ser Gly Gly Met Arg Gly Ile 1 5 10 15Arg2748DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 27ctggttgcgc tcaacagccc cctgtcaggc
ggcatgcggg gcatccgc 482816PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 28Leu Val Ala
Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 10
152945DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 29gttgcgctca acagccccct
gtcaggcggc atgcggggca tccgc 453015PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 30Val Ala
Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 10
153142DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 31gcgctcaaca gccccctgtc
aggcggcatg cggggcatcc gc 423214PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 32Ala Leu Asn
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 103339DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 33ctcaacagcc ccctgtcagg cggcatgcgg
ggcatccgc 393413PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 34Leu Asn Ser Pro Leu
Ser Gly Gly Met Arg Gly Ile Arg 1 5 103536DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 35aacagccccc tgtcaggcgg catgcggggc atccgc
363612PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 36Asn Ser Pro Leu Ser Gly Gly Met
Arg Gly Ile Arg 1 5 103778DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
37cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca
60ggcggcatgc ggggcatc 783826PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 38His Ser His
Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser
Pro Leu Ser Gly Gly Met Arg Gly Ile 20 253972DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 39cacagccacc gcgacttcca gccggtgctc
cacctggttg cgctcaacag ccccctgtca 60ggcggcatgc gg
724024PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 40His Ser His Arg Asp Phe Gln Pro
Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu Ser Gly Gly
Met Arg 204169DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
41cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca
60ggcggcatg 694223PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 42His Ser His Arg Asp
Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu
Ser Gly Gly Met 204366DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
43cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca
60ggcggc 664422PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 44His Ser His Arg Asp
Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu
Ser Gly Gly 204563DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
45cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca
60ggc 634621PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 46His Ser His Arg Asp
Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu
Ser Gly 204760DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
47cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca
604820PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 48His Ser His Arg Asp Phe Gln Pro
Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu Ser
204957DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 49cacagccacc gcgacttcca
gccggtgctc cacctggttg cgctcaacag ccccctg 575019PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 50His Ser His Arg Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn 1 5 10 15Ser Pro Leu5154DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 51cacagccacc gcgacttcca gccggtgctc
cacctggttg cgctcaacag cccc 545218PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 52His Ser
His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
15Ser Pro5351DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
53cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag c
515417PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 54His Ser His Arg Asp Phe Gln Pro
Val Leu His Leu Val Ala Leu Asn 1 5 10 15Ser5548DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 55cacagccacc gcgacttcca gccggtgctc
cacctggttg cgctcaac 485616PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 56His Ser His
Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
155745DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 57cacagccacc gcgacttcca
gccggtgctc cacctggttg cgctc 455815PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 58His Ser
His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu 1 5 10
155942DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 59cacagccacc gcgacttcca
gccggtgctc cacctggttg cg 426014PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 60His Ser His
