U.S. patent application number 15/018106 was filed with the patent office on 2016-06-16 for compositions and methods for detecting or eliminating senescent cells to diagnose or treat disease.
The applicant listed for this patent is Unity Biotechnology, Inc.. Invention is credited to Shayne Squires.
Application Number | 20160166718 15/018106 |
Document ID | / |
Family ID | 40824946 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160166718 |
Kind Code |
A1 |
Squires; Shayne |
June 16, 2016 |
COMPOSITIONS AND METHODS FOR DETECTING OR ELIMINATING SENESCENT
CELLS TO DIAGNOSE OR TREAT DISEASE
Abstract
Disclosed are agents (e.g., peptides, polypeptides, proteins,
small molecules, antibodies, and antibody fragments that target
senescent cells) and methods of their use for imaging senescent
cells in vivo and for treating or preventing cancer, age-related
disease, tobacco-related disease, or other diseases and disorders
related to or caused by cellular senescence in a mammal. The
methods include administering one or more of the agents of the
invention to a mammal, e.g., a human. The agents, which
specifically bind to senescent cells, can be labeled with a
radioactive label or a therapeutic label, e.g., a cytotoxic
agent.
Inventors: |
Squires; Shayne; (Cedarburg,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Unity Biotechnology, Inc. |
San Francisco |
CA |
US |
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|
Family ID: |
40824946 |
Appl. No.: |
15/018106 |
Filed: |
February 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14557316 |
Dec 1, 2014 |
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15018106 |
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12809952 |
Feb 21, 2012 |
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PCT/US2008/013913 |
Dec 19, 2009 |
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14557316 |
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61015416 |
Dec 20, 2007 |
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Current U.S.
Class: |
424/1.53 ;
424/172.1; 435/188; 435/7.23; 506/9; 514/44R; 530/389.1; 530/391.5;
530/391.9 |
Current CPC
Class: |
C07K 16/28 20130101;
A61K 49/0056 20130101; A61K 49/0004 20130101; A61K 35/12 20130101;
C07K 2317/24 20130101; C07K 2317/73 20130101; G01N 33/57492
20130101; A61K 38/16 20130101; C12N 15/86 20130101; A61K 51/1027
20130101; A61K 47/64 20170801; G01N 2800/50 20130101; A61K 47/6849
20170801; A61K 38/168 20130101; A61K 38/10 20130101; C07K 14/00
20130101; A61K 51/08 20130101; C12N 2710/10043 20130101; C07K
2317/92 20130101; C12N 15/1037 20130101; A61K 51/088 20130101; G01N
33/5032 20130101; A61K 47/6803 20170801; A61K 2039/505 20130101;
A61K 47/6851 20170801; C07K 2317/76 20130101 |
International
Class: |
A61K 51/10 20060101
A61K051/10; C07K 16/28 20060101 C07K016/28; A61K 47/48 20060101
A61K047/48; G01N 33/574 20060101 G01N033/574 |
Claims
1. An agent comprising a peptide, polypeptide, antibody, or
fragment thereof comprising an amino acid sequence set forth in any
one of SEQ ID NOs:1-3 and 5-8 or a peptide, polypeptide, antibody,
antibody fragment, or small molecule capable of specifically
binding to an antigen comprising an amino acid sequence having at
least 20 amino acids with at least 80% sequence identity to any one
of the amino acid sequences set forth in SEQ ID NOS:11-23.
2. The agent of claim 1, wherein said antigen comprises an amino
acid sequence having at least 20 amino acids of any one of the
amino acid sequences set forth in SEQ ID NOS:11-23.
3. The agent of claim 1, wherein said antigen comprises any one of
the amino acid sequences set forth in SEQ ID NOS:11-23.
4. The agent of claim 1, further comprising one or more of a
detectable label, a therapeutic agent, a chelating agent, or a
linker moiety.
5. The agent of claim 4, wherein said agent is indirectly attached
to said detectable label.
6. The agent of claim 4, wherein said agent is directly attached to
said detectable label.
7. The agent of claim 4, wherein said linker moiety comprises the
amino acid sequence GGGC, GGGS, or GG.
8. The agent of claim 4, wherein said detectable label is a
radioactive agent, a fluorescent agent, a bioluminescent molecule,
an epitope tag, or a heavy metal.
9. The agent of claim 8, wherein said radioactive agent is an
iodine, astatine, or bromine label that is attached to an amino
acid of said agent.
10. The agent of claim 8, wherein said radioactive agent is
technetium-99m.
11. The agent of claim 8, wherein said fluorescent agent is
fluorescein isothiocyanate (FITC), allophycocyanin (APC),
phycoerythrin (PE), rhodamine, tetramethyl rhodamine isothiocyanate
(TRITC), fluorescent protein (GFP), enhanced GFP (eGFP), yellow
fluorescent protein (YFP), cyan fluorescent protein (CFP), red
fluorescent protein (RFP), or dsRed.
12. The agent of claim 8, wherein said bioluminescent molecule is
luciferase.
13. The agent of claim 8, wherein said epitope tag is c-myc,
hemagglutinin, or a histidine tag.
14. The agent of claim 4, wherein said therapeutic agent is a
cytotoxic agent.
15. The agent of claim 14, wherein said cytotoxic agent is an
alkylating agent, an antibiotic, an antineoplastic agent, an
antimetabolic agent, a ribosomal activity inhibitor, an
antiproliferative agent, a tubulin inhibitor, a topoisomerase I or
II inhibitor, a growth factor, an hormonal agonist or antagonists,
an apoptotic agent, an immunomodulator, a radioactive agent, a
phospholipase, or a cytotoxic peptide or lysin.
16. The agent of claim 14, wherein said cytotoxic agent is selected
from the group consisting of the following compounds and their
derivatives: ricin, doxorubicin, methotrexate, camptothecin,
homocamptothecin, thiocolchicine, colchicine, combretastatin,
combretastin A-4, podophyllotoxin, rhizoxin, rhizoxin-d,
dolistatin, paclitaxel, CC 1065, ansamitocin p3, maytansinoid,
streptolysin O, stoichactis toxin, phallolysin, staphylococcus
alpha toxin, holothurin A, digitonin, melittin, lysolecithin,
cardiotoxin, and cerebratulus A toxin.
17. The agent of claim 4, wherein said chelating agent is an
ininocarboxylic reactive group, a polyaminopolycarboxylic reactive
group, diethylenetriaminepentaacetic acid (DTPA), or 1, 4,7,1
O-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
18. The agent of claim 1, wherein said agent further comprises a
pharmaceutically acceptable carrier.
19. The agent of claim 1, wherein said agent specifically binds to
a senescent cell.
20. The agent of claim 19, wherein said senescent cell comprises a
senescent lung, breast, colon, prostate, gastric, hepatic, ovarian,
esophageal, or bronchial epithelial or stromal cell, a senescent
skin epithelial or stromal cell; a senescent glial cell; or a
senescent vascular endothelial or stromal cell.
21. A method of imaging a senescent cell-containing region in a
mammal in vivo, said method comprising the steps of: (a)
administering to said mammal the agent of claim 1 and a detectable
label; (b) allowing said agent to bind senescent cells and allowing
unbound agent to be cleared from the body of said mammal; and (c)
obtaining an image of said senescent cell-containing region.
22. The method of claim 21, wherein said mammal is a human.
23. The method of claim 21, wherein said region is the breast,
prostate, gastrointestinal tract, liver, lungs, intracranial space,
nasopharynx, oropharynx, larynx, esophagus, mediastinum, abdomen
and pelvis, any region of the body containing peripheral
vasculature, or the entire body.
24. The method of claim 21, wherein said image is obtained using
scintigraphy.
25. A method of predicting cancer risk in a mammal, said method
comprising the steps of: (a) administering to said mammal the agent
of claim 1 and a detectable label; (b) predicting an elevated
cancer risk in said mammal by detecting binding of said agent to a
senescent cell of said mammal.
26. The method of claim 25, wherein said mammal is a human.
27. The method of claim 25, wherein said cancer is prostate cancer,
colon cancer, lung cancer, squamous cell cancer of the head and
neck, esophageal cancer, hepatocellular carcinoma, gastric cancer,
pancreatic cancer, ovarian cancer, or breast cancer.
28. A method of treating or preventing disease in a mammal, said
method comprising administering to said mammal the agent of claim
14.
29. The method of claim 28, wherein said mammal is a human.
30. The method of claim 28, wherein said disease is cancer,
age-related disease, tobacco-related disease, or skin wrinkles.
31. The method of claim 30, wherein said cancer is prostate cancer,
colon cancer, lung cancer, squamous cell cancer of the head and
neck, esophageal cancer, hepatocellular carcinoma, gastric cancer,
pancreatic cancer, ovarian cancer, or breast cancer.
32. The method of claim 30, wherein said age-related or
tobacco-related disease is cardiovascular disease, cerebrovascular
disease, peripheral vascular disease, Alzheimer's disease,
osteoarthritis, cardiac diastolic dysfunction, benign prostatic
hypertrophy, aortic aneurysm, or emphysema.
33. A method of treating or preventing disease in a mammal, said
method comprising administering to said mammal a nucleic acid
molecule encoding a cytotoxic agent and the agent of claim 1.
34. The method of claim 33, wherein said mammal is a human.
35. The method of claim 33, wherein said disease is cancer,
age-related disease, or tobacco-related disease.
36. The method of claim 31, wherein said cancer is prostate cancer,
colon cancer, lung cancer, squamous cell cancer of the head and
neck, esophageal cancer, hepatocellular carcinoma, gastric cancer,
pancreatic cancer, ovarian cancer, or breast cancer.
37. The method of claim 35, wherein said age-related or
tobacco-related disease is cardiovascular disease, cerebrovascular
disease, peripheral vascular disease, Alzheimer's disease,
osteoarthritis, cardiac diastolic dysfunction, benign prostatic
hypertrophy, aortic aneurysm, or emphysema.
38. The method of claim 33, wherein said nucleic acid molecule is
administered in a vector.
39. The method of claim 38, wherein said vector is an adenoviral
vector.
40. The method of claim 33, wherein said cytotoxic agent and said
agent are expressed as a single polypeptide chain.
41. A method of identifying a peptide or polypeptide capable of
detecting senescent cells comprising the steps of: (a) culturing
cells to produce senescent cells; (b) exposing said senescent cells
to a phage peptide library; (c) recovering said senescent cells and
phage from said phage peptide library bound to said senescent
cells; (d) eluting and amplifying said bound phage to produce
amplified phage; (e) repeating steps (a)-(d) one or more times; and
(f) recovering a peptide or polypeptide expressed by said amplified
phage that is capable of detecting senescent cells.
42. A method for identifying an antibody or antibody fragment
capable of specifically binding to a senescent cell-specific
antigen, said method comprising the steps of: (a) contacting an
antibody or antibody fragment with a polypeptide comprising at
least 20 amino acids comprising at least 80% amino acid sequence
identity to any one of the amino acid sequences set forth in SEQ ID
NOS:11-23; and (b) identifying an antibody or antibody fragment
that binds said polypeptide with a dissociation constant of less
than 10.sup.-7 M.
43. A method for making an antibody or antibody fragment capable of
specifically binding to a senescent cell-specific antigen, said
method comprising the steps of: (a) administering a polypeptide
comprising at least 20 amino acids comprising at least 80% amino
acid sequence identity to any one of the amino acid sequences set
forth in SEQ ID NOS:11-23 to a mammal; (b) allowing said mammal to
generate a humoral immune response to said polypeptide; and (c)
isolating from said mammal an antibody or antibody fragment that
binds said polypeptide with a dissociation constant of less than
10.sup.-7 M.
44. The method of claim 43, wherein said mammal is a mouse,
hamster, rat, guinea pig, chicken, goat, sheep, cow, horse,
non-human primate, or human.
45. A method for identifying a small molecule capable of
specifically binding to a senescent cell-specific antigen, said
method comprising the steps of: (a) contacting a small molecule
with a polypeptide comprising at least 20 amino acids comprising at
least 80% amino acid sequence identity to any one of the amino acid
sequences set forth in SEQ ID NOS:11-23; and (b) identifying a
small molecule that binds said polypeptide with a dissociation
constant of less than 10.sup.-7 M.
46. A method of making an antibody or antibody fragment, said
method comprising recombinantly expressing a nucleic acid sequence
that encodes an amino acid sequence comprising one or more of SEQ
ID NOs:1-3 and 5-8, wherein said antibody or antibody fragment
specifically binds a senescent cell.
47. A method of cellular therapy comprising administering to a
mammal in need thereof the agent of claim 1 prior to, concurrent
with, or following administration of a cellular therapeutic.
48. The method of claim 47, wherein said mammal is a human.
49. A method of cellular therapy comprising contacting the agent of
claim 1 to a donor cell, tissue, or organ prior to, concurrent
with, or following administration of said cell, tissue, or organ to
a mammal.
50. The method of claim 49, wherein said mammal is a human.
51. The method of claim 49, wherein said donor cell, tissue, or
organ comprises an autologous, allogeneic, syngeneic, or xenogeneic
cell, tissue, or organ.
52. The method of claim 49, wherein said cell comprises a stem cell
population.
53. The method of claim 52, wherein said stem cell is selected from
a hematopoietic, umbilical cord blood, totipotent, multipotent, or
pluripotent stem cell.
54. A kit comprising: (a) the agent of claim 1; and (b) one or more
of a detectable label, a therapeutic agent, a chelating agent, or a
linker moiety.
55. A method of treating a disease related to or caused by cellular
senescence in a mammal comprising administering to the mammal a
pharmaceutical composition comprising: (a) a peptide, a
polypeptide, or an antibody or antibody fragment thereof, wherein
the peptide, the polypeptide, or the antibody or antibody fragment
thereof specifically binds to a senescent cell; and (b) a
pharmaceutically acceptable carrier.
56. The method of claim 55, wherein the peptide, the polypeptide,
or the antibody or antibody fragment thereof specifically binds to
a senescent cell-specific peptide, polypeptide, or
glycoprotein.
57. The method of claim 55, wherein the peptide, the polypeptide,
or the antibody or the antibody fragment thereof administered to
the mammal is coupled to a therapeutic agent.
58. The method of claim 57, wherein the therapeutic agent is a
cytotoxic agent.
59. The method of claim 55, wherein the disease is a cancer, an
age-related disease, or a tobacco-related disease.
60. A method of treating a disease related to or caused by cellular
senescence in a mammal comprising administering to the mammal a
pharmaceutical composition comprising: (a) a small molecule that
specifically binds to a senescent cell; and (b) a pharmaceutically
acceptable carrier.
61. The method of claim 60, wherein the small molecule specifically
binds to a senescent cell-specific peptide, polypeptide, or
glycoprotein.
62. The method of claim 60, wherein the disease is a cancer, an
age-related disease, or a tobacco-related disease.
63. A method for selectively killing a senescent cell in a mammal
comprising administering to the mammal a pharmaceutical composition
comprising (a) a peptide, a polypeptide, or an antibody or antibody
fragment thereof, wherein the peptide, the polypeptide, or the
antibody or antibody fragment thereof specifically binds to the
senescent cell; and (b) a pharmaceutically acceptable carrier.
64. The method of claim 63, wherein the peptide, the polypeptide,
or the antibody or antibody fragment thereof specifically binds to
a senescent cell-specific peptide, polypeptide, or
glycoprotein.
65. The method of claim 63, wherein the peptide, the polypeptide,
or the antibody or the antibody fragment that is administered to
the mammal is coupled to a therapeutic agent.
66. The method of claim 65, wherein the therapeutic agent is a
cytotoxic agent.
67. The method of claim 63, wherein the senescent cell is a
senescent lung cell, a senescent breast cell, a senescent colon
cell, a senescent prostate cell, a senescent gastric cell, a
senescent hepatic cell, a senescent ovarian cell, a senescent
esophageal cell, a senescent bronchial epithelial or stromal cell,
a senescent skin epithelial or stromal cell, a senescent glial
cell, or a senescent vascular endothelial or stromal cell.
68. The method of claim 63, wherein the pharmaceutical composition
comprises the peptide, and wherein the peptide comprises (a) the
amino acid sequence selected from any one of SEQ ID NOS:1-3; or (b)
an amino acid sequence at least 90% identical to the amino acid
sequence selected from any one of SEQ ID NOS:1-3.
69. The method of claim 63, wherein the pharmaceutical composition
comprises the antibody or antibody fragment thereof, and wherein
the antibody or antibody fragment thereof comprises (a) the amino
acid sequence selected from any one of SEQ ID NOS:1-3 in a
complementarity determining region; or (b) an amino acid sequence
at least 90% identical to the amino acid sequence selected from any
one of SEQ ID NOS:1-3 in a complementarity determining region.
70. The method of claim 64, wherein the senescent cell-specific
peptide, polypeptide, or glycoprotein is a senescent cell-specific
antigen comprising the amino acid sequence set forth in any one of
SEQ ID NOS:11-23.
71. A method for selectively killing a senescent cell in a mammal
comprising administering to the mammal a pharmaceutical composition
comprising (a) a small molecule that specifically binds to the
senescent cell; and (b) a pharmaceutically acceptable carrier.
72. The method of claim 71, wherein the small molecule specifically
binds to a senescent cell-specific peptide, polypeptide, or
glycoprotein.
73. The method of claim 71, wherein the senescent cell is a
senescent lung cell, a senescent breast cell, a senescent colon
cell, a senescent prostate cell, a senescent gastric cell, a
senescent hepatic cell, a senescent ovarian cell, a senescent
esophageal cell, a senescent bronchial epithelial or stromal cell,
a senescent skin epithelial or stromal cell, a senescent glial
cell, or a senescent vascular endothelial or stromal cell.