Arg Asp Phe Gln Pro Val Leu His Leu Val Ala 1 5 106139DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 61cacagccacc gcgacttcca gccggtgctc
cacctggtt 396213PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 62His Ser His Arg Asp
Phe Gln Pro Val Leu His Leu Val 1 5 106336DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 63cacagccacc gcgacttcca gccggtgctc cacctg
366412PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 64His Ser His Arg Asp Phe Gln Pro
Val Leu His Leu 1 5 106578DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
65actcatcagg actttcagcc agtgctccac ctggtggcac tgaacacccc cctgtctgga
60ggcatgcgtg gtatccgt 786626PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 66Thr His Gln
Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Thr 1 5 10 15Pro
Leu Ser Gly Gly Met Arg Gly Ile Arg 20 256775DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 67catcaggact ttcagccagt gctccacctg
gtggcactga acacccccct gtctggaggc 60atgcgtggta tccgt
756825PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 68His Gln Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn Thr Pro 1 5 10 15Leu Ser Gly Gly Met Arg
Gly Ile Arg 20 256972DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
69caggactttc agccagtgct ccacctggtg gcactgaaca cccccctgtc tggaggcatg
60cgtggtatcc gt 727024PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 70Gln Asp Phe
Gln Pro Val Leu His Leu Val Ala Leu Asn Thr Pro Leu 1 5 10 15Ser
Gly Gly Met Arg Gly Ile Arg 207169DNAArtificial SequenceDescription
of Artificial Sequence DNA encoding exemplary anti-angiogenic
peptide 71gactttcagc cagtgctcca cctggtggca ctgaacaccc ccctgtctgg
aggcatgcgt 60ggtatccgt 697223PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 72Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn Thr Pro Leu Ser 1 5 10 15Gly
Gly Met Arg Gly Ile Arg 207367DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
73atttcagcca gtgctccacc tggtggcact gaacaccccc ctgtctggag gcatgcgtgg
60tatccgt 677422PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 74Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn Thr Pro Leu Ser Gly 1 5 10 15Gly Met Arg
Gly Ile Arg 207563DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
75cagccagtgc tccacctggt ggcactgaac acccccctgt ctggaggcat gcgtggtatc
60cgt 637621PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 76Gln Pro Val Leu His Leu Val Ala Leu Asn
Thr Pro Leu Ser Gly Gly 1 5 10 15Met Arg Gly Ile Arg
207760DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 77ccagtgctcc acctggtggc
actgaacacc cccctgtctg gaggcatgcg tggtatccgt 607820PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 78Pro Val Leu His Leu Val Ala Leu Asn Thr
Pro Leu Ser Gly Gly Met 1 5 10 15Arg Gly Ile Arg
207957DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 79gtgctccacc tggtggcact
gaacaccccc ctgtctggag gcatgcgtgg tatccgt 578019PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 80Val Leu His Leu Val Ala Leu Asn Thr Pro
Leu Ser Gly Gly Met Arg 1 5 10 15Gly Ile Arg8154DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 81ctccacctgg tggcactgaa cacccccctg
tctggaggca tgcgtggtat ccgt 548218PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 82Leu His
Leu Val Ala Leu Asn Thr Pro Leu Ser Gly Gly Met Arg Gly 1 5 10
15Ile Arg8351DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
83cacctggtgg cactgaacac ccccctgtct ggaggcatgc gtggtatccg t
518417PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 84His Leu Val Ala Leu Asn Thr Pro
Leu Ser Gly Gly Met Arg Gly Ile 1 5 10 15Arg8548DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 85ctggtggcac tgaacacccc cctgtctgga
ggcatgcgtg gtatccgt 488616PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 86Leu Val Ala
Leu Asn Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 10
158745DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 87gtggcactga acacccccct
gtctggaggc atgcgtggta tccgt 458815PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 88Val Ala
Leu Asn Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 10
158942DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 89gcactgaaca cccccctgtc
tggaggcatg cgtggtatcc gt 429014PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 90Ala Leu Asn
Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 1 5 109139DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 91ctgaacaccc ccctgtctgg aggcatgcgt
ggtatccgt 399213PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 92Leu Asn Thr Pro Leu