74. A method for depleting senescent cells from donor cells, a
donor tissue, or a donor organ, said method comprising (a)
contacting the donor cells, the donor tissue, or the donor organ
with a peptide, a polypeptide, a small molecule, or an antibody or
antibody fragment thereof, wherein the peptide, the polypeptide,
the small molecule, or the antibody or antibody fragment thereof
specifically binds to a senescent cell; and (b) administering the
donor cells, the donor tissue, or the donor organ to the mammal
prior to, concurrent with, or subsequent to the step of
contacting.
75. The method of claim 74, wherein the peptide, the polypeptide,
small molecule, or the antibody or antibody fragment thereof is
administered to the mammal prior to, concurrent with, or subsequent
to administration of the donor cells, the donor tissue, or the
donor organ to the mammal.
76. The method of claim 74, wherein the peptide, the polypeptide,
or the antibody or the antibody fragment thereof contacting the
donor cells, tissue, or organ is coupled to a therapeutic
agent.
77. The method of claim 76 wherein the therapeutic agent is a
cytotoxic agent.
78. A method of imaging a senescent cell-containing region in a
mammal in vivo, said method comprising the steps of: (a)
administering to the mammal a peptide, a polypeptide, a small
molecule, or an antibody or antibody fragment thereof coupled to a
detectable label, wherein the detectably labeled peptide,
polypeptide, small molecule, or antibody or antibody fragment
thereof specifically binds to a senescent cell; (b) allowing the
detectably labeled peptide, polypeptide, small molecule, or
antibody or antibody fragment thereof to bind to the senescent cell
and allowing unbound detectably labeled peptide, polypeptide, small
molecule, or antibody or antibody fragment thereof to be cleared
from the body of said mammal; and (c) obtaining an image of the
senescent cell-containing region.
79. A method of predicting cancer risk in a mammal, said method
comprising: (a) administering to the mammal a peptide, a
polypeptide, a small molecule, or an antibody or antibody fragment
thereof coupled to a detectable label, wherein the detectably
labeled peptide, polypeptide, small molecule, or antibody or
antibody fragment thereof specifically binds to a senescent cell;
and (b) detecting binding of the detectably labeled peptide,
polypeptide, small molecule, or antibody or antibody fragment
thereof to the senescent cell of the mammal, thereby predicting an
elevated cancer risk in the mammal.
80. A method for identifying a peptide or polypeptide that binds
specifically to a senescent cell, said method comprising: (a)
culturing cells to produce senescent cells; (b) exposing the
senescent cells to a phage peptide library; (c) recovering the (i)
senescent cells and (ii) the phage from the phage peptide library
that is bound to the senescent cells; (d) eluting and amplifying
the bound phage to produce amplified phage; (e) repeating steps
(a)-(d) one or more times; and (f) recovering a peptide or
polypeptide expressed by the amplified phage, which peptide or
polypeptide is capable of identifying senescent cells.
81. A method for identifying a small molecule that specifically
binds to a senescent cell, said method comprising (a) contacting a
plurality of small molecule candidates with a senescent cell; and
(b) determining specific binding of the small molecule candidates
to the senescent cell, thereby identifying a small molecule that
specifically binds to the senescent cell.
82. A pharmaceutical composition comprising: (a) a peptide, a
polypeptide, or an antibody or antibody fragment thereof coupled to
a therapeutic agent, wherein the peptide, the polypeptide, or the
antibody or antibody fragment thereof specifically binds to a
senescent cell-specific peptide, polypeptide, or glycoprotein, and
wherein the therapeutic agent is capable of killing a senescent
cell; and (b) a pharmaceutically acceptable carrier.
83. The pharmaceutical composition of claim 82, wherein the
composition comprises the peptide coupled to the therapeutic agent,
and wherein the peptide comprises (a) the amino acid sequence
selected from any one of SEQ ID NOS:1-3; or (b) an amino acid
sequence at least 90% identical to the amino acid sequence selected
from any one of SEQ ID NOS:1-3.
84. The pharmaceutical composition of claim 82, wherein the
composition comprises the antibody or antibody fragment thereof
coupled to the therapeutic agent, and wherein the antibody or
antibody fragment thereof comprises (a) the amino acid sequence
selected from any one of SEQ ID NOS:1-3 in a complementarity
determining region; or (b) an amino acid sequence at least 90%
identical to the amino acid sequence selected from any one of SEQ
ID NOS:1-3 in a complementarity determining region.
85. The pharmaceutical composition of claim 82, wherein the
senescent cell-specific peptide, polypeptide, or glycoprotein is a
senescent cell-specific antigen comprising the amino acid sequence
set forth in any one of SEQ ID NOS:11-23.
86. A pharmaceutical composition comprising: (a) a small molecule
that specifically binds to a senescent cell-specific peptide,
polypeptide, or glycoprotein; and (b) a pharmaceutically acceptable
carrier.
Description
STATEMENT REGARDING SEQUENCE LISTING
[0001] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is 200201_401D1
SEQUENCE_LISTING.txt. The text file is 70.5 KB, was created on Nov.
29, 2014, and is being submitted electronically via EFS-Web.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods for the
detection and treatment of cancer, age-related diseases,
tobacco-related diseases, and other diseases and disorders related
to or caused by cellular senescence.
BACKGROUND OF THE INVENTION
[0003] The definition of cellular senescence has undergone some
revision since the phenomenon was described by Leonard Hayflick as
cessation of replication of cultured human cells after a finite
number of population doublings (Hayflick et al., Exp. Cell Res.
37:585-621, 1961). Senescent cells remain metabolically active but
do not divide and resist apoptosis for long periods of time
(Goldstein, Science 249:1129-1133, 1990). Cellular senescence is
characterized by growth cycle arrest in the G1 phase, absence of S
phase, and lifespan control by multiple dominant genes
(Stanulis-Praeger, Mech. Ageing Dev. 38:1-48, 1987).
[0004] Cellular senescence differs from quiescence and terminal
differentiation in several important aspects. Senescent cells have
characteristic morphological changes such as enlargement,
flattening, and increased granularity (Dimri et al., Proc. Nat.
Acad. Sci. USA 92:9363-9367, 1995). Senescent cells do not divide
even if stimulated by mitogens (Campisi, Trends Cell Biol.
11:S27-S31, 2001). Senescence involves activation of p53 and/or Rb
and their regulators such as p16.sup.INIK4a, p21, and ARF. Except
when p53 or Rb is inactivated, senescence is irreversible.
Senescent cells express increased levels of plasminogen activator
inhibitor (PAI) and exhibit staining for .beta.-galactosidase
activity at pH 6 (Sharpless et al., J. Clin. Invest. 113:160-168,
2004). Irreversible G1 arrest is mediated by inactivation of cyclin
dependent kinase (CdK) complexes which phosphorylate Rb. P21
accumulates in aging cells and inhibits CdK4-CdK6. P16 inhibits
CdK4-CdK6 and accumulates proportionally with .beta.-galactosidase
activity and cell volume (Stein et al., Mol. Cell. Biol.
19:2109-2117, 1999). P21 is expressed during initiation of
senescence but need not persist; p16 expression helps maintain
senescence once initiated.
[0005] Replicative senescence, the type of senescence originally
observed by Hayflick, is related to the progressive shortening of
telomeres with each cell division. Senescence is induced when
certain chromosomal telomeres reach a critical length (Mathon and
Lloyd, Nat. Rev. Cancer 3:203-213, 2001; Martins, U.M. Exp Cell
Res. 256:291-299, 2000). Senescence can be abrogated by the
expression of telomerase which lengthens telomeres; human
fibroblasts undergo replication indefinitely when transfected to
express telomerase. Most cancers express telomerase in order to
maintain telomere length and replicate indefinitely. The minority
of cancers that do not express telomerase have alternative
lengthening of telomere (ALT) mechanisms.
[0006] Indirect evidence suggests some relationship between
replicative senescence and aging. Cultured cells from old donors
exhibit senescence after fewer population doublings than cells from
young donors (Martin et al., Lab. Invest. 23:86-92, 1970; Schneider
et al., Proc. Nat. Acad. Sci. USA 73:3584-3588, 1976). Cells from
short-lived species senesce after fewer population doublings than
cells from long-lived species (Rohme, D., Proc. Nat. Acad. Sci. USA
78:5009-3320, 1981). Cultured cells from donors with hereditary
premature aging syndromes such as Werner's syndrome show senescence
after fewer replications than cells from age-matched controls
(Goldstein, Genetics of Aging, 171-224, 1978; Martin, Genetic
Effects on Aging, 5-39, 1990). Whether replicative senescence
actually contributes to aging or age-related symptoms in vivo is
questionable on the basis of theoretical estimates of the number of
cell divisions that occur in vivo and the absence of strong
empirical evidence.
[0007] There are, however, other pathways to senescence than
replication. Collectively, these are often referred to as
stress-induced premature senescence (SIPS). Oxidative stress can
shorten telomeres (von Zglinicki, Trends Biochem. Sci. 27:339-344,
2002) and hyperoxia has been shown to induce senescence. Gamma
irradiation of human fibroblasts in early to mid G1 phase causes
senescence in a p53-dependent manner (Di Leonardo et al., Genes
Dev. 8:2540-2551, 1994). Ultraviolet radiation also induces
cellular senescence. Other agents that can induce cellular
senescence include hydrogen peroxide (Krtolica et al., Proc. Nat.
Acad. Sci. USA 98:12072-12077, 2001), sodium butyrate,
5-azacytadine, and transfection with the Ras oncogene (Tominaga,
Mech. Ageing Dev. 123(8):927-936, 2002). Chemotherapeutic agents
including doxorubicin, cisplatin, and a host of others have been
shown to induce senescence in cancer cells (Roninson, Cancer Res.
63:2705-2715, 2003). 5-bromodeoxyuridine treatment results in
cellular senescence in both normal and malignant cells (Michishita
et al., J. Biochem. 126:1052-1059, 1999). Generally speaking,
agents that damage DNA can cause cellular senescence. The existence
of cellular senescence in vivo has been demonstrated. In a study by
Dimri et al., published in 1995, senescent fibroblasts were shown
to exhibit staining for .beta.-galactosidase activity at pH 6.
These cells failed to incorporate tritiated thymidine and retained
.beta.-galactosidase activity after replating but did not divide.
Quiescent fibroblasts did not show staining Keratinocytes,
umbilical vein endothelial cells, and mammary epithelial cells all
showed increased staining with increased population doublings.
Immortalized cells and terminally differentiated keratinocytes did
not show staining. Staining was performed on skin biopsies to test
whether senescence is observed in vivo. An age-dependent pattern in
which an increased number of cells showed staining with increased
donor age was observed in the dermis and epidermis (Dimri et al.,
Proc. Nat. Acad. Sci. USA 92:9363-9367, 1995). The existence of an
increase in the number of senescent fibroblasts has been shown in
the lungs of subjects with emphysema relative to subjects without
emphysema (Muller et al., Resp. Res. 7:32-41, 2006).
[0008] Cellular senescence confers a number of functional changes
on the cell that likely have clinical relevance. Senescent
endothelial cells secrete elevated levels of plasminogen activator
inhibitor 1 (PAI-1; Kletsas et al., Ann. N.Y Acad. Sci. 908:11-25,
2000). Senescent fibroblasts over express collagenase and under
express collagenase inhibitors (West et al., Exp Cell Res
184:138-147, 1989). Serial passages of human fibroblasts from a 25
year old donor showed increased elastase endopeptidase type
activity (Homsy et al., Journal of Investigative Dermatology
91:472-477, 1988). Endothelial cells obtained from tissue overlying
atherosclerotic plaques were observed to have a senescent
morphology and express increased levels of PAI-1 and intracellular
adhesion molecule 1 and decreased levels of nitric oxide (Davis et
al., British Heart Journal 60:459-464, 1988; Arterioscler. Thromb.
11:1678-1689, 1991; Finn et al., Circulation 105:1541-1544, 1976;
Comi et al., Exp. Cell Res. 219:304-308, 1995; Chang et al., Proc.
Nat. Acad. Sci. USA 92:11190-11194, 1995). Indirect evidence that
cellular senescence may play a role in cardiovascular disease also
is provided by the observation that shorter leukocyte telomere
length is associated with an increased risk of premature myocardial
infarction (Brouilette et al., Arteriosclerosis, Thrombosis, and
Vascular Biology 23:842-846, 2003).
[0009] Cancer cells are immortal, meaning that they can replicate
indefinitely without exhibiting senescence. A preponderance of
opinion in the scientific community says that the teleological
purpose of senescence is to prevent cancer by limiting the number
of cell divisions that can occur. This view is supported by
experiments in mice showing that p53 knockout results in increased
cancer incidence and severity. Indirect evidence that senescence
suppresses cancer occurrence includes the observations that
oncogenes immortalize or extend cellular lifespan and tumor
suppressors Rb and p53, which are critical for senescence, suffer a
loss of function mutation in cancer (Shay et al., Biochem. Biophys.
Acta. 1071:1-7, 1991).
[0010] Senescent cells can also promote tumorigenesis. Senescent
stromal cells express tumor promoting as well as tumor suppressing
factors that exert a paracrine effect on neighboring epithelial
cells; such effects include mitogenicity and antiapoptosis (Chang
et al., Proc. Nat. Acad. Sci. USA 97(8):4291-4296, 2000). Senescent
fibroblasts have been shown to stimulate premalignant and malignant
epithelial cells but not normal epithelial cells to form tumors in
mice; this occurred when as few as 10% of the fibroblasts were
senescent (Krtolica et al., Proc. Nat. Acad. Sci. USA
98:12072-12077, 2001). Tumor promoting factors secreted by
senescent cells are partly mediated by p21.sup.waf1/cip1/sdi1
(Roninson, Cancer Res. 63:2705-2715, 2003). A threshold of
senescent stromal cells appears to provide a milieu allowing
adjacent premalignant epithelial cells to survive, migrate, and
divide (Campisi, Nat. Rev. Cancer 3:339-349, 2003).
[0011] In summary, cellular senescence does occur in vivo and is a
likely sequel to environmental insults. Its prevalence increases
with age at least in some tissue compartments. Senescence confers
functional changes on the cell which have been associated to some
degree with various age-related diseases (Chang et al., Proc. Nat.
Acad. Sci. USA 97(8):4291-4296, 2000). Senescent cells also
contribute to tumor formation. There exists a need for agents that
are capable of detecting senescent cells in vivo and for treating
or preventing diseases and disorders related to or caused by
cellular senescence.
SUMMARY OF THE INVENTION
[0012] This invention features compositions and methods for the
detection and imaging of senescent cells in vitro or in vivo in
order to predict the risk of diseases or disorders related to or
caused by cellular senescence (e.g., cancer occurrence, cancer
metastasis, cardiovascular disease, cerebrovascular disease,
Alzheimer's disease, and emphysema). The invention also features
compositions and methods for treating, preventing, or inhibiting
the development or progression of diseases or disorders related to
or caused by cellular senescence (e.g., by administering an agent
that results in the death and removal of one or more, all, or
substantially all senescent cells or cells expressing one or more
senescence markers from an organism). The compositions and methods
can be administered in order to prevent, ameliorate, inhibit the
development of, or treat diseases or disorders related to or caused
by cellular senescence (e.g., cancer, cardiovascular disease,
cerebrovascular disease, Alzheimer's disease, emphysema,
osteoarthritis, and other age-related diseases). The invention
features peptides which bind with higher affinity to senescent
cells than non-senescent cells.
[0013] In a first aspect, the invention features a peptide,
polypeptide, antibody, or antibody fragment agent having an amino
acid sequence set forth in any one of SEQ ID NOS:1-3 and 5-8 or a
peptide, polypeptide, antibody, antibody fragment, or small
molecule agent that is capable of specifically binding to an
antigen having an amino acid sequence having at least 20 amino
acids with at least 80% sequence identity to any one of the amino
acid sequences set forth in SEQ ID NOS:11-23. In one embodiment,
the antigen has an amino acid sequence having at least 20 amino
acids of any one of the amino acid sequences set forth in SEQ ID
NOS:11-23. In another embodiment, the antigen has any one of the
amino acid sequences set forth in SEQ ID NOS:11-23.
[0014] In embodiments of the first aspect of the invention, the
agent includes a detectable label, a therapeutic agent, a chelating
agent, or a linker moiety. An agent of the first aspect of the
invention can be indirectly or directly attached to the detectable
label. The linker moiety can have the amino acid sequence GGGC (SEQ
ID NO:9), GGGS (SEQ ID NO:10), or GG. A detectable label can be a
radioactive agent, fluorescent agent, bioluminescent molecule,
epitope tag, or heavy metal. Radioactive agents include iodine,
astatine, and bromine labels that are attached to an amino acid of
an agent of the first aspect of the invention. A radioactive agent
can also be technetium-99m. Fluorescent agents include fluorescein
isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE),
rhodamine, tetramethyl rhodamine isothiocyanate (TRITC),
fluorescent protein (GFP), enhanced GFP (eGFP), yellow fluorescent
protein (YFP), cyan fluorescent protein (CFP), red fluorescent
protein (RFP), and dsRed. A bioluminescent molecule can be
luciferase. Epitope tags include c-myc, hemagglutinin, and
histidine tags. A therapeutic agent can be a cytotoxic agent, such
as an alkylating agent, an antibiotic, an antineoplastic agent, an
antimetabolic agent, a ribosomal activity inhibitor, an
antiproliferative agent, a tubulin inhibitor, a topoisomerase I or
II inhibitor, a growth factor, an hormonal agonist or antagonists,
an apoptotic agent, an immunomodulator, a radioactive agent, a
phospholipase, or a cytotoxic peptide or lysin. A cytotoxic agent
can also be ricin, doxorubicin, methotrexate, camptothecin,
homocamptothecin, thiocolchicine, colchicine, combretastatin,
combretastin A-4, podophyllotoxin, rhizoxin, rhizoxin-d,
dolistatin, paclitaxel, CC1065, ansamitocin p3, maytansinoid,
streptolysin O, stoichactis toxin, phallolysin, staphylococcus
alpha toxin, holothurin A, digitonin, melittin, lysolecithin,
cardiotoxin, cerebratulus A toxin, or any derivative thereof. A
chelating agent joined to an agent of the first aspect of the
invention can be an ininocarboxylic reactive group, a
polyaminopolycarboxylic reactive group,
diethylenetriaminepentaacetic acid (DTPA), or
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In
any embodiment, the agent of the first aspect of the invention can
further include a pharmaceutically acceptable carrier. An agent of
the first aspect of the invention can specifically bind to a
senescent cell, such as a senescent lung, breast, colon, prostate,
gastric, hepatic, ovarian, esophageal, or bronchial epithelial or
stromal cell, a senescent skin epithelial or stromal cell; a
senescent glial cell; or a senescent vascular endothelial or
stromal cell.
[0015] In a second aspect, the invention features a method of
imaging a senescent cell-containing region in a mammal, such as a
human, in vivo, by administering to the mammal an agent of the
first aspect of the invention that is joined to a detectable label,
allowing the agent to bind senescent cells and allowing unbound
agent to be cleared from the body of said mammal, and obtaining an
image of the senescent cell-containing region. Senescent
cell-containing regions include the breast, prostate,
gastrointestinal tract, liver, lungs, intracranial space,
nasopharynx, oropharynx, larynx, esophagus, mediastinum, abdomen
and pelvis, any region of the body containing peripheral
vasculature, and the entire body. An image of the senescent
cell-containing region can be obtained by scintigraphy.
[0016] In a third aspect, the invention features a method of
predicting cancer risk in a mammal, such as a human, by
administering to the mammal an agent of the first aspect of the
invention that is joined to a detectable label and predicting an
elevated cancer risk in the mammal by detecting binding of the
agent to a senescent cell of the mammal. The method can be used to
predict the risk of prostate cancer, colon cancer, lung cancer,
squamous cell cancer of the head and neck, esophageal cancer,
hepatocellular carcinoma, gastric cancer, pancreatic cancer,
ovarian cancer, and breast cancer.
[0017] In a fourth aspect, the invention features a method of
treating or preventing disease in a mammal, such as a human, by
administering to the mammal an agent of the first aspect of the
invention joined to a cytotoxic agent.
[0018] In a fifth aspect, the invention features a method of
treating or preventing disease in a mammal, such as a human, by
administering to the mammal a nucleic acid molecule encoding a
cytotoxic agent and an agent of the first aspect of the
invention.
[0019] The nucleic acid molecule can be administered in a vector,
such as an adenoviral vector. In one embodiment, the cytotoxic
agent and agent of the first aspect of the invention are expressed
as a single polypeptide chain.
[0020] In either the fourth or fifth aspects of the invention,
diseases that can be treated or prevented include cancer,
age-related diseases, tobacco-related diseases, and skin wrinkles
Cancers that can be treated or prevented include prostate cancer,
colon cancer, lung cancer, squamous cell cancer of the head and
neck, esophageal cancer, hepatocellular carcinoma, gastric cancer,
pancreatic cancer, ovarian cancer, and breast cancer. Age-related
or tobacco-related diseases include cardiovascular disease,
cerebrovascular disease, peripheral vascular disease, Alzheimer's
disease, osteoarthritis, cardiac diastolic dysfunction, benign
prostatic hypertrophy, aortic aneurysm, and emphysema.
[0021] In a sixth aspect, the invention features a method for
identifying a peptide or polypeptide capable of detecting senescent
cells by (a) culturing cells to produce senescent cells, (b)
exposing the senescent cells to a phage peptide library, (c)
recovering the senescent cells and phage from said phage peptide
library bound to said senescent cells, (d) eluting and amplifying
bound phage to produce amplified phage, (e) repeating steps (a)-(d)
one or more times, and (f) recovering a peptide or polypeptide
expressed by the amplified phage that is capable of detecting
senescent cells.
[0022] In a seventh aspect, the invention features a method for
identifying an antibody or antibody fragment capable of
specifically binding to a senescent cell-specific antigen by
contacting an antibody or antibody fragment with a polypeptide
having at least 20 amino acids having at least 80% amino acid
sequence identity to any one of the amino acid sequences set forth
in SEQ ID NOS:11-23, and identifying an antibody or antibody
fragment that binds the polypeptide with a dissociation constant of
less than 10.sup.-7 M.
[0023] In an eighth aspect, the invention features a method for
identifying an antibody or antibody fragment capable of
specifically binding to a senescent cell-specific antigen by
administering a polypeptide having at least 20 amino acids having
at least 80% amino acid sequence identity to any one of the amino
acid sequences set forth in SEQ ID NOS:11-23 to a mammal, allowing
the mammal to generate a humoral immune response to the
polypeptide, and identifying an antibody or antibody fragment that
binds the polypeptide with a dissociation constant of less than
10.sup.-7 M. Mammals suitable for the identification of antibodies
that specifically bind senescent cell-specific antigens include
mice, hamsters, rats, guinea pigs, chickens, goats, sheep, cows,
horses, non-human primates, and humans.
[0024] In an ninth aspect, the invention features a method for
identifying a small molecule capable of specifically binding to a
senescent cell-specific antigen by contacting a small molecule with
a polypeptide having at least 20 amino acids having at least 80%
amino acid sequence identity to any one of the amino acid sequences
set forth in SEQ ID NOS:11-23 and identifying a small molecule that
binds the polypeptide with a dissociation constant of less than
10.sup.-7 M.
[0025] In an tenth aspect, the invention features a method of
making an antibody or antibody fragment by recombinantly expressing
a nucleic acid sequence that encodes an amino acid sequence having
one or more of SEQ ID NOs:1-3 and 5-8, wherein the antibody or
antibody fragment specifically binds a senescent cell.
[0026] In an eleventh aspect, the invention features a method of
cellular therapy performed by administering to a mammal, such as a
human, in need thereof an agent of the invention prior to,
concurrent with, or following administration of a cellular
therapeutic.
[0027] In an twelfth aspect, the invention features a method of
cellular therapy performed by contacting an agent of the invention
to a donor cell, tissue, or organ prior to, concurrent with, or
following administration of the cell, tissue, or organ to a mammal,
such as a human. A donor cell, tissue, or organ can be an
autologous, allogeneic, syngeneic, or xenogeneic cell, tissue, or
organ. A donor cell can be a stem cell, such as a hematopoietic,
umbilical cord blood, totipotent, multipotent, or pluripotent stem
cell.
[0028] In a thirteenth aspect, the invention features a kit
containing an agent of the invention and one or more of a
detectable label, a therapeutic agent, a chelating agent, or a
linker moiety.
[0029] The term "about" is used herein to mean a value that is
.+-.10% of the recited value.
[0030] By "administration" or "administering" is meant a method of
providing a dosage of an agent of the invention to a mammal (e.g.,
a human), where the route is, e.g., topical, oral, parenteral
(e.g., intravenous, intraperitoneal, intrarterial, intradermal,
intramuscular, or subcutaneous injection, inhalation, optical
drops, or implant), nasal, vaginal, rectal, or sublingual
application in admixture with a pharmaceutically acceptable carrier
adapted for such use. The preferred method of administration can
vary depending on various factors, e.g., the components of the
pharmaceutical composition, site of the potential or actual disease
(e.g., the location of lung, breast, colon, prostate, liver, brain,
heart, etc.), and the severity of disease.
[0031] By "analog" is meant an agent that differs from, but is
structurally, functionally, and/or chemically related to the
reference agent. The analog may retain the essential properties,
functions, or structures of the reference agent. Most preferably,
the analog retains at least one biological function of the
reference agent. Generally, differences are limited so that the
structure or sequence of the reference agent and the analog are
similar overall. For example, a peptide analog and its reference
peptide may differ in amino acid sequence by one or more amino acid
substitutions, additions, and/or deletions, or the presence of one
or more non-naturally occurring amino acid residues, in any
combination. An analog of a peptide or polypeptide of the invention
may be naturally occurring, such as an allelic variant, or it may
be a variant that is not known to occur naturally. Non-naturally
occurring analogs of peptides may be made by direct synthesis, by
modification, or by mutagenesis techniques.
[0032] By "chelating agent" is meant a molecule that forms multiple
chemical bonds with a single metal atom. Prior to forming the
bonds, the chelating agent has more than one pair of unshared
electrons. The bonds are formed by sharing pairs of electrons with
the metal atom. Chelating agents include, for example, an
iminodicarboxylic group or a polyaminopolycarboxylic group.
Chelating agents may be attached to an agent of the invention using
the methods generally described in Liu et al., Bioconjugate Chem.
12(4):653, 2001; Alter et al., U.S. Pat. No. 5,753,627; and PCT
Publication No. WO 91/01144; each of which is hereby incorporated
by reference). An agent of the invention may be complexed, through
its attached chelating agent, to a detectable label, thereby
resulting in an agent that is indirectly labeled. Similarly,
cytotoxic or therapeutic agents, may also be attached via a
chelating group to an agent of the invention.
[0033] By "coupled" is meant the characteristic of a first molecule
being joined to a second molecule by a covalent bond or through
noncovalent intermolecular attraction.
[0034] By "cytotoxic agent" is meant any naturally occurring,
modified, or synthetic compound that is toxic to cells. Such agents
are useful in the treatment of neoplasms, and in the treatment of
other symptoms or diseases characterized by cell proliferation or a
hyperactive cell population. Cytotoxic agents can also be used to
target undesirable cells or tissues other than neoplastic cells or
tissues, e.g., senescent cells. Cytotoxic agents include, but are
not limited to, alkylating agents, antibiotics, antimetabolites,
tubulin inhibitors, topoisomerase I and II inhibitors, hormonal
agonists or antagonists, immunomodulators, or agents that cause
cell lysis including naturally occurring or synthetic peptides.
Cytotoxic agents may be cytotoxic when activated by light or
infrared (Photofrin, IR dyes; Nat. Biotechnol. 19(4):327-331,
2001), may operate through other mechanistic pathways, or be
supplementary potentiating agents.
[0035] By "detectable label" is meant any type of label which, when
attached to an agent of the invention, renders the agent
detectable. A detectable label may be toxic or non-toxic, and may
have one or more of the following attributes, without restriction:
fluorescence (Kiefer et al., WO 9740055), color, toxicity (e.g.,
radioactivity, e.g., a .gamma.-emitting radionuclide,
Auger-emitting radionuclide, .beta.-emitting radionuclide, an
.alpha.-emitting radionuclide, or a positron-emitting
radionuclide), radiosensitivity, or photosensitivity. Although a
detectable label may be directly attached, for example, to an amino
acid residue of an agent of the invention, or indirectly attached,
for example, by being complexed with a chelating group that is
attached (e.g., linked via a covalent bond or indirectly linked) to
an amino acid residue of an agent of the invention. A detectable
label may also be indirectly attached to an agent of the invention
by the ability of the label to be specifically bound by a second
molecule. One example of this type of an indirectly attached label
is a biotin label that can be specifically bound by a second
molecule, streptavidin. The second molecule may also be linked to a
moiety that allows neutron capture (e.g., a boron cage as described
in, for example, Kahl et al., Proc. Natl. Acad. Sci. USA
87:7265-7269, 1990).
[0036] A detectable label may also be a metal ion from heavy
elements or rare earth ions, such as Gd.sup.3+, Fe.sup.3+,
Mn.sup.3+, or Cr.sup.2+ (see, e.g., Curter, Invest. Radiol.
33(10):752-761, 1998). Preferred radioactive detectable labels are
radioactive iodine labels (e.g., .sup.122I, .sup.123I, .sup.124I,
.sup.125I, or .sup.131I) that are capable of being coupled to each
D- or L-Tyr or D- or L-4-amino-Phe residues present in the agents
of the invention. Preferred non-radioactive detectable labels are
the many known dyes that are capable of being coupled to
NH.sub.2-terminal amino acid residues.
[0037] Preferred examples of detectable labels that may be toxic to
cells include ricin, diphtheria toxin, and radioactive detectable
labels (e.g., .sup.122I, .sup.123I, .sup.124I, .sup.125I,
.sup.131I, .sup.177Lu, .sup.64Cu, .sup.67Cu, .sup.153Sm,
.sup.166Ho, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Bi,
.sup.225Ac, .sup.67Ga, .sup.68Ga, .sup.75Br, .sup.76Br, .sup.77Br,
.sup.117mSn, .sup.47Sc, .sup.109Pd, .sup.89Sr, .sup.159Gd,
.sup.149Pm, .sup.142Pr, .sup.111Ag, .sup.165Dy, .sup.213Bi,
.sup.111In, .sup.114mIn, .sup.201Ti, .sup.195mPt, .sup.193Pt,
.sup.86Y and .sup.90Y). These compounds, and others described
herein may be directly or indirectly attached to an agent of the
invention or its analogs. A toxic detectable label may also be a
chemotherapeutic agent (e.g., camptothecins, homocamptothecins,
5-fluorouracil or adriamycin), or may be a radiosensitizing agent
(e.g., paclitaxel, gemcitabine, fluoropyrimidine, metronitozil, or
the deoxycytidine analog 2',2' difluoro-2'-deoxycytidine (dFdCyd)
to which is directly or indirectly attached an agent or analog
thereof of the present invention.
[0038] A detectable label, when coupled to an agent of the
invention emits a signal that can be detected by a signal
transducing machine. In some cases, the detectable label can emit a
signal spontaneously, such as when the detectable label is a
radionuclide. In other cases the detectable label emits a signal as
a result of being stimulated by an external field such as when the
detectable label is a relaxivity metal. Examples of signals
include, without limitation, gamma rays, X-rays, visible light,
infrared energy, and radio waves. Examples of signal transducing
machines include, without limitation, gamma cameras including
SPECT/CT devices, PET scanners, fluorimeters, and Magnetic
Resonance Imaging (MRI) machines.
[0039] By "diagnostically effective amount" is meant a dose of
detectably-labeled agent that, when administered internally to a
mammal, is quantitatively sufficient to be detected by a signal
transducing machine external to the mammal (e.g., a gamma camera
used in gamma scintigraphy) but typically is quantitatively
insufficient to produce a pharmacological effect.
[0040] By "imaging agent" is meant a compound that, when
administered to a living subject, such as a mammal (e.g., a human),
allows the visualization of internal structures (e.g., cells,
tissues, and organs) and, in some cases can provide information as
to the function of a cell, tissue, or organ in the subject.
[0041] By "linker moiety" is meant a sequence of amino acid
residues, e.g., at least one, two, three, four, five, six, seven,
eight, nine, ten, fifteen, twenty, thirty, forty, fifty, or more
residues, that couples an agent of the invention (e.g., a peptide,
polypeptide, protein, small molecule, antibody, or antibody
fragment that target senescent cells) to, e.g., one or more of a
detectable label, a therapeutic agent, and a chelating agent.
[0042] By "senescent cell" is meant a cell that is metabolically
active but is permanently withdrawn from the cell cycle (see, e.g.,
Campisi, Cell 120:513-522, 2005). Senescent cells do not replicate
and possess one or more of the following additional characteristics
attributed to senescent cells: cell cycle arrest in the G1 phase;
an enlarged, flattened morphology; increased granularity; staining
for .beta.-galactosidase activity at pH 6; senescence associated
heterochromatic foci; and characteristic gene expression that is in
part regulated by p16 and p21. Alternatively, a senescent cell is a
cell that can be induced to become senescent (e.g., by stress) or
that expresses cell-surface markers characteristic of senescent
cells; these markers include senescent cell-specific antigens
having the polypeptide sequences set forth in SEQ ID NOS:11-23.
Senesecent cell-specific antigens are those peptides, polypeptides,
or glycoproteins that are expressed on the cell surface of
senescent cells, but are absent or only weakly expressed on the
cell surface of non-senescent cells.
[0043] By "peptide" is meant a polymer that includes two or more
amino acids joined to each other by a peptide bond or a modified
peptide bond. "Peptide" refers to both short chain polymers,
commonly referred to as peptides, oligopeptides, or oligomers,
having, e.g., about 10-50 linked amino acid residues, and to longer
polymers having up to about 100 amino acid residues in length.
Peptides may contain amino acids other than the 20 gene-encoded
amino acids, and linkages other than peptide bonds and may include
cyclic or branched peptides. "Peptides" include amino acid
sequences modified either by natural processes, or by chemical
modification techniques that are well known in the art.
Modifications may occur anywhere in a polypeptide, including the
peptide backbone, the amino acid side-chains, and the amino or
carboxyl termini.
[0044] The notations used herein for the peptide amino acid
residues are those abbreviations commonly used in the art. The less
common abbreviations Abu, Ava, .beta.-Ala, hSer, Nle, Nva, Pal,
Dab, and Dap stand for 2-amino-butyric acid, amino valeric acid,
beta-aminopropionic acid, homoserine, norleucine, norvaline, (2, 3,
or 4) 3-pyridyl-Ala, 1,4-diaminobutyric acid, and
1,3-diaminopropionic acid, respectively. In all aspects of the
invention, it is noted that when amino acids are not designated as
either D- or L-amino acids, the amino acid is either an L-amino
acid or could be either a D- or L-amino acid. Examples of peptides
of the invention include those peptides having the sequence of, or
a sequence substantially identical to, the sequences set forth in
SEQ ID NOs:1-3 and 5-8 and related sequences. These peptide
sequences can also be incorporated into a polypeptide, protein,
antibody, or antibody fragment.