Ser Gly Gly Met Arg Gly Ile Arg 1 5 109336DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 93aacacccccc tgtctggagg catgcgtggt atccgt
369412PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 94Asn Thr Pro Leu Ser Gly Gly Met
Arg Gly Ile Arg 1 5 109578DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
95catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac ccccctgtct
60ggaggcatgc gtggtatc 789626PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 96His Thr His
Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr
Pro Leu Ser Gly Gly Met Arg Gly Ile 20 259775DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 97catactcatc aggactttca gccagtgctc
cacctggtgg cactgaacac ccccctgtct 60ggaggcatgc gtggt
759825PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 98His Thr His Gln Asp Phe Gln Pro
Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser Gly Gly
Met Arg Gly 20 259972DNAArtificial SequenceDescription of
Artificial Sequence DNA encoding exemplary anti-angiogenic peptide
99catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac ccccctgtct
60ggaggcatgc gt 7210024PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 100His Thr
His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
15Thr Pro Leu Ser Gly Gly Met Arg 2010169DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 101catactcatc aggactttca gccagtgctc
cacctggtgg cactgaacac ccccctgtct 60ggaggcatg 6910223PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 102His Thr His Gln Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser Gly Gly Met
2010366DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 103catactcatc aggactttca
gccagtgctc cacctggtgg cactgaacac ccccctgtct 60ggaggc
6610422PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 104His Thr His Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser Gly
Gly 2010563DNAArtificial SequenceDescription of Artificial Sequence
DNA encoding exemplary anti-angiogenic peptide 105catactcatc
aggactttca gccagtgctc cacctggtgg cactgaacac ccccctgtct 60gga
6310621PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 106His Thr His Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser Gly
2010760DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 107catactcatc aggactttca
gccagtgctc cacctggtgg cactgaacac ccccctgtct 6010820PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 108His Thr His Gln Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser
2010957DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 109catactcatc aggactttca
gccagtgctc cacctggtgg cactgaacac ccccctg 5711019PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 110His Thr His Gln Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu11154DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 111catactcatc aggactttca gccagtgctc
cacctggtgg cactgaacac cccc 5411218PRTArtificial SequenceDescription
of Artificial Sequence Exemplary anti-angiogenic peptide 112His Thr
His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
15Thr Pro11351DNAArtificial SequenceDescription of Artificial
Sequence DNA encoding exemplary anti-angiogenic peptide
113catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac c
5111417PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 114His Thr His Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr11548DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 115catactcatc aggactttca gccagtgctc
cacctggtgg cactgaac 4811616PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 116His Thr
His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
1511745DNAArtificial SequenceDescription of Artificial Sequence DNA
encoding exemplary anti-angiogenic peptide 117catactcatc aggactttca
gccagtgctc cacctggtgg cactg 4511815PRTArtificial
SequenceDescription of Artificial Sequence Exemplary
anti-angiogenic peptide 118His Thr His Gln Asp Phe Gln Pro Val Leu
His Leu Val Ala Leu 1 5 10 1511942DNAArtificial SequenceDescription
of Artificial Sequence DNA encoding exemplary anti-angiogenic
peptide 119catactcatc aggactttca gccagtgctc cacctggtgg ca
4212014PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 120His Thr His Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala 1 5 1012139DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 121catactcatc aggactttca gccagtgctc
cacctggtg 3912213PRTArtificial SequenceDescription of Artificial
Sequence Exemplary anti-angiogenic peptide 122His Thr His Gln Asp
Phe Gln Pro Val Leu His Leu Val 1 5 1012336DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 123catactcatc aggactttca gccagtgctc cacctg