[0045] By "reducing agent" is meant a chemical compound used to
reduce another chemical compound by donating electrons, thereby
becoming oxidized.
[0046] By "specifically binds" is meant that an agent of the
invention recognizes and binds to a target (e.g., a senescent
cell), but does not substantially recognize and bind to a
non-target (e.g., non-senescent cells), both in vivo and in a
sample, e.g., an in vitro biological sample, that includes, e.g.,
senescent cells. A desirable agent of the invention specifically
binds to senescent cells. Preferably, the agents of the invention
bind senescent cells with at least 2, 5, 10, 20, 100, or 1000 fold
greater affinity than they bind to non-senescent cells.
Alternatively, agents of the invention specifically bind to
senescent cells with a dissociation constant less than 10.sup.-6M,
more preferably less than 10.sup.-7M, 10.sup.-8M, 10.sup.-9M,
10.sup.-10 M, 10.sup.- M, or 10.sup.-12M, and most preferably less
than 10.sup.-13M, 10.sup.-14M, or 10.sup.-15M.
[0047] By "substantial sequence identity" or "substantially
identical" is meant that a nucleic acid or amino acid sequence
exhibits at least 50%, preferably 60%, 70%, 75%, or 80%, more
preferably 85%, 90% or 95%, and most preferably 99% identity to a
reference amino acid sequence (e.g., one or more of the sequences
set forth in SEQ ID NOs:1-3 and 5-8). For amino acid sequences, the
length of comparison sequences will generally be at least 5 amino
acids, preferably at least 10 contiguous amino acids, more
preferably at least 15, 20, 25, 30, 40, 50, 60, 80, 90, 100, 150,
200, 250, 300, or 350 contiguous amino acids, and most preferably
the full-length amino acid sequence. Sequence identity is typically
measured using BLAST.RTM. (Basic Local Alignment Search Tool) or
BLAST.RTM.2 with the default parameters specified therein (see,
Altschul et al., J. Mol. Biol. 215:403-410, 1990); and Tatiana et
al., FEMS Microbiol. Lett. 174:247-250, 1999). This software
program matches similar sequences by assigning degrees of homology
to various substitutions, deletions, and other modifications.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine, valine, isoleucine,
leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
[0048] By "therapeutic agent" is meant any compound that is used in
the detection, diagnosis or treatment of disease. Such compounds
may be naturally-occurring, modified, or synthetic. A therapeutic
agent may be, for example, an agent that causes apoptosis or
necrosis of a cell (e.g., a senescent cell) in an organism (e.g., a
mammal, such as a human), thereby reducing the number of such cells
in the organism. Therapeutic agents that reduce the number of
senescent cells in an organism may be, e.g., alkylating agents,
antibiotics, antimetabolites, hormonal agonists or antagonists,
anti- or pro-apoptotic agents, immunomodulators, or supplementary
potentiating agents.
[0049] By "treating, stabilizing, or preventing cancer" is meant
causing a reduction in the size of a tumor or in the number of
cancer cells, slowing or preventing an increase in the size of a
tumor or in cancer cell proliferation, increasing the disease-free
survival time between the disappearance of a tumor or other cancer
and its reappearance, preventing an initial or subsequent
occurrence of a tumor or other cancer, or reducing an adverse
symptom associated with a tumor or other cancer. In a desired
embodiment, the percent of tumor or cancerous cells surviving the
treatment is at least 20, 40, 60, 80, or 100% lower than the
initial number of tumor or cancerous cells, as measured using any
standard assay, such as those described herein. Desirably, the
decrease in the number of tumor or cancerous cells induced by
administration of a compound of the invention is at least 2, 5, 10,
20, or 50-fold greater than the decrease in the number of non-tumor
or non-cancerous cells. Desirably, the methods of the present
invention result in a decrease of 20, 40, 60, 80, or 100% in the
size of a tumor or number of cancerous cells as determined using
standard methods. Desirably, at least 20, 40, 60, 80, 90, or 95% of
the treated subjects have a complete remission in which all
evidence of the tumor or cancer disappears. Desirably, the tumor or
cancer does not reappear after more than 5, 10, 15, or 20
years.
[0050] By "treating, stabilizing, or preventing age-related
diseases" is meant causing a reduction in the number of symptoms, a
decrease in severity of any, all, or substantially all of the
symptoms, a complete resolution of any, all, or substantially all
symptoms, or preventing the occurrence of any, all, or
substantially all symptoms associated with one or more of the
age-related diseases including, but not limited to, cardiovascular
disease, cerebrovascular disease, peripheral vascular disease,
Alzheimer's disease, osteoarthritis, cardiac diastolic dysfunction,
benign prostatic hypertrophy, and cancers that increase in
incidence and prevalence with increasing patient age such as
cancer, e.g., breast cancer, prostate cancer, and colon cancer.
[0051] By "treating, stabilizing, or preventing tobacco-related
diseases" is meant causing a reduction in the number of symptoms, a
decrease in severity of any, all, or substantially all of the
symptoms, a complete resolution of any, all, or substantially all
symptoms, or preventing the occurrence of any, all, or
substantially all symptoms associated with one or more of the
diseases associated with the use of smoking tobacco as a risk
factor, including, but are not limited to, cardiovascular disease,
cerebrovascular disease, peripheral vascular disease, aortic
aneurysms, emphysema, esophageal cancer, lung cancer, and squamous
cell cancers of the head and neck. Tobacco-related diseases also
include diseases that are associated with the use of chewing
tobacco, such as squamous cell cancers of the mouth.
[0052] Other features and advantages of the invention will be
apparent from the following description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 is a graph showing the cytotoxicity of SenL (SEQ ID
NO:4) when tested on senescent fibroblasts, non-senescent
fibroblasts, and immortalized prostate epithelial cells. No cell
death was observed in immortalized epithelium.
[0054] FIG. 2 is a graph showing the results of a WST assay used to
measure cell proliferation following treatment with SenL on
senescent fibroblasts, non-senescent fibroblasts, and immortalized
prostate epithelial cells. The WST-1 assay depends on mitochondrial
dehydrogenase levels, and senescent cells have higher mitochondrial
mass than their non-senescent counterparts (Martin-Ruiz et al., J.
Biol. Chem. 279(17):17826-33, 2004). Consequently, baseline values
for senescent cells are higher than for the other cell types.
Treatment with SenL showed no significant effect on metabolic
activity for any of the three cell types and therefore did not
affect cell proliferation rates.
[0055] FIG. 3 is a graph showing the cytotoxicity of SenC (SEQ ID
NO:5) conjugated to ricin A subunit as tested in senescent
fibroblasts, non-senescent fibroblasts, and immortalized prostate
epithelial cells. Significantly more senescent cells were killed
than non-senescent cells, and no effect was observed on
immortalized epithelium.
[0056] FIGS. 4 (A and B) are fluorescent micrographs showing the
specific binding of a peptide agent of the invention. Senescent
cell binding peptide SenC (SEQ ID NO:5) was conjugated to
fluorescein and contacted with senescent fibroblasts (FIG. 4A) and
non-senescent fibroblasts (FIG. 4B). Both images were acquired
using 1/60 second exposure time. Senescent cells show perinuclear
and cytoplasmic staining, indicating significant internalization.
Only faint surface staining is visible on the non-senescent
cells.
[0057] FIGS. 5 (A and B) are immunofluorescent micrographs showing
the cell surface expression of cathepsin B on senescent cells (FIG.
5A) and lack of expression on non-senescent cells (FIG. 5B).
DETAILED DESCRIPTION OF THE INVENTION
[0058] The invention features agents of the invention (e.g.,
peptides, polypeptides, proteins, small molecules, antibodies, or
antibody fragments that target senescent cells) capable of
specifically binding to senescent cells. Thus, agents of the
invention can include, e.g., detectable labels, therapeutic agents,
chelating agents, and cytotoxic agents, and can be used in the
detection and treatment of diseases and conditions associated with
cellular senescence.
[0059] In an embodiment, agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can be used to produce
imaging agents by conjugation to a detectable label and used for
medical imaging. Images obtained using an imaging agent of the
invention specifically show tissues, organs, and structures in the
body (e.g., a human body) that contain senescent cells by means of,
e.g., a signal emitted from the location of the cells, tissues,
organs, and structures upon specific binding of the imaging agent
to the senescent cells. The signal so obtained indicates the
presence of senescent cells and allows semi-quantitative estimation
of the relative senescent cell content of tissues, organs, or
structures. Structures, tissues, or organs that contain a threshold
content of senescent cells are at an elevated risk for the
subsequent development of diseases or disorders (e.g., cancer).
Typically, a subject deemed at risk for the subsequent development
of diseases or disorders (e.g., cancer) exhibit an increase of at
least 0.5%, more preferably 1, 1.5, 2, 2.5, 5, or 10%, and more
preferably at least 15% or more in the level of senescent cells
relative to a patient that does not have the disease or disorder.
Use of one or more of the imaging agents of the invention allows
for the determination of a patient's risk for developing a disease
or disorder, such as cancer or a neurodegenerative disease.
Patients determined to have an elevated risk of developing cancer
or other diseases and disorders related to or caused by cellular
senescence by the use of the imaging agents of the invention can be
aggressively monitored, and their cellular senescence related
disease or disorder (e.g., a cancer) can thereby be detected
earlier, facilitating early treatment. Surgical prophylactic
treatment can also be administered if necessary.
[0060] One or more of the imaging agents of the invention can also
be used to determine the senescent cell content in the brain, which
may include, e.g., senescent glial cells and senescent
cerebrovascular cells, in order to predict the patient's risk of
developing, e.g., Alzheimer's disease or cerebrovascular disease.
One or more of the imaging agents of the invention can also be used
to predict the onset of vascular disease, including cardiovascular
disease, by detecting the senescent cell content in one or more
vascular structures in a patient. One or more of the imaging agents
of the invention can also be used to predict the risk of developing
emphysema by determining the senescent cell content in the lungs of
a smoker.
[0061] One or more of the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can also be used to prepare
compositions for therapeutic administration (e.g., to treat,
stabilize, inhibit the development or progression of, or prevent
age-related diseases, tobacco-related diseases, cancer,
neurodegenerative diseases, and other diseases and disorders
related to or caused by cellular senescence) by coupling of the
agents of the invention to, e.g., therapeutic and/or cytotoxic
agents. Senescent cells have been observed in skin to make up only
a fraction of the stromal cell compartment even in old donors
(Dimri et al., Proc. Nat. Acad. Sci. USA 92:9363-9367, 1995) and up
to 16% of stromal fibroblasts in patients with emphysema (Bergner,
Respiratory Res. 7:32, 2006), but senescent cells can exert
clinically significant paracrine effects even when they compose
only 10% of the content of stromal cells (Krtolica et al., Proc.
Nat. Acad. Sci. USA 98:12072-12077, 2001). Thus, eliminating
senescent cells while leaving intact the majority of the cellular
compartment from which the senescent cells originate can ablate the
harmful paracrine effects of senescent cells. The secretion of
elevated levels of collagenase and elastase and depressed levels of
collagenase inhibitors by senescent stromal cells implicates them
in diseases or conditions that feature breakdown or decrease in
structural integrity of the extra cellular matrix, including
emphysema, aortic aneurysms, vascular disease, osteoarthritis, and
skin wrinkling Elimination of some, all, or substantially all
senescent stromal cells in a patient (e.g., in a tissue or organ of
a patient) can attenuate the progression, decrease the symptoms, or
prevent the occurrence of these diseases and conditions. Senescent
stromal cells have the ability to stimulate tumorigenesis and, by
weakening the extracellular matrix, facilitate metastasis. Thus,
the elimination of some, all, or substantially all of the senescent
stromal cells in a patient (e.g., in a tissue or organ of a
patient) can decrease the risk of tumorigenesis and metastasis
(e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or
more).
[0062] The senescent cell targeting agents of the invention (e.g.,
peptides, polypeptides, proteins, small molecules, antibodies, or
antibody fragments that target senescent cells) can be prepared by
amino acid coupling using solid phase peptide synthesis (SPPS). As
is known in the art, the amino acids to be used as substrates to
form the agents of the invention are Fmoc-protected prior to
incorporation into a peptide chain (see, e.g., Chanand White, FMOC
Solid Phase Peptide Synthesis, A Practical Approach, Oxford
University Press, New York, 2003); incorporated herein by reference
in its entirety). The standard coupling techniques used to couple
the amino acids in order to form the agents of the invention are
known in the art (see, e.g., Chan and White, supra). For example, a
polyamide-Rink resin can be prepared by loading a polyamide resin
with Fmoc-Rink using chemical protocols well known in the art (see,
e.g., Chan and White, supra). The first amino acid in the peptide
sequence is coupled to the resin after removing Fmoc from the
N-terminal amine of the resin using piperidine. Once the coupling
is complete, the resin is washed and Fmoc is removed from the
coupled amino acid using piperidine. The resin is washed again, and
the next amino acid in the sequence is coupled to the previously
coupled amino acid. This process is repeated using the necessary
amino acids until the desired peptide is formed. Following the
coupling of the final amino acid, the Fmoc group is removed using
piperidine. The terminal amine can be left as a free amine, or it
can be acetylated. The peptide can be cleaved from the resin using
trifluoroacetic acid (TFA), triisopropylsilane, and water according
to techniques known in the art (see, e.g., Chan and White, supra).
The cleaved peptide is then separated from the residue by
filtration. The TFA is typically evaporated to dryness followed by
precipitation of the peptide with diethyl ether. Typically, the
final peptide product is purified using HPLC. Mass spectrometry is
used to verify that the desired peptide is obtained. The agents of
the invention (e.g., peptides, polypeptides, proteins, small
molecules, antibodies, or antibody fragments that target senescent
cells) can be readily prepared by automated solid phase peptide
synthesis using any one of a number of well-known, commercially
available automated synthesizers, such as the Applied Biosystems
ABI 433A peptide synthesizer.
[0063] The agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can be labeled for fluorescence detection
by labeling the agent with a fluorophore, such as rhodamine or
fluorescein, using techniques well known in the art (see, e.g.,
Lohse et al., Bioconj. Chem. 8:503-509, 1997). The senescent cell
targeting agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can also be labeled with a radioactive
metal or a relaxivity metal by coupling the agent to a metal
chelating agent that chelates a radioactive metal or relaxivity
metal. Examples of chelating agents include, but are not limited
to, ininocarboxylic and polyaminopolycarboxylic reactive groups,
diethylenetriaminepentaacetic acid (DTPA), and
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
The chelating agent can be coupled via its amino acid side chain
directly to the agents of the invention. Alternatively, an
intervening amino acid sequence can be coupled using SPPS to both
the agents of the invention and the chelating agent.
Senescent Cell-Specific Antigens
[0064] The inventors have discovered that senescent cells express
senescent cell-specific antigens. These antigens can be
specifically targeted and bound by the diagnostic or therapeutic
(e.g., cytotoxic) agents of the invention, as discussed herein. The
specific binding of the diagnostic or therapeutic agents of the
invention only to senescent cells and not non-senescent cells
reduces the incidence of inaccurate diagnoses (e.g., using
diagnostic agents) or bystander cell injury (e.g., resulting from
cytotoxic agents).
[0065] Senescent cell-specific antigens that can be targeted by
agents of the invention include, e.g., mutant beta-actin (SEQ ID
NO:11; GI: 28336) and beta-actin (ACTB) protein (SEQ ID NO:12; GI:
15277503), which were identified as cell-surface,
senescent-specific antigens occurring in both replicatively
senescent and stress-induced prematurely senescent cells, drug
resistance-related protein LRP (SEQ ID NO:13; GI: 1097308) and
major vault protein (MVP; SEQ ID NO:14; GI: 19913410), which were
identified as senescence-specific surface proteins in the
replicatively senescent cells only, and thyroid hormone binding
protein precursor (SEQ ID NO:15; GI: 339647), unnamed protein
product (SEQ ID NO:20; GI: 35655), prolyl 4-hydroxylase, beta
subunit precursor (P4HB; SEQ ID NO:16; GI: 20070125), chain A,
human protein disulfide isomerase (PDI; SEQ ID NO:17; GI:
159162689), electron-transfer-flavoprotein, beta polypeptide (ETFB;
SEQ ID NO:18; GI: 4503609), unnamed protein product (SEQ ID NO:21;
GI: 158257194), unnamed protein product (SEQ ID NO:22; GI:
158259937), ATP synthase, H' transporting, mitochondrial F1
complex, alpha subunit precursor (SEQ ID NO:19; GI: 4757810), and
cathepsin B (CTSB; SEQ ID NO:23), which were identified on the cell
surface of stress induced prematurely senescent cells.
[0066] Antibodies that specifically bind to the senescent
cell-specific antigens described herein are listed in Table 1.
These antibodies, and any other polypeptide, antibody, antibody
fragment, or small molecule that specifically binds a senescent
cell-specific antigen or fragment thereof, can be incorporated in a
diagnostic or therapeutic agent of the invention, as discussed
herein. In addition, the antibodies listed in Table 1 can be
modified, e.g., by humanization, according to known methods, as is
discussed below, for use in the diagnosis and treatment of disease
in a mammal, e.g., a human. Alternatively, antibodies against the
senescent cell-specific antigens described herein for use in the
diagnosis and treatment of disease in a mammal, e.g., a human, can
be produced according to methods known in the art, as is discussed
below.
TABLE-US-00001 TABLE 1 Senescent Cell-Specific SEQ ID Antigen NO:
Anti-Senescent Cell-Specific Antigen Antibodies Mutant beta-actin
11 See, e.g., Leavitt et al., Mol Cell Biol. 7:2467-2476 (1987);
Lin et al., PNAS USA 82:6995-6999 (1985). Beta-actin (ACTB) protein
12 Clone AC-15 (mouse anti-human beta-actin antibody); Clone mAbcam
8226 (mouse anti-human beta-actin antibody). Drug
resistance-related protein 13 Clone 1032 (mouse anti-human LRP
antibody); LRP rabbit anti-human LRP antibody (see, e.g., Kitazono
et al., J. Natl. Cancer Inst. 91:1647-1653 (1999)). Major vault
protein 14 Clone 1014 (mouse anti-human major vault protein (MVP)
antibody); clone 2Q431 (mouse anti-human major vault protein
antibody). Thyroid hormone binding protein 15 See, e.g., Cheng et
al., J. Biol. Chem. 262:11221-11227 precursor (1987). Prolyl
4-hydroxylase, beta subunit 16 Clone P4HB (Abcam Cat. No. ab70415;
mouse anti-human precursor (P4HB) prolyl 4-hydroxylase, beta
subunit precursor antibody). Chain A, human protein disulfide 17
Abcam Cat. No. ab48167 (rabbit anti-human PDI isomerase (PDI)
antibody); clone 1D3 (Santa Cruz Cat. No. sc-59640; mouse
anti-human PDI antibody). Electron-transfer-flavoprotein, 18 Abcam
Cat. No. ab73986 (rabbit anti-human ETFP beta polypeptide antibody.
(ETFP) ATP synthase, H.sup.+ transporting, 19 Clone 15H4 (Abcam
Cat. No. ab14748; mouse anti- mitochondrial F1 complex, human
ATP5A); see also, e.g., Vantourout et al., alpha subunit precursor
Mol. Immunol. 45:485492 (2008). Unnamed protein 20 See, e.g.,
Pihlajaniemi et al., EMBO J. 6:643-649 product (GI: 35655) (1987).
Unnamed protein product 21 See, e.g., Altschul et al., Nucleic
Acids Res. 25:3389-3402 (GI: 158257194) (1997) Unnamed protein
product 22 See e.g., Heidebrecht et al., Mol Cancer Res. 1:271-9
(2003) (GI: 158259937) Cathepsin B (CTSB) 23 Clone ZZ12 (mouse
anti-human cathepsin B antibody); clone S-12 (goat anti-human
cathepsin B antibody).
Small Molecules of the Invention
[0067] The invention also features small molecules that can be used
diagnostically or therapeutically based on their ability to
specifically bind senescent cells. For example, small molecules of
the invention include those capable of specifically binding to one
or more of the senescent cell-specific antigens described herein
and those capable of mimicking the physical, chemical, biological,
and/or targeting characteristics of SEQ ID NOs:1-3 and 5-8 and
peptides that are substantially identical. Small molecules of the
invention can be labeled or fused to diagnostic or therapeutic
linkers, markers, cytotoxic agents, or other agents of the
invention to aid in the diagnosis or treatment of diseases or
disorders related to or caused by cellular senescence. Furthermore,
small molecules of the invention can specifically bind a
polypeptide that has at least 80% (80%, 90%, 95%, 99%, or 100%)
amino acid sequence identity to any one of the amino acid sequences
set forth in SEQ ID NOS:11-23, or a fragment thereof.
Methods of Screening Small Molecules for Binding to Senescent
Cells
[0068] The invention also features methods for the high throughput
screening (HTS) of candidate small molecule agents of the invention
for their ability to bind senescent cells or senescent
cell-specific antigens, including, but not limited to, the
polypeptides set forth in SEQ ID NOS:11-23, or fragments thereof.
Candidate small molecules will also be screened for their ability
to specifically bind senescent cells. In general, candidate small
molecules preferably bind target sequences with a dissociation
constant less than 10.sup.-6 M for further consideration as an
agent of the invention.
[0069] Senescent cells of any origin can be used in HTS binding
assays and methods. In general, fluorescence and luminescence based
assays (e.g., ELISA, colorimetric assays) are used to measure
binding affinities of candidate small molecules contacted against
single or multiple target senescent cells. Upon the identification
of a candidate small molecule from a first screening process,
further scrutiny of the binding affinity and ability of the
candidate small molecule can be by means of a second, different HTS
assay. This could be accomplished, for example, by contacting the
candidate small molecule with alternate senescent cell populations
to more precisely determine the binding affinity of the molecule. A
discussion of HTS methodologies is found in, e.g., Verkman, "Drug
discovery in academia," Am. J. Physiol. Cell Physiol. 286,
C465-C474 (2004) and Dove, "Screening for content--the evolution of
high throughput," Nat. Biotechnol. 21:859-864 (2003). Examples of
HTS screening methods for the discovery of useful small molecule
agents are found in, e.g., U.S. Pat. Nos. 7,279,286 and 7,276,346,
which are incorporated by reference herein.
[0070] Candidate small molecules that have undergone HTS screening
may be further modified to empirically improve senescent cell
binding affinities according to the design considerations discussed
below.
Small Molecule Design
[0071] Small molecules of the invention can also be generated
according to the principles of rational design. Computer modeling
technology allows visualization of the three-dimensional atomic
structure of a selected molecule and the design of new compounds
that will interact with senescent cells or senescent cell-specific
antigens (e.g., the senescent cell-specific antigens discussed
above (set forth in SEQ ID NOS:11-23), or fragments thereof). The
three-dimensional construct typically depends on data from x-ray
crystallographic analyses or NMR imaging of the selected molecule
or epitope. A computer graphics system enables prediction of how a
candidate small molecule compound will bind to the target senescent
cell and allows experimental manipulation of the structures of the
small molecule and target protein to perfect binding specificity. A
prediction of what the molecule-protein interaction will be when
small changes are made in one or both can be determined by using
molecular mechanics software and computationally intensive
computers. An example of a molecular modeling system described
generally above includes the CHARMm and QUANTA programs (Polygen
Corporation, Waltham, Mass.). CHARMm performs the energy
minimization and molecular dynamics functions, while QUANTA
performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules that intact with each other. Another molecular modeling
program that can be used to identify small molecules for use in the
methods of the invention is DOCK (Kuntz Laboratory, UCSF).
Small Molecule Synthesis
[0072] Small molecules of the invention can be organic or inorganic
compounds, and even nucleic acids. Specific binding to senescent
cells can be achieved by including chemical groups having the
correct spatial location and charge in the small molecule. In a
preferred embodiment, compounds are designed with hydrogen bond
donor and acceptor sites arranged to be complementary to the
targeted molecule or epitope. An agent is formed with chemical side
groups ordered to yield the correct spatial arrangement of hydrogen
bond acceptors and donors when the agent is in a specific
conformation induced and stabilized by binding to the target
molecule or epitope. Additional binding forces such as ionic bonds
and Van der Waals interactions can also be considered when
synthesizing a small molecule of the invention. The likelihood of
forming the desired conformation can be refined and/or optimized
using molecular computational programs.
[0073] Organic compounds can be designed to be rigid, or to present
hydrogen bonding groups on edge or plane, which can interact with
complementary sites. Rebek, Science 235, 1478-1484 (1987) and Rebek
et al., J. Am. Chem. Soc. 109, 2426-2431 (1987) have summarized
these approaches and the mechanisms involved in binding of
compounds to regions of proteins.
[0074] Synthetic methods can be used by one skilled in the art to
make small molecules that interact with functional groups of
proteins, glycoproteins, or epitopes present on the exterior or in
the interior of senescent cells.
Preparation of Antibody Compositions of the Invention
[0075] The invention also provides antibody compositions that
include one or more of the sequences set forth in SEQ ID NOS:1-3
and 5-8, and peptides substantially identical thereto, which can be
included in, e.g., one or more of the complementarity determining
regions (CDRs) of the antibody compositions. The invention further
provides antibody compositions that specifically bind a senescent
cell-specific antigen having at least 80% (e.g., 80%, 90%, 95%,
99%, or 100%) amino acid sequence identity to any one of the
senescent cell-specific antigens set forth in SEQ ID NOS:11-23, or
a fragment thereof.
[0076] The antibodies of the invention may be recombinant (e.g.,
chimeric or humanized), synthetic, or naturally occurring.
Antibodies or antibody fragments of the invention can be
recombinantly produced, or identified and isolated from a mammal
that has been induced to produce or that naturally-produces
antibodies that specifically bind to senescent cell-specific
antigens. For example, a polypeptide having at least 80% (e.g.,
85%, 90%, 95%, 99%, or 100%) amino acid sequence identity to one of
the senescent cell-specific antigens set forth in SEQ ID NOS:11-23
can be administered (e.g., by injection) to a mammal (e.g., a
mouse, rat, hamster, guinea pig, sheep, goat, cow, horse, non-human
primate, or human) to provoke a humoral immune response against the
immunizing senescent cell-specific antigen. Blood, plasma, or
ascites from the immunized mammal can be screened to identify or
harvest antibodies that specifically bind the immunizing
senenescent cell-specific antigen. If desired, the amino acid
and/or nucleic acid sequences of the antibodies produced can be
determined. Furthermore, all or a portion of the nucleic acid
sequences of the antibodies can be incorporated into a vector
(e.g., an expression vector or a viral vector) for expression of
the antibody in a cell of a subject (e.g., a mammal, such as a
human) following administration of the vector to the subject, or
any other uses consistent with the methods described herein.
[0077] The invention features complete antibodies, diabodies,
bi-specific antibodies, antibody fragments, Fab fragments, F(ab')2
molecules, single chain Fv (scFv) molecules, tandem scFv molecules,
or antibody fusion proteins. Antibodies of the invention include,
e.g., the IgG, IgA, IgM, IgD, and IgE isotypes. Antibodies of the
invention contain one or more CDRs or binding peptides that bind to
proteins, glycoproteins, or epitopes present on the exterior or in
the interior of senescent cells.
[0078] Many of the antibodies, or fragments thereof, described
herein can undergo non-critical amino-acid substitutions, additions
or deletions in both the variable and constant regions without loss
of binding specificity or effector functions, or intolerable
reduction of binding affinity (i.e., below about 10.sup.-7 M).
Usually, an antibody incorporating such alterations exhibits
substantial sequence identity to a reference antibody from which it
is derived. Occasionally, a mutated antibody can be selected having
the same specificity and increased affinity compared with a
reference antibody from which it was derived. Phage-display
technology offers powerful techniques for selecting such
antibodies. See, e.g., Dower et al. (WO 91/17271), McCafferty et
al. (WO 92/01047), and Huse (WO 92/06204), each of which is
incorporated by reference herein.
Antibody Fragments
[0079] In another embodiment of the invention, an agent of the
invention is a fragment of an intact antibody described herein.
Antibody fragments include separate variable heavy chains, variable
light chains, Fab, Fab', F(ab').sub.2, Fabc, and Fv. Fragments can
be produced by enzymatic or chemical separation of intact
immunoglobulins. For example, a F(ab').sub.2 fragment can be
obtained from an IgG molecule by proteolytic digestion with pepsin
at pH 3.0-3.5 using standard methods such as those described in
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Pubs., N.Y. (1988). Fab fragments may be obtained from
F(ab').sub.2 fragments by limited reduction, or from whole antibody
by digestion with papain in the presence of reducing agents.
Fragments can also be produced by recombinant DNA techniques.
Segments of nucleic acids encoding selected fragments are produced
by digestion of full-length coding sequences with restriction
enzymes, or by de novo synthesis. Often fragments are expressed in
the form of phage-coat fusion proteins. This manner of expression
is advantageous for affinity-sharpening of antibodies.
Humanized Antibodies
[0080] The invention also includes humanized antibodies in which
one or more of the CDRs are derived from a non-human antibody
sequence (e.g., those antibodies described in Table 1), and one or
more, but preferably all, of the CDRs bind specifically to a
protein, glycoprotein, or epitope present on the exterior or in the
interior of a senescent cell (e.g., the senescent cell-specific
antigens described above (SEQ ID NOS:11-23), or a fragment
thereof).
[0081] A humanized antibody contains constant framework regions
derived substantially from a human antibody (termed an acceptor
antibody), as well as, in some instances, a majority of the
variable region derived from a human antibody. One or more of the
CDRs (all or a portion thereof, as well as discreet amino acids
surrounding one or more of the CDRs) are provided from a non-human
antibody, such as a mouse antibody. The constant region(s) of the
antibody, may or may not be present. Humanized antibodies provide
several advantages over non-humanized antibodies for therapeutic or
diagnostic use in humans. These include: [0082] 1) The human immune
system should not recognize the framework or constant region of the
humanized antibody as foreign, and therefore the antibody response
against such an injected antibody should be less than against a
totally foreign mouse antibody or a partially foreign chimeric
antibody;
[0083] 2) Because the effector portion of the humanized antibody is
human, it may interact better with other parts of the human immune
system; and [0084] 3) Injected mouse antibodies have been reported
to have a much shorter half-life in the human circulation than the
half-life exhibited by normal human antibodies (see, e.g., Shaw et
al., J. Immunol. 138:4534-4538 (1987)). Injected humanized
antibodies have a half-life essentially equivalent to naturally
occurring human antibodies, allowing smaller and less frequent
doses.
[0085] The substitution of one or more, e.g., mouse CDRs into a
human variable domain framework is most likely to result in
retention of their correct spatial orientation if the human
variable domain framework adopts the same or similar conformation
to the mouse variable framework from which the CDRs originated.
This is achieved by obtaining the human variable domains from human
antibodies whose framework sequences exhibit a high degree of
sequence identity with the murine variable framework domains from
which the CDRs were derived. The heavy and light chain variable
framework regions can be derived from the same or different human
antibody sequences. The human antibody sequences can be the
sequences of naturally occurring human antibodies or can be
consensus sequences of several human antibodies. See, e.g.,
Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger
et al., Protein Engineering 6:971 (1993).
[0086] Suitable human antibody sequences are identified by computer
comparisons of the amino acid sequences of the mouse variable
regions with the sequences of known human antibodies. The
comparison is performed separately for heavy and light chains but
the principles are similar for each.
[0087] Methods of preparing chimeric and humanized antibodies and
antibody fragments are described in U.S. Pat. Nos. 4,816,567,
5,530,101, 5,622,701, 5,800,815, 5,874,540, 5,914,110, 5,928,904,
6,210,670, 6,677,436, and 7,067,313 and U.S. Patent Application
Nos. 2002/0031508, 2004/0265311, and 2005/0226876. Preparation of
antibody or fragments thereof is further described in U.S. Pat.
Nos. 6,331,415, 6,818,216, and 7,067,313. Each of these patents is
incorporated herein by reference.
Diagnostic Agents Coupled to Agents of the Invention
[0088] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can be joined or coupled to any molecule
that can be used to label, detect, identify, screen, or diagnose
senescent cells in a mammal (e.g., a human). Diagnostic and
therapeutic (e.g., cytotoxic) agents of the invention that include
a detectable label can be administered in a pharmaceutical compound
to a subject (e.g., a cancer patient) or these compositions can be
encoded by a vector (e.g., a viral vector) and expressed in a cell
of the subject following administration.
[0089] An agent of the invention that incorporates a detectable
label can be used, for example, to type, grade, and track the
metastasis of a tumor in a subject having cancer. The diagnostic
agent can also include a therapeutic agent (e.g., cytotoxic) of the
invention. In this case, the diagnostic agent facilitates the
detection or measurement, or binding to senescent cells, of the
therapeutic agent after administration to a subject.
[0090] A diagnostic or therapeutic agent of the invention that
includes a detectable label can be produced recombinantly in vitro.
In one embodiment of the invention, the detectable label is capable
of detection based on an intrinsic property, e.g., fluorescence and
bioluminescence, or based on its ability to bind with another
molecule capable of being detected (e.g., an epitope tag, such as
c-myc, hemagglutinin, and histamine tags). Peptides, polypeptides,
or proteins with diagnostic properties can be altered (e.g., by
making amino acid substitutions, mutations, truncations, or
additions) to facilitate incorporation into an agent of the
invention. Desirable alterations include, for example, changes to
the amino acid sequence that facilitate protein expression,
longevity, cell secretion, and detectability.
[0091] The invention also features a nucleic acid molecule encoding
a peptidic detectable label as a fusion protein with an agent of
the invention; the nucleic acid molecule can be incorporated into a
vector (e.g., an expression vector), such that, upon expression of
the agent of the invention in a cell transfected or transduced with
the vector, expression of the detectable label and agent of the
invention are operably linked (e.g., fused, contiguously-joined, or
tethered together).
[0092] Molecules that can be used as the detectable label as
discussed herein include, but are not limited to, radioactive
agents, fluorescent agents, bioluminescent molecules, epitope tags,
and heavy metals, each of which is discussed in detail below.
[0093] Fluorescent agents include fluorochromes, such as
fluorescein isothiocyanate (FITC), allophycocyanin (APC),
phycoerythrin (PE), rhodamine, and tetramethyl rhodamine
isothiocyanate (TRITC). Other fluorescent molecules include green
fluorescent protein (GFP; SEQ ID NO: 27), enhanced GFP (eGFP),
yellow fluorescent protein (SEQ ID NO: 28; YFP), cyan fluorescent
protein (SEQ ID NO: 29; CFP), and red fluorescent protein (SEQ ID
NO: 30; RFP or DsRed). Each of these fluorescent molecules can be
used as detectable label in a diagnostic agent of the invention.