3612412PRTArtificial SequenceDescription of Artificial Sequence
Exemplary anti-angiogenic peptide 124His Thr His Gln Asp Phe Gln
Pro Val Leu His Leu 1 5 1012521PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 125His Ser
His Arg Asp Phe Val Ala Leu Asn Ser Pro Leu Ser Gly Gly 1 5 10
15Met Arg Gly Ile Arg 2012621PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 126His Ser
His Arg Asp Phe Gln Pro Val Leu His Leu Leu Ser Gly Gly 1 5 10
15Met Arg Gly Ile Arg 2012721PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 127Gln Pro
Val Leu His Leu Val Ala Leu Asn Thr Pro Leu Ser Gly Gly 1 5 10
15Met Arg Gly Ile Arg 2012821PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 128His Thr
His Gln Asp Phe Val Ala Leu Asn Thr Pro Leu Ser Gly Gly 1 5 10
15Met Arg Gly Ile Arg 2012921PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 129His Thr
His Gln Asp Phe Gln Pro Val Leu His Leu Leu Ser Gly Gly 1 5 10
15Met Arg Gly Ile Arg 2013027PRTArtificial SequenceDescription of
Artificial Sequence Consensus anti-angiogenic peptide 130His Xaa
His Xaa Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10
15Xaa Pro Leu Ser Gly Gly Met Arg Gly Ile Arg 20
2513121PRTArtificial SequenceDescription of Artificial Sequence
Consensus anti-angiogenic peptide 131His Xaa His Xaa Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Xaa Pro Leu Ser Gly
20132699DNAHomo sapiens 132gagcccaaat cttgtgacaa aactcacaca
tgcccaccgt gcccagcacc tgaactcctg 60gggggaccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 120acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
240tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 300ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 360atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 420gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
540cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 600aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 660tacacgcaga agagcctctc cctgtctccg ggtaaatga
699133232PRTHomo sapiens 133Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala 1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105
110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser
Leu Ser Leu Ser Pro Gly Lys225 23013448DNAArtificial
SequenceDescription of Artificial Sequence DNA encoding exemplary
anti-angiogenic peptide 134tctagaggtg gtctagtgcc gcgcggcagc
ggttcccccg ggttgcag 4813516PRTArtificial SequenceDescription of
Artificial Sequence Exemplary anti-angiogenic peptide 135Ser Arg
Gly Gly Leu Val Pro Arg Gly Ser Gly Ser Pro Gly Leu Gln 1 5 10
1513625PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic mouse peptide 136Met Arg Gly Ile Arg
Gly Ala Asp Phe Gln Ala Phe Gln Gln Ala Arg 1 5 10 15Ala Val Gly
Leu Ser Gly Thr Phe Arg 20 2513725PRTArtificial SequenceDescription
of Artificial Sequence Synthesized anti-angiogenic mouse peptide
137Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile
1 5 10 15Val Arg Arg Ala Asp Arg Gly Ser Val 20
2513825PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic mouse peptide 138Gly Ser Val Pro Ile
Val Asn Leu Lys Asp Glu Val Leu Ser Pro Ser 1 5 10 15Trp Asp Ser
Leu Phe Ser Gly Ser Gln 20 2513925PRTArtificial SequenceDescription
of Artificial Sequence Synthesized anti-angiogenic mouse peptide
139Gly Ser Gln Gly Gln Val Gln Pro Gly Ala Arg Ile Phe Ser Phe Asp
1 5 10 15Gly Arg Asp Val Leu Arg His Pro Ala 20
2514024PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic mouse peptide 140His Pro Ala Trp Pro
Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser 1 5 10 15Gly Arg Arg
Leu Met Glu Ser Tyr 2014125PRTArtificial SequenceDescription of
Artificial Sequence Synthesized anti-angiogenic mouse peptide
141Glu Thr Trp Arg Thr Glu Thr Thr Gly Ala Thr Gly Gln Ala Ser Ser
1 5 10 15Leu Leu Ser Gly Arg Leu Leu Glu Gln 20
2514226PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic mouse peptide 142Lys Ala Ala Ser Ala
His Asn Ser Tyr Ile Val Leu Ala Ile Glu Asn 1 5 10 15Ser Phe Met
Thr Ser Phe Ser Lys Lys Lys 20 2514325PRTArtificial
SequenceDescription of Artificial Sequence Synthesized
anti-angiogenic human peptide 143Met Arg Gly Ile Arg Gly Ala Asp
Phe Gln Ala Phe Gln Gln Ala Arg 1 5 10 15Ala Val Gly Leu Ala Gly
Thr Phe Arg 20 2514425PRTArtificial SequenceDescription of
Artificial Sequence Synthesized anti-angiogenic human peptide
144Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile
1 5 10 15Val Arg Arg Ala Asp Arg Ala Ala Val 20
2514525PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic human peptide 145Ala Ala Val Pro Ile
Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser 1 5 10 15Trp Glu Ala
Leu Phe Ser Gly Ser Glu 20 2514625PRTArtificial SequenceDescription
of Artificial Sequence Synthesized anti-angiogenic human peptide
146Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp
1 5 10 15Gly Lys Asp Val Leu Arg His Pro Thr 20
2514724PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic human peptide 147His Pro Thr Trp Pro
Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn 1 5 10 15Gly Arg Arg
Leu Thr Glu Ser Tyr 2014825PRTArtificial SequenceDescription of
Artificial Sequence Synthesized anti-angiogenic human peptide
148Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
1 5 10 15Leu Leu Gly Gly Arg Leu Leu Gly Gln 20
2514928PRTArtificial SequenceDescription of Artificial Sequence
Synthesized anti-angiogenic human peptide 149Leu Gly Gln Ser Ala
Ala Ser Ala His His Ala Tyr Ile Val Leu Ala 1 5 10 15Ile Glu Asn
Ser Phe Met Thr Ala Ser Lys Lys Lys 20 2515027PRTArtificial
SequenceDescription of Artificial Sequence Synthesized
anti-angiogenic mutant mouse peptide 150Ala Thr Ala Gln Asp Phe Gln
Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15Thr Pro Leu Ser Gly
Gly Met Arg Gly Ile Arg 20 25151552DNAHomo sapiens 151cacagccacc
gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60ggcggcatgc
ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg
120gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct
gtacagcatc 180gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc
tcaaggacga gctgctgttt 240cccagctggg aggctctgtt ctcaggctct
gagggtccgc tgaagcccgg ggcacgcatc 300ttctccttta acggcaagga
cgtcctgacc caccccacct ggccccagaa gagcgtgtgg 360catggctcgg
accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg
420gaggctccct cggccacggg ccaggcctac tcgctgctgg ggggcaggct
cctggggcag 480agtgccgcga gctgccatca cgcctacatc gtgctatgca
ttgagaacag cttcatgact 540gcctccaagt ag 552152183PRTHomo sapiens
152His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1
5 10 15Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe
Gln 20 25 30Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe
Arg Ala 35 40 45Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val
Arg Arg Ala 50 55 60Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp
Glu Leu Leu Phe65 70 75 80Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser
Glu Gly Pro Leu Lys Pro 85 90 95Gly Ala Arg Ile Phe Ser Phe Asn Gly
Lys Asp Val Leu Thr His Pro 100 105 110Thr Trp Pro Gln Lys Ser Val
Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125Arg Leu Thr Glu Ser
Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser 130 135 140Ala Thr Gly
Gln Ala Tyr Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln145 150 155
160Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175Ser Phe Met Thr Ala Ser Lys 180153552DNAMus musculus
153catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac
ccccctgtct 60ggaggcatgc gtggtatccg tggagcagat ttccagtgct tccagcaagc
ccgagccgtg 120gggctgtcgg gcaccttccg ggctttcctg tcctctaggc
tgcaggatct ctatagcatc 180gtgcgccgtg ctgaccgggg gtctgtgccc
atcgtcaacc tgaaggacga ggtgctatct 240cccagctggg actccctgtt
ttctggctcc cagggtcaac tgcaacccgg ggcccgcatc 300ttttcttttg
acggcagaga tgtcctgaga cacccagcct ggccgcagaa gagcgtatgg
360cacggctcgg accccagtgg gcggaggctg atggagagtt actgtgagac
atggcgaact 420gaaactactg gggctacagg tcaggcctcc tccctgctgt
caggcaggct cctggaacag 480aaagctgcga gctgccacaa cagctacatc
gtcctgtgca ttgagaatag cttcatgacc 540tctttctcca aa 552154184PRTMus
musculus 154His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala
Leu Asn1 5 10 15Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala
Asp Phe Gln 20 25 30Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ser Gly
Thr Phe Arg Ala 35 40 45Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser
Ile Val Arg Arg Ala 50 55 60Asp Arg Gly Ser Val Pro Ile Val Asn Leu
Lys Asp Glu Val Leu Ser65 70 75 80Pro Ser Trp Asp Ser Leu Phe Ser
Gly Ser Gln Gly Gln Leu Gln Pro 85 90 95Gly Ala Arg Ile Phe Ser Phe
Asp Gly Arg Asp Val Leu Arg His Pro 100 105 110Ala Trp Pro Gln Lys
Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125Arg Leu Met
Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly 130 135 140Ala
Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln145 150
155 160Lys Ala Ala Ser Cys His Asn Ser Tyr Ile Val Leu Cys Ile Glu
Asn 165 170 175Ser Phe Met Thr Ser Phe Ser Lys 180
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