Peptidic fluorescent molecules can be recombinantly expressed in a
cell (e.g., a blood cell, such as a lymphocyte) following
transfection or transduction of the cell with an expression vector
that encodes the nucleotide sequence of the fluorescent molecule.
Upon exposure of the fluorescent molecule to a stimulating
frequency of light, the fluorescent molecule will emit light at a
low, medium, or high intensity that can be observed by eye under a
microscope or by an optical imaging device. Exemplary fluorescent
molecules suitable for use as the detectable label in a diagnostic
agent of the invention are described in, e.g., U.S. Pat. Nos.
7,417,131 and 7,413,874.
[0094] Bioluminescent molecules can also be used as a detectable
label incorporated into a diagnostic agent of the invention.
Bioluminescent molecules, such as luciferase (e.g., firefly (SEQ ID
NO: 31), Renilla (SEQ ID NO: 32), and Omphalotus luciferase) and
aequorin, emit light as part of a chemical reaction with a
substrate (e.g., luciferin and coelenterazine). In one embodiment
of the invention, a vector encoding a luciferase gene provides for
the in vivo, in vitro, or ex vivo detection of cells (e.g., blood
cells, such as lymphocytes) that have been transduced or
transfected with an agent of the invention. Exemplary
bioluminescent molecules suitable for use as the detectable label
in a diagnostic agent of the invention, and methods for their use
are described in, e.g., U.S. Pat. Nos. 5,292,658, 5,670,356,
6,171,809, and 7,183,092.
[0095] Epitope tags are short amino acid sequences, e.g., 5-20
amino acid residues in length, that can be incorporated into an
agent of the invention as a detectable label to allow for detection
once expressed in a cell, secreted from the cell, or bound to a
target cell (e.g., a senescent cell). An agent of the invention
that incorporates an epitope tag as a diagnostic agent can be
detected by virtue of its interaction with an antibody, antibody
fragment, or other binding molecule specific for the epitope tag.
Nucleotide sequences encoding the epitope tag are produced either
by cloning appropriate portions of natural genes or by synthesizing
a polynucleotide that encodes the epitope tag. An antibody,
antibody fragment, or other binding molecule that binds an epitope
tag can directly incorporate its own detectable label (e.g., a
fluorochrome, radiolabel, heavy metal, or enzyme such as
horseradish peroxidase) or serve as a target for a secondary
antibody, antibody fragment, or other binding molecule that
incorporates such a label. Exemplary epitope tags that can be used
as a detectable label include c-myc (SEQ ID NO: 24), hemagglutinin
(HA; SEQ ID NO: 25), and histidine tag (His.sub.6; SEQ ID NO: 26).
Furthermore, fluorescent (e.g., GFP) and bioluminescent molecules,
discussed above, can also serve as epitope tags, as antibodies,
antibody fragments, and other binding molecules are commercially
available for the detection of these moieties.
[0096] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can also be coupled to a chelating agent to
form diagnostic agents of the invention. Diagnostic agents can be
prepared by various methods depending upon the chelator chosen. A
portion of an agent of the invention (e.g., a portion of a peptide,
polypeptide, protein, small molecule, antibody, or antibody
fragment) can be isolated from a natural source, recombinantly
produced, or synthesized. The portion of an agent of the invention
is most conveniently prepared by techniques generally established
in the art of peptide synthesis, such as the solid-phase approach.
Solid-phase synthesis involves the stepwise addition of amino acid
residues to a growing peptide chain that is linked to an insoluble
support or matrix, such as polystyrene. The C-terminus residue of
the peptide is first anchored to a commercially available support
with its amino group protected with an N-protecting agent such as a
t-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl
(FMOC) group. The amino-protecting group is removed with suitable
deprotecting agents such as TFA in the case of tBOC or piperidine
for FMOC and the next amino acid residue (in N-protected form) is
added with a coupling agent such as dicyclocarbodiimide (DCC). Upon
formation of a peptide bond, the reagents are washed from the
support. After addition of the final residue, the agent is cleaved
from the support with a suitable reagent, such as trifluoroacetic
acid (TFA) or hydrogen fluoride (HF).
[0097] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) and chelator components can be coupled to
form a conjugate by reacting the free amino group of the threonine
residue of a peptide portion of an agent of the invention with an
appropriate functional group of the chelator, such as a carboxyl
group or activated ester. For example, a conjugate may incorporate
the chelator ethylenediaminetetraacetic acid (EDTA), common in the
art of coordination chemistry, when functionalized with a carboxyl
substituent on the ethylene chain. Synthesis of EDTA derivatives of
this type are reported in Arya et al., Bioconjugate Chemistry,
2:323, 1991), wherein the four coordinating carboxyl groups are
each blocked with a t-butyl group while the carboxyl substituent on
the ethylene chain is free to react with the amino group of a
peptide portion of the agent of the invention, thereby forming a
conjugate.
[0098] A conjugate may incorporate a metal chelator component that
is peptidic, i.e., compatible with solid-phase peptide synthesis.
In this case, the chelator may be coupled to the agent of the
invention (e.g., peptides, polypeptides, proteins, small molecules,
antibodies, or antibody fragments that target senescent cells) in
the same manner as EDTA described above or more conveniently the
chelator and agent of the invention are synthesized in toto
starting from the C-terminal residue of the agent and ending with
the N-terminal residue of the chelator.
[0099] Conjugates may further incorporate a linking group component
that serves to couple the agent of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) to the chelator while not
adversely affecting either the targeting function of the agent or
the metal binding function of the chelator. Suitable linking groups
include amino acid chains and alkyl chains functionalized with
reactive groups for coupling to both the agent and the chelator. An
amino acid chain is the preferred linking group when the chelator
is peptidic so that the conjugate can be synthesized in toto by
solid-phase techniques.
[0100] An alkyl chain linking group may be incorporated in the
conjugate by reacting the amino group of the threonine residue of a
peptide portion of an agent of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) with a first functional
group on the alkyl chain, such as a carboxyl group or an activated
ester. Subsequently the chelator is attached to the alkyl chain to
complete the formation of the conjugate by reacting a second
functional group on the alkyl chain with an appropriate group on
the chelator. The second functional group on the alkyl chain is
selected from substituents that are reactive with a functional
group on the chelator while not being reactive with the threonine
residue of the agent. For example, when the chelator incorporates a
functional group such as a carboxyl group or an activated ester,
the second functional group of the alkyl chain linking group can be
an amino group. It will be appreciated that formation of the
conjugate may require protection and deprotection of the functional
groups present in order to avoid formation of undesired products.
Protection and deprotection are accomplished using protecting
groups, reagents, and protocols common in the art of organic
synthesis. Particularly, protection and deprotection techniques
employed in solid phase peptide synthesis described above may be
used.
[0101] An alternative chemical linking group to an alkyl chain is
polyethylene glycol (PEG), which is functionalized in the same
manner as the alkyl chain described above for incorporation in the
conjugates. It will be appreciated that linking groups may
alternatively be coupled first to the chelator and then to the
agent of the invention (e.g., peptides, polypeptides, proteins,
small molecules, antibodies, or antibody fragments that target
senescent cells).
[0102] In accordance with one aspect of the invention,
agent-chelator conjugates of the invention incorporate a
diagnostically useful metal capable of forming a complex. Suitable
metals include, e.g., radionuclides, such as technetium and rhenium
in their various forms (e.g., .sup.99 mTCO.sup.3+, .sup.99
mTCO.sub.2.sup.+, ReO.sup.3+, and ReO.sub.2.sup.+). Incorporation
of the metal within the conjugate can be achieved by various
methods common in the art of coordination chemistry. When the metal
is technetium-99 m, the following general procedure may be used to
form a technetium complex. An agent-chelator conjugate solution is
formed initially by dissolving the conjugate in aqueous alcohol
such as ethanol. The solution is then degassed to remove oxygen
then thiol-protecting groups are removed with a suitable reagent,
for example, with sodium hydroxide, and then neutralized with an
organic acid, such as acetic acid (pH 6.0-6.5). In the labeling
step, a stoichiometric excess of sodium pertechnetate, obtained
from a molybdenum generator, is added to a solution of the
conjugate with an amount of a reducing agent such as stannous
chloride sufficient to reduce technetium and heated. The labeled
conjugate may be separated from contaminants .sup.99
mTcO.sub.4.sup.- and colloidal .sup.99 mTcO.sub.2
chromatographically, for example, with a C-18 Sep Pak
cartridge.
[0103] In an alternative method, labeling of the agent of the
invention can be accomplished by a transchelation reaction. The
technetium source is a solution of technetium complexed with labile
ligands facilitating ligand exchange with the selected chelator.
Suitable ligands for transchelation include tartarate, citrate, and
heptagluconate. In this instance the preferred reducing reagent is
sodium dithionite. It will be appreciated that the conjugate may be
labeled using the techniques described above, or alternatively the
chelator itself may be labeled and subsequently coupled to the
agent of the invention to form the conjugate; a process referred to
as the "prelabeled ligand" method.
[0104] Another approach for labeling agents of the invention (e.g.,
peptides, polypeptides, proteins, small molecules, antibodies, or
antibody fragments that target senescent cells) involves
immobilizing the agent-chelator conjugate on a solid-phase support
through a linkage that is cleaved upon metal chelation. This is
achieved when the chelator is coupled to a functional group of the
support by one of the complexing atoms. Preferably, a complexing
sulfur atom is coupled to the support which is functionalized with
a sulfur protecting group such as maleimide.
[0105] When labeled with a diagnostically useful metal,
agent-chelator conjugates of the invention can be used to detect
tissue at risk of developing cancer (e.g., lung cancer, breast
cancer, colon cancer, and prostate cancer), age-related diseases
(e.g., cardiovascular disease, cerebrovascular disease, or
Alzheimer's disease), tobacco-related diseases (e.g., emphysema,
aortic aneurysms, esophageal cancer, or squamous cell cancer of the
head and neck), or other diseases and disorders relating to or
caused by cellular senescence by procedures established in the art
of diagnostic imaging. An agent labeled with a radionuclide metal,
such as technetium-99 m, may be administered to a mammal (e.g., a
human) by intravenous injection in a pharmaceutically acceptable
solution such as isotonic saline, or by other methods described
herein. The amount of a labeled agent of the invention (e.g.,
peptides, polypeptides, proteins, small molecules, antibodies, or
antibody fragments that target senescent cells) appropriate for
administration is dependent upon the distribution profile of the
chosen conjugate in the sense that a rapidly cleared agent may be
administered at higher doses than one that clears less rapidly.
Unit doses acceptable for imaging tissues that contain senescent
cells are in the range of, e.g., 5-40 mCi for a 70 kg individual.
In vivo distribution and localization can be tracked by standard
techniques described herein at an appropriate time subsequent to
administration; typically between 30 minutes and 180 minutes
depending upon the rate of accumulation at the target site with
respect to the rate of clearance at non-target tissue.
Therapeutic or Cytotoxic Agents Coupled to Agents of the
Invention
[0106] Any of the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can be coupled to any known
cytotoxic or therapeutic moiety. Examples include, e.g.,
antineoplastic agents such as: Acivicin; Aclarubicin; Acodazole
Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin;
Altretamine; Ambomycin; A. metantrone Acetate; Aminoglutethimide;
Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;
Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;
Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;
Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;
Busulfan; Cactinomycin; Calusterone; Camptothecin; Caracemide;
Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride;
Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin;
Cladribine; Combretestatin A-4; Crisnatol Mesylate;
Cyclophosphamide; Cytarabine; Dacarbazine; DACA
(N-[2-(Dimethyl-amino) ethyl] acridine-4-carboxamide);
Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine;
Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone;
Docetaxel; Dolasatins; Doxorubicin; Doxorubicin Hydrochloride;
Droloxifene; Droloxifene Citrate; Dromostanolone Propionate;
Duazomycin; Edatrexate; Eflornithine Hydrochloride; Ellipticine;
Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin
Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine;
Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131;
Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride;
Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate;
Fluorouracil; 5-FdUMP; Fluorocitabine; Fosquidone; Fostriecin
Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;
Homocamptothecin; Hydroxyurea; Idarubicin Hydrochloride;
Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b;
Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a;
Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride;
Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole
Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone
Hydrochloride; Masoprocol; Maytansine; Mechlorethamine
Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;
Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine; PeploycinSulfate; Perfosfamide; Pipobroman;
Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin;
Rhizoxin; Rhizoxin D; Riboprine; Rogletimide; Safingol; Safingol
Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;
Sparsomycin; Spirogermanium Hydrochloride; Spiromustine;
Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89;
Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur;
Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;
Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq;
Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride;
Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate;
Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole
Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin;
Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate;
Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate
Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine
Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;
Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2' Deoxyformycin;
9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic
acid; 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine;
2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;
CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor
ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide;
busulfan; N-methyl-Nnitrosourea (MNU);
N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N'
cyclohexyl-N-nitrosourea (CCNU);
N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea
(MeCCNU); N-(2-chloroethyl)-N'-(diethyl)
ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin;
diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa;
mitomycin C; AZQ; adozelesin; Cisplatin; Carboplatin; Ormaplatin;
Oxaliplatin; C1-973; DWA 2114R; JM216; JM335; Bis (platinum);
tomudex; azacitidine; cytarabine; gemcitabine; 6-Mercaptopurine;
6-Thioguanine; Hypoxanthine; teniposide 9-amino camptothecin;
Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin;
mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);
amsacrine; pyrazoloacridine; all-trans retinol;
14-hydroxy-retro-retinol; all-trans retinoic acid;
N-(4-Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl
TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or
2-chlorodeoxyadenosine (2-Cda).
[0107] Other therapeutic compounds include, but are not limited to,
20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;
aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin
1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine;
baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2;
bropirimine; budotitane; buthionine sulfoximine; calcipotriol;
calphostin C; camptothecin derivatives (e.g.,
10-hydroxy-camptothecin); canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'
deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane;
dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; discodermolide; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epothilones (A, R=H; B, R=Me); epithilones;
epristeride; estramustine analogue; estrogen agonists; estrogen
antagonists; etanidazole; etoposide; etoposide 4'-phosphate
(etopofos); exemestane; fadrozole; fazarabine; fenretinide;
filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;
fludarabine; fluorodaunorunicin hydrochloride; forfenimex;
formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium
nitrate; galocitabine; ganirelix; gelatinase inhibitors;
gemcitabine; glutathione inhibitors; hepsulfam; heregulin;
hexamethylene bisacetamide; homoharringtonine (HHT); hypericin;
ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;
insulin-like growth factor-1 receptor inhibitor; interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin;
ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole;
isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maytansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; rnerbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; ifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mithracin; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; podophyllotoxin; porfimer
sodium; porfiromycin; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thalidomide; thiocoraline; thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist;
thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine; titanocene dichloride; topotecan; topsentin;
toremifene; totipotent stem cell factor; translation inhibitors;
tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived
growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin B; vector system, erythrocyte gene therapy;
velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;
vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and
zinostatin stimalamer.
[0108] One or more of the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can also be coupled to a
lytic peptide. Such lytic peptides induce cell death and include,
but are not limited to, streptolysin O; stoichactis toxin;
phallolysin; staphylococcus alpha toxin; holothurin A; digitonin;
melittin; lysolecithin; cardiotoxin; and cerebratulus A toxin (Kem
et al., J. Biol. Chem. 253(16):5752-5757, 1978). Agents of the
invention can also be coupled to a synthetic peptide that shares
some sequence homology or chemical characteristics with any of the
naturally occurring peptide lysins; such characteristics include,
but are not limited to, linearity, positive charge, amphipathicity,
and formation of alpha-helical structures in a hydrophobic
environment (Leuschner et al., Biology of Reproduction 73:860-865,
2005). Agents of the invention can also be coupled to an agent that
induces complement-mediated cell lysis such as, for example, the
immunoglobulin F.sub.c subunit. Agents of the invention can also be
coupled to any member of the phospholipase family of enzymes
(including phospholipase A, phospholipase B, phospholipase C, or
phospholipase D) or to a catalytically-active subunit thereof.
[0109] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, or antibody fragments that target
senescent cells) can also be coupled to a radioactive agent,
including, but not limited to: Fibrinogen .sup.125I;
Fludeoxyglucose .sup.18F; Fluorodopa .sup.18F; Insulin .sup.125I;
Insulin .sup.131I; Iobenguane .sup.123I; Iodipamide Sodium
.sup.131I; Iodoantipyrine .sup.131I; Iodocholesterol .sup.131I;
Iodohippurate Sodium .sup.123I; Iodohippurate Sodium .sup.125I;
Iodohippurate Sodium .sup.131I; Iodopyracet .sup.125I; Iodopyracet
.sup.131I; Iofetamine Hydrochloride .sup.123I; Iomethin .sup.125I;
Iomethin .sup.131I; Iothalamate Sodium .sup.125I; Iothalamate
Sodium .sup.131I; tyrosine .sup.131I; Liothyronine .sup.125I;
Liothyronine .sup.131I; Merisoprol Acetate .sup.197Hg; Merisoprol
Acetate .sup.203Hg; Merisoprol .sup.197Hg; Selenomethionine
.sup.75Se; Technetium .sup.99mTc Antimony Trisulfide Colloid;
Technetium .sup.99mTc Bicisate; Technetium .sup.99mTc Disofenin;
Technetium .sup.99mTc Etidronate; Technetium .sup.99mTc
Exametazine; Technetium .sup.99mTc Furifosmin; Technetium
.sup.99mTc Gluceptate; Technetium .sup.99mTc Lidofenin; Technetium
.sup.99mTc Mebrofenin; Technetium .sup.99mTc Medronate; Technetium
.sup.99mTc Medronate Disodium; Technetium .sup.99mTc Mertiatide;
Technetium .sup.99mTc Oxidronate; Technetium .sup.99mTc Pentetate;
Technetium .sup.99mTc Pentetate Calcium Trisodium; Technetium
.sup.99mTc Sestamibi; Technetium .sup.99mTc Siboroxime; Technetium
.sup.99mTc; Succimer; Technetium .sup.99mTc Sulfur Colloid;
Technetium .sup.99mTc Teboroxime; Technetium .sup.99mTc
Tetrofosmin; Technetium .sup.99mTc Tiatide; Thyroxine .sup.125I;
Thyroxine .sup.131I; Tolpovidone .sup.131I; Triolein .sup.125I; or
Triolein .sup.131I.
[0110] Therapeutic or cytotoxic agents may further include, for
example, anti-cancer Supplementary Potentiating Agents, including,
but not limited to: Tricyclic anti-depressant drugs (e.g.,
imipramine, desipramine, amitryptyline, clomipramine, trimipramine,
doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline);
non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone,
and citalopram); Ca.sup.2+ antagonists (e.g., verapamil,
nifedipine, nitrendipine, and caroverine); Calmodulin inhibitors
(e.g., prenylamine, trifluoroperazine, and clomipramine);
Amphotericin B; Triparanol analogs (e.g., tamoxifen);
antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs
(e.g., reserpine); Thiol depleters (e.g., buthionine and
sulfoximine) and Multiple Drug Resistance reducing agents such as
Cremaphor EL.
[0111] The agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can also be administered with cytokines,
such as granulocyte colony stimulating factor, or anticancer agents
used in anti-cancer cocktails including, e.g., those shown with
their MTDs in parentheses: gemcitabine (1000 mg/m.sup.2);
methotrexate (15 gm/m.sup.2 i.v.+leuco.<500 mg/m.sup.2 i.v. w/o
leuco); 5-FU (500 mg/m.sup.2/day.times.5 days); FUDR (100
mg/kg.times.5 in mice, 0.6 mg/kg/day in human i.a.); FdUMP;
Hydroxyurea (35 mg/kg/d in man); Docetaxel (60-100 mg/m.sup.2);
discodermolide; epothilones; vincristine (1.4 mg/m.sup.2);
vinblastine (escalating: 3.3-11.1 mg/m.sup.2, or rarely to 18.5
mg/m.sup.2); vinorelbine (30 mg/m.sup.2/wk); meta-pac; irinotecan
(50-150 mg/m.sup.2, 1.times./wk depending on patient response);
SN-38 (-100 times more potent than Irinotecan); 10-OH campto;
topotecan (1.5 mg/m.sup.2/day in humans, 1.times.iv LD1Omice=75
mg/m.sup.2); etoposide (100 mg/m.sup.2 in man); adriamycin;
flavopiridol; Cis-Pt (100 mg/m.sup.2 in man); carbo-Pt (360
mg/m.sup.2 in man); bleomycin (20 mg/m2); mitomycin C (20
mg/m.sup.2); mithramycin (30 sug/kg); capecitabine (2.5 g/m.sup.2
orally); cytarabine (100 mg/m.sup.2/day); 2-C1-2'deoxyadenosine;
Fludarabine-P04 (25 mg/m.sup.2/day, .times.5 days); mitoxantrone
(12-14 mg/m.sup.2); mitozolomide (>400 mg/m.sup.2); Pentostatin;
or Tomudex.
[0112] Any of the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can be coupled to an
antimetabolic agent. Antimetabolic agents include, but are not
limited to, the following compounds and their derivatives:
azathioprine, cladribine, cytarabine, dacarbazine, fludarabine
phosphate, fluorouracil, gencitabine chlorhydrate, mercaptopurine,
methotrexate, mitobronitol, mitotane, proguanil chlorohydrate,
pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane,
vinblastine sulfate, vincristine sulfate, etc. In other
embodiments, the agents of the invention can be coupled to a folic
acid-type antimetabolite, a class of agents that includes, for
example, methotrexate, proguanil chlorhydrate, pyrimethanime,
trimethoprime, or trimetrexate glucuronate, or derivatives of these
compounds.
[0113] In another embodiment, any of the agents of the invention
(e.g., peptides, polypeptides, proteins, small molecules,
antibodies, or antibody fragments that target senescent cells) can
be coupled to a member of the anthracycline family of neoplastic
agents, including but not limited to aclarubicine chlorhydrate,
daunorubicine chlorhydrate, doxorubicine chlorhydrate, epirubicine
chlorhydrate, idarubicine chlorhydrate, pirarubicine, or zorubicine
chlorhydrate; a camptothecin, or its derivatives or related
compounds, such as 10, 11 methylenedioxycamptothecin; or a member
of the maytansinoid family of compounds, which includes a variety
of structurally-related compounds, e.g., ansamitocin P3,
maytansine, 2'-N-demethylmaytanbutine, and maytanbicyclinol.
[0114] Any of the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can be modified or labeled
to facilitate diagnostic or therapeutic uses. Detectable labels
such as a radioactive, fluorescent, heavy metal, or other agents
may be bound to any of the agents of the invention. Single, dual,
or multiple labeling of an agent may be advantageous. For example,
dual labeling with radioactive iodination of one or more residues
combined with the additional coupling of, for example, .sup.90Y via
a chelating group to amine-containing side or reactive groups,
would allow combination labeling. This may be useful for
specialized diagnostic needs such as identification of widely
dispersed small neoplastic cell masses.
[0115] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells), or analogs thereof, can also be modified,
for example, by halogenation of the tyrosine residues of the
peptide component. Halogens include fluorine, chlorine, bromine,
iodine, and astatine. Such halogenated agents may be detectably
labeled, e.g., if the halogen is a radioisotope, such as, for
example .sup.18F, .sup.75Br, .sup.77Br, .sup.122I, .sup.123I,
.sup.124I, .sup.125I, .sup.129I, .sup.131I, or .sup.211A.
Halogenated agents of the invention contain a halogen covalently
bound to at least one amino acid, and preferably to D-Tyr residues
in each agent molecule. Other suitable detectable modifications
include binding of other compounds (e.g., a fluorochrome such as
fluorescein) to a lysine residue of the agent of the invention, or
analog, particularly an agent or analog having a linker including
lysines.
[0116] Radioisotopes for radiolabeling any of the agents of the
invention (e.g., peptides, polypeptides, proteins, small molecules,
antibodies, or antibody fragments that target senescent cells)
include any radioisotope that can be covalently bound to a residue
of the peptide component of the agent of the invention or analog
thereof. The radioisotopes can be selected from radioisotopes that
emit either beta or gamma radiation, or alternatively, any of the
agents of the invention can be modified to contain chelating groups
that, for example, can be covalently bonded to lysine residue(s) of
the analog. The chelating groups can then be modified to contain
any of a variety of radioisotopes, such as gallium, indium,
technetium, ytterbium, rhenium, or thallium (e.g., .sup.125I,
.sup.67Ga, .sup.111In, .sup.99mTc, .sup.169Yb, .sup.186Re).
[0117] When one or more of the agents of the invention (e.g.,
peptides, polypeptides, proteins, small molecules, antibodies, or
antibody fragments that target senescent cells) is modified by
attachment of a radioisotope, preferable radioisotopes are those
having a radioactive half-life corresponding to, or longer than,
the biological half-life of the agent used. More preferably, the
radioisotope is a radioisotope of a halogen atom (e.g. a
radioisotope of fluorine, chlorine, bromine, iodine, and astatine),
even more preferably .sup.75Br, .sup.77Br, 76Br, .sup.122I,
.sup.123I, .sup.124I, .sup.125I, .sup.129I, .sup.131I, or
.sup.211At.
[0118] Agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) coupled to radioactive metals are useful in
radiographic imaging or radiotherapy. Preferred radioisotopes also
include .sup.99mTc, .sup.51Cr, .sup.67Ga, .sup.68Ga, .sup.111In,
.sup.168Yb, .sup.140La, .sup.90Y, .sub.88Y, .sup.153Sm .sup.156Ho,
.sup.165Dy, .sup.64Cu, .sup.97Ru, .sup.186Re, .sup.188Re,
.sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi, .sup.214Bi. The
choice of metal can be determined based on the desired therapeutic
or diagnostic application.
[0119] The agents of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells), when coupled to a metal component, are
useful as diagnostic and/or therapeutic agents. A detectable label
may be a metal ion from heavy elements or rare earth ions, such as
Gd.sup.3+, Fe.sup.3+, Mn.sup.3+, or Cr.sup.2+. Conjugates that
include paramagnetic or superparamagnetic metals are useful as
diagnostic agents in MRI imaging applications. Paramagnetic metals
that can be coupled to the agents of the invention include, but are
not limited to, chromium (III), manganese (II), iron (II), iron
(III), cobalt (II), nickel (II), copper (II), praseodymium (III),
neodymium (III), samarium (III), gadolinium (III), terbium (III),
dysprosium (III), holmium (III), erbium (III), and ytterbium (III).
Preferably, the polymer has a relaxtivity of at least 10, 12, 15,
or 20 mM.sup.-1 sec.sup.-1 Z.sup.-1, wherein Z is the concentration
of paramagnetic metal.
[0120] Chelating groups may be used to indirectly couple detectable
labels or other molecules to an agent of the invention (e.g., a
peptide, polypeptide, protein, small molecule, antibody, or
antibody fragment). Chelating groups can link agents of the
invention with radiolabels, such as a bifunctional stable chelator,
or can be linked to one or more terminal or internal amino acid
reactive groups. Conjugates can be linked via an isothiocyanate
.beta.-Ala or appropriate non .alpha.-amino acid linker which
prevents Edman degradation. Examples of chelators known in the art
include, for example, the ininocarboxylic and
polyaminopolycarboxylic reactive groups, ininocarboxylic and
polyaminopolycarboxylic reactive groups,
diethylenetriaminepentaacetic acid (DTPA), and
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA).
[0121] An agent of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells) can also be coupled directly to a cytotoxic
or therapeutic agent using known chemical methods, or the two
moieties can be coupled via an indirect linkage, such as through a
chelating group. For example, the agent can be attached to a
chelating group that is attached to the cytotoxic or therapeutic
agent. Chelating groups include, but are not limited to,
ininocarboxylic and polyaminopolycarboxylic reactive groups,
diethylenetriaminepentaacetic acid (DTPA), and
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
For general methods, see, e.g., Liu et al., Bioconjugate Chem.
12(4):653, 2001; Cheng et al., WO 89/12631; Kieffer et al., WO
93/12112; Albert et al., U.S. Pat. No. 5,753,627; and WO 91/01144
(each of which are hereby incorporated by reference).
[0122] When coupled to a therapeutic or cytotoxic agent, specific
targeting by the agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) allows selective destruction
of senescent cells. For example, the agents of the invention can be
used to target and destroy senescent cells of the lung, breast,
prostate, and colon in order to prevent, stabilize, inhibit the
progression of, or treat cancers originating in these organs. Also,
for example, the agents of the invention can be used to target and
destroy senescent cells of the vasculature, brain, liver, kidney,
heart, lung, prostate, colon, nasopharynx, oropharynx, larynx,
bronchus, and skin in order to prevent, stabilize, inhibit the
progression of, or treat age-related diseases or tobacco-related
diseases or conditions relating to these organs. Any of the agents
of the invention (e.g., peptides, polypeptides, proteins, small
molecules, antibodies, or antibody fragments that target senescent
cells) can be administered to a mammalian subject, such as a human,
directly or in combination with any pharmaceutically acceptable
carrier or salt known in the art. Pharmaceutically acceptable salts
may include non-toxic acid addition salts or metal complexes that
are commonly used in the pharmaceutical industry. Examples of acid
addition salts include organic acids such as acetic, lactic,
pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,
palmitic, suberic, salicylic, tartaric, methanesulfonic,
toluenesulfonic, or trifluoroacetic acids or the like; polymeric
acids such as tannic acid, carboxymethyl cellulose, or the like;
and inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid phosphoric acid, or the like. Metal complexes include
zinc, iron, and the like. One exemplary pharmaceutically acceptable
carrier is physiological saline. Other physiologically acceptable
carriers and their formulations are known to one skilled in the art
and described, for example, in Remington's Pharmaceutical Sciences,
(18th edition), ed. A. Gennaro, 1990, Mack Publishing Company,
Easton, Pa.
Agents of the Invention for Use in Cellular Therapy
Applications
[0123] One or more agents of the invention (e.g., peptides,
polypeptides, proteins, small molecules, antibodies, or antibody
fragments that target senescent cells) can be used to improve the
efficacy of a cellular therapy provided to a patient (e.g., a
mammal, such as a human) in need thereof by depleting (i.e.,
killing) one or more, all, or substantially all (e.g., 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 99% or more) senescent cells in the transferred donor
cells, tissue, or organ prior to, concurrent with, or following
administration of the cellular therapy. Alternatively, a patient
(e.g., a mammal, such as a human) may be treated with one or more
agents of the invention prior to, concurrent with, or following
administration of the cellular therapy. Cellular therapies include,
without limitation, the autologous, allogeneic, syngeneic, or
xenogeneic transplantation or engraftment of cells (e.g., blood,
pancreatic islet cells, and stem cells (e.g., hematopoietic stem
cells (HSC), umbilical cord blood stem cells (see, e.g., US Patent
Application Publication 2002/0164794), pluripotent stem cells
(e.g., hox11-expressing pluripotent stem cells described in US
Patent Application Publication 2005/0158288), multipotent stem
cells, and totipotent stem cells), tissues (e.g., skin and adipose
tissue), or organs (e.g., kidney, liver, heart, and lung) to a
patient (e.g., a mammal, such as a human) in need thereof. Cellular
therapies (e.g., cell (e.g., stem cells), tissue, or organ
transplantation or engraftment) are currently being developed for a
wide range of therapeutic indications for degenerative and
pathologic diseases, such as osteoarthritis (see, e.g., U.S. Patent
Application Publication No. 2007/0264238), ischemia and cardiac
tissue damage, such as that caused by myocardial infarction (see,
e.g., Dzau et al., U.S. Patent Application Publication No.
2007/0259425), type I and type II diabetes (see, e.g., Uchida et
al., U.S. Patent Application Publication No. 2007/0212732), renal
dysfunction and multi-organ failure (see, e.g., Westenfelder, U.S.
Patent Application Publication No. 2007/0178071), and
neurodegenerative diseases (see, e.g., Kim et al., U.S. Patent
Application Publication No. 2007/0054399). The preceding U.S.
patent application Publications are hereby incorporated by
reference in their entirety.
Administration and Dosage
[0124] Pharmaceutical formulations of a therapeutically effective
amount of an agent of the invention (e.g., peptides, polypeptides,
proteins, small molecules, antibodies, or antibody fragments that
target senescent cells), or pharmaceutically acceptable
salt-thereof, can be administered orally, parenterally (e.g.,
intramuscular, intraperitoneal, intravenous, or subcutaneous
injection, inhalation, intradermally, optical drops, or implant),
nasally, vaginally, rectally, sublingually, or topically. The
pharmaceutical formulation can include the agent of the invention
in admixture with a pharmaceutically acceptable carrier adapted for
the route of administration.
[0125] Methods well known in the art for making pharmaceutical
formulations can be found, for example, in Remington's
Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack
Publishing Company, Easton, Pa. Compositions intended for oral use
may be prepared in solid or liquid forms according to any method
known to the art for the manufacture of pharmaceutical
compositions. The compositions may optionally contain sweetening,
flavoring, coloring, perfuming, and/or preserving agents in order
to provide a more palatable preparation. Solid dosage forms for
oral administration include capsules, tablets, pills, powders, and
granules. In such solid forms, the active compound is admixed with
at least one inert pharmaceutically acceptable carrier or
excipient. These may include, for example, inert diluents, such as
calcium carbonate, sodium carbonate, lactose, sucrose, starch,
calcium phosphate, sodium phosphate, or kaolin. Binding agents,
buffering agents, and/or lubricating agents (e.g., magnesium
stearate) may also be used. Tablets and pills can additionally be
prepared with enteric coatings.
[0126] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and soft gelatin capsules. These forms contain inert
diluents commonly used in the art, such as water or an oil medium.
Besides such inert diluents, compositions can also include
adjuvants, such as wetting agents, emulsifying agents, and
suspending agents.
[0127] Formulations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, or emulsions.
Examples of suitable vehicles include propylene glycol,
polyethylene glycol, vegetable oils, gelatin, hydrogenated
naphthalenes, and injectable organic esters, such as ethyl oleate.
Such formulations may also contain adjuvants, such as preserving,
wetting, emulsifying, and dispersing agents. Biocompatible,
biodegradable lactide polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for the agents of the invention include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes.
[0128] Liquid formulations can be sterilized by, for example,
filtration through a bacteria-retaining filter, by incorporating
sterilizing agents into the compositions, or by irradiating or
heating the compositions. Alternatively, they can also be
manufactured in the form of sterile, solid compositions which can
be dissolved in sterile water or some other sterile injectable
medium immediately before use.
[0129] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to active
substances, excipients such as coca butter or a suppository wax.
Compositions for nasal or sublingual administration are also
prepared with standard excipients known in the art. Formulations
for inhalation may contain excipients, for example, lactose, or may
be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
may be oily solutions for administration in the form of nasal drops
or spray, or as a gel.
[0130] The amount of active ingredient in the compositions of the
invention can be varied. One skilled in the art will appreciate
that the exact individual dosages may be adjusted somewhat
depending upon a variety of factors, including the agent being
administered, the time of administration, the route of
administration, the nature of the formulation, the rate of
excretion, the nature of the subject's conditions, and the age,
weight, health, and gender of the patient. In addition, the
severity of the condition targeted by an agent of the invention
will also have an impact on the dosage level. Generally, dosage
levels of an agent of the invention of between 0.1 .mu.g/kg to 100
mg/kg of body weight are administered daily as a single dose or
divided into multiple doses. Preferably, the general dosage range
is between 250 .mu.g/kg to 5.0 mg/kg of body weight per day. Wide
variations in the needed dosage are to be expected in view of the
differing efficiencies of the various routes of administration. For
instance, oral administration generally would be expected to
require higher dosage levels than administration by intravenous
injection. Variations in these dosage levels can be adjusted using
standard empirical routines for optimization, which are well known
in the art. In general, the precise therapeutically effective
dosage can be determined by the attending physician in
consideration of the above-identified factors.
[0131] An agent of the invention (e.g., a peptide, polypeptide,
protein, small molecule, antibody, or antibody fragment that
targets senescent cells) can be administered in a sustained release
composition, such as those described in, for example, U.S. Pat. No.
5,672,659 and U.S. Pat. No. 5,595,760. The use of immediate or
sustained release compositions depends on the type of condition
being treated. If the condition consists of an acute or over-acute
disorder, a treatment with an immediate release form will be
preferred over a prolonged release composition. Alternatively, for
preventative or long-term treatments, a sustained released
composition will generally be preferred.
[0132] An agent of the invention (e.g., a peptide, polypeptide,
protein, small molecule, antibody, or antibody fragment that
targets senescent cells) can be prepared in any suitable manner.
The agent may be isolated from naturally-occurring sources,
recombinantly produced, or produced synthetically, identified from
a library of small molecules, or produced by a combination of these
methods. The synthesis of short peptides is well known in the art.
See e.g., Stewart et al., Solid Phase Peptide Synthesis (Pierce
Chemical Co., 2d ed., 1984). A peptide portion of any of the agents
of the invention can be synthesized according to standard peptide
synthesis methods known in the art.
[0133] The present invention is illustrated by the following
examples, which are in no way intended to be limiting of the
invention.
EXAMPLES
Example 1
Discovery of Agents that Bind Senescent Cells
Phage Selection Technique
[0134] Normal skin cell line CCD-1070Sk was obtained from American
Type Culture Collection (Bethesda, Md.). The cells were grown in
Eagle's Minimal Essential medium with Earle's BSS, 2 mM
L-glutamine, 1.0 mM Sodium pyruvate, 0.1 mM nonessential amino
acids and 1.5 g/L sodium bicarbonate supplemented with 10% fetal
bovine serum.
[0135] Cells were sub-cultured every 4-5 days till they became
senescent. Cell senescence was confirmed by senescence-associated
beta-galactosidase staining.
[0136] An M13 phage peptide library, Ph.D.-12, was obtained from
New England BioLabs (Beverly, Mass.), which displays random 12-mer
peptides.
[0137] For the selection step for round 1, an aliquot (10 .mu.L) of
the Ph.D.-12 complete phage library was incubated with
5.times.10.sup.5 cells of senescent fibroblasts in 1 mL PBS/0.5%
BSA for .about.3.5 hours at room temperature with slow shaking on
Lab-Quake. At the end of the incubation, the cells were pelleted in
a microcentrifuge at 1500 RPM for 2 minutes and the supernatant
removed. Cells were washed with PBS/1.0% BSA/0.5% Tween (wash
buffer) for a total of 4 washes using fresh tubes between washes.
Phage that bound to the target cells were eluted with 200 .mu.L of
0.2 M glycine (pH 2.2) for 8 minutes then neutralized with 30 .mu.L
of 1 M Tris-HCl (pH 9.0). The number of phage bound was determined
and the remaining eluate was amplified.
[0138] From the amplified eluate from Round 1, an aliquot
(2.times.10.sup.11 phage) was used for subtraction panning against
normal skin fibroblasts. The phage were incubated with
2.times.10.sup.6 cells of normal skin fibroblast (CC D 1070 sk) at
room temperature for 60 minutes at room temperature with slow
shaking in PBS with 0.5% BSA. At the end of incubation, the cells
were pelleted in a microcentrifuge at 1500 RPM for 2 minutes and
the supernatant recovered. The supernatant was used to resuspend
another 2.times.10.sup.6 subtraction cells. This subtraction step
was repeated 3 times for each round.
[0139] After the final subtraction step in each round, the
recovered phage supernatant was used to suspend 5.times.10.sup.5
selection cells. The bound phage were recovered and amplified as
described above. The process was repeated for a total of 5 rounds
of selection. After the fifth round of selection, phage were
titered and 20 well-separated plaques were picked, amplified, and
sequenced.
Results:
[0140] Three consensus sequences were obtained from the phage
display selection procedure. These correspond to the SEQ ID
NOS:1-3. SEQ ID NO:1 represented 9/20 clones, SEQ ID NO:2
represented 6/20 clones, and SEQ ID NO:3 represented 2/20
clones.
Example 2
Human Study to Test the Prognostic Use of a Senescent Cell Binding
Agent Labeled with a Radioisotope; Using a Senescent Cell Detecting
Radiotracer to Predict Cancer Risk
[0141] This study is designed to show that noninvasive, in vivo
imaging of senescent cell content can be used to predict cancer
risk in smokers. Six-hundred subjects will be enrolled. Eighty
percent of these subjects will be smokers; twenty percent will be
nonsmokers. Subjects less than 18 years of age, subjects with a
history of cancer, and pregnant subjects will be excluded. The
anticipated mean age of study subjects is 55 years. Each study
subject will undergo scintigraphic imaging using a radio-labeled
peptide of the invention (SEQ ID NO:1) as the radiopharmaceutical.
The radiopharmaceutical will be prepared by reacting
peptide-gly-gly-gly-ser-DTPA with 111-In chloride. Each study
subject will receive an intravenously administered dose of 5 mCi of
111-In labeled peptide. Anterior and posterior whole body planar
images of the subjects will be obtained at 24 and 48 hours
following administration of the radio-labeled peptide. SPECT
imaging of the chest will also be obtained at 24 and 48 hours
following administration of the radio-labeled peptide.
Scintigraphic images will be acquired on a SPECT/CT camera using a
medium energy collimator. Two radiologists will read the SPECT/CT
study of each subject. Regions of interest will be drawn around
each lung and the mediastinum. The activity within each region will
be determined for each subject. Subjects will be followed for two
years and monitored for the incidence of lung cancer. Kaplan-Meier
curves will be drawn for the study population based upon cancer
free survival. An optimal threshold of activity within the regions
of interest will be determined such as to divide subjects that
develop cancer from subjects who don't; a Kaplan-Meier curve will
be drawn for the set of subjects with activity levels above the
threshold and a Kaplan-Meier curve will be drawn for subjects with
activity levels below the threshold level. We expect to observe
development of lung cancer in approximately twenty subjects.
Activity in pulmonary regions of interest will be significantly
higher in smokers than nonsmokers. Smokers who develop lung cancer
will have significantly higher activity in pulmonary regions of
interest than smokers who do not develop lung cancer. Cancer-free
survival curves will be significantly different for subjects with a
pulmonary region of interest activity level above the threshold
value than for subjects with activities below the threshold
value.
Example 3
Using Senescent Cell Binding Agents Coupled to Cytotoxic Agents to
Eliminate Senescent Cells: Effect on Subsequent Development of
Cancer
[0142] The purpose of this study is to show that elimination of
some senescent cells from an organism using the agents of the
invention (e.g., peptides, polypeptides, proteins, small molecules,
antibodies, or antibody fragments that target senescent cells) will
reduce the risk of subsequently developing cancer. For example, a
senescent cell binding peptide. (SEQ ID NO:1) will be conjugated to
a cytotoxic peptide having the sequence KFAKFAKKFAKFAKKFAKFAK (SEQ
ID NO:4; Leuschner and Hansel, Biology of Reproduction 73:860-865)
via an amino acid linker sequence (e.g., a linker sequence selected
from GGGC (SEQ ID NO:9), GGGS (SEQ ID NO:10), and GG) at the C
terminus of the senescent cell binding peptide. Study subjects will
consist of 60 BALB/c mice, mean age 6 mo which includes 30
experimental animals and 30 controls. Experimental animals will
receive an intravenously administered dose of 0.2 mg/Kg of body
weight of senescent cell binding peptide conjugate once every three
months for one year. Control animals will receive an equal dose of
a 37 amino acid control peptide. Kaplan-Meier curves representing
tumor free survival will be constructed for each group of mice.
Approximately 30% of control mice will develop tumors by the end of
the study period. The study is expected to show significantly
longer tumor free survival in the experimental group compared to
the control group.
Example 4
Reduction of Senescent Cell Content in Diabetic Mice by Treatment
with a Senescent Cell Cytotoxic Agent
[0143] Diabetes is induced in female CD-1 mice, 5-7 weeks old and
25-35 g in body weight, by intraperitoneal injection of 200 mg/Kg
body weight of streptozotocin dissolved in sodium citrate saline
buffer (pH 4.5). Tail vein blood glucose will be measured 5 days
after injection to ensure induction of diabetes. Diabetic mice will
be maintained at constant temperature (23.degree. C.) with 12 hour
light and 12 hour dark cycles for 16 weeks following confirmation
of diabetes. Seven diabetic mice will receive a weekly tail vein
injection of a senescent cell cytotoxic agent (SenL; SEQ ID NO:6;
GVYHFAPLTPTPGGGSKFAKFAKKFAKFAK; 300 .mu.g/dose), comprising a
senescent cell binding sequence linked to a lytic peptide sequence
during the 16 weeks. Seven control animals will receive an
equivalent volume TV injection of normal saline. At the end of 16
weeks, all animals will be sacrificed. Tissue cross sections will
be prepared from aorta, lung, liver, and heart from snap frozen
tissue. Tissue samples will be stained for SA-.beta.-gal activity
using the method of Campisi et al. Percentage of SA-.beta.-gal
positive cells will be determined for each tissue sample from each
animal by counting 1000 cells in each of four random microscopic
fields for each tissue sample. A two-tailed t-test will be used to
evaluate the loss of senescent cells in the tissues of diabetic
mice treated with senescent cell cytotoxic agent relative to the
loss of senescent cells in the tissues of diabetic control
mice.
Example 5
Enhancement of Stem Cell Treatments by Pre-Treatment with Senescent
Cell Binding Agents Coupled to Cytotoxic Agents: Effects on
Subsequent Stem Cell Engraftment
[0144] The following experiment can be used to show that
exogenously administered stem cells engraft at higher rates into
damaged tissue if the treated organism undergoes pretreatment with
a senescent cell cytotoxic agent of the invention (e.g., a peptide
agent) to reduce the content of senescent cells in the damaged
tissue compartment. Removal of senescent cells increases the
engraftment of stem cells into damaged tissue.
[0145] Balb-C mice will undergo left anterior descending (LAD)
artery ligation for 60 minutes to induce myocardial infarction. The
mice will be pre-anesthetized in an isoflurane inhalation chamber
and receive an i.p. injection of sodium pentobarbital (25 mg/kg).
The animals will be intubated and ventilated for the duration of
the procedure. The LAD artery will be identified following left
lateral thoracotomy and pericardectomy. Ligation will be performed
on the proximal 2 mm portion of the LAD using a 9-0 ethilon stitch.
Mice will be maintained at 23.degree. C. with 12 hour light and 12
hour dark cycles for 6 days. Seven surviving mice will be used as
experimental animals and seven will be used as control animals.
Experimental mice will receive tail vein injections of a senescent
cell cytotoxic agent conjugated to a lytic peptide sequence (SEQ ID
NO:8; GVYHFAPLTPTPGGKFAKFAKKFAKFAK; 300 .mu.g/dose) every second
day for six days.
[0146] Murine hematopoietic stem cells (HSC) will be obtained from
StemCell Technologies Inc. Cells will be transfected to express
enhanced green fluorescent protein (EGFP). Plasmid pEF-1 a-EGFP,
containing an EGFP gene under the control of human EF1, a promoter,
and a neomycin-resistance cassette, will be constructed as follows:
(1) the promoter region of pEGFP-N3 (Clontech, Palo Alto, Calif.)
will be removed by cutting out the AseI-NheI DNA fragment and
joining the blunt-ended termini, and (2) human EF1, a promoter from
pEF-BOS (a fragment of HindIII and EcoRI DNA) will be inserted into
the HindIII-EcoRI site of the plasmid. Murine HSC will be
transfected with pEF-1 a-EGFP by electroporation and selected in
the presence of G418. A single clone that brightly expresses EGFP
will be chosen and used for the experiments. The clone will be
adapted to feeder-free conditions and maintained on gelatin-coated
dishes in Dulbecco's Modified Eagle's Media supplemented with 15%
fetal calf serum, 2 mM sodium pyruvate, 2 mM L-glutamine, 1.times.
nonessential amino acids, 1,000 units of 0.1 mM 2-mercaptoethanol
per mL, along with 100 units of streptomycin and 100 .mu.g of
penicillin per mL. Cells will be collected after trypsinization
with EDTA and placed in aliquots of the medium described above for
mouse tail vein injection 1 hour later.
[0147] HSC (10.sup.6) will be injected via tail vein into each
experimental and control mouse. Ten days later, each animal will be
sacrificed. Hearts will be excised and fixed in 2% paraformaldehyde
in phosphate-buffered solution (PBS) for 2 hours and cryoprotected
in 30% sucrose overnight. Tissue will be embedded in optimum
cutting temperature medium and sectioned at 5 .mu.m on a cryostat.
Serial sections will be stained with hematoxylin and eosin
(H&E). Tissue will be examined with a fluorescent microscope.
Percentage of GFP positive cells will be determined for each
cardiac tissue sample from each animal by counting 1000 cells in
each of four random microscopic fields for each tissue sample. A
two-tailed t-test will be used to evaluate the hypothesis that
exogenously administered HSC engraft at a higher rate into mice
treated with senescent cell cytotoxic agent relative to untreated
controls. Special attention will be paid to cardiac tissue in the
LAD territory (anterior wall) of each heart.
Example 6
In Vitro Validation of the Use of Senescent Cell Binding Agents as
Agents to Deliver Molecular Cargo to the Cytoplasm of Senescent
Cells
Cytotoxicity of Senescent Cell Binding Peptide Conjugated to a
Lytic Peptide Sequence
[0148] Cytotoxic peptide SenL (i.e., SEQ ID NO:6) was synthesized
by conjugating a senescent cell binding agent (SEQ ID NO:1) to a
lytic peptide sequence (KFAKFAKKFAKFAK; SEQ ID NO:4) via a 4
residue linker (GGGS; SEQ ID NO:10) and tested for differential
cytotoxic activity in senescent fibroblasts, prostate epithelial
cells, and non-senescent fibroblasts. Senescent fibroblasts
exhibited dose-dependent cell death at a significantly higher rate
than either non-senescent fibroblasts or prostate epithelial cells
(FIG. 1). The effect of SenL on cell proliferation was also
assessed using the Cell Proliferation Assay WST-1 (Roche, Mannheim,
Germany). As shown in FIG. 2, no change in cell proliferation was
seen in any of the three cell types in response to treatment with
SenL. The proliferation assay uses WST-1 as a reagent; the reaction
is catalyzed by mitochondrial dehydrogenases. Senescent cells have
higher mitochondrial mass than their non-senescent counterparts
(Lee et al., J. Biomed. Sci. 9:517-26, 2002); consequently,
baseline WST assay values are higher for senescent cells. The
occurrence of cell death in response to treatment with SenL and
absence of change in proliferation rate indicate that SenL causes
cell death in non-proliferating cells, e.g. senescent cells.
[0149] It is worth noting that each cultured cell population
contains a mixture of senescent and non-senescent cells at all
population doublings, but that the relative proportion of senescent
cells within the population increases stochastically with each
population doubling (Martin-Ruiz et al., J. Biol. Chem.
279(17):17826-33, 2004). Thus, the "senescent cells" used in this
cell killing experiment are predicted to contain a subpopulation of
non-senescent cells. Likewise, a fraction of the "non-senescent
cells" used in this experiment are predicted to be senescent.
Therefore, the observed difference in cell killing between the two
populations is reduced by the impure composition of each population
with regard to senescence. This explains why some cytotoxicity is
observed in the "non-senescent" fibroblast population. It also
explains why the prostate epithelial cells (RWPE-1), which are not
predicted to have any senescent cells due to immortalization
through HPV-18 transduction, show no cell death at all.
In Vitro Cytotoxicity: Conjugation to Ricin-A
[0150] A senescent cell binding agent (i.e., SEQ ID NO:1) was
conjugated to the ricin A subunit via a 4 peptide linker (GGGC; SEQ
ID NO:9) to produce SenR (SEQ ID NO:7). Senescent fibroblasts,
non-senescent fibroblasts, and prostate epithelial cells were then
incubated with the peptide-ricin A conjugate (SenR). Increased cell
death was observed in the case of senescent cells treated with SenR
than in the other cell types (FIG. 3). For example, an
approximately 3 fold increase in cell death was measured in
senescent fibroblast when compared to non-senescent fibroblast when
treated with 50 .mu.M of SenR.
Binding of Peptide to Senescent Cells Versus Non-Senescent
Cells
[0151] In the absence of unlabeled peptide, radio-labeled SenC
bound to prostate epithelium, normal fibroblasts, and senescent
cells at an average of 0.06%, 0.08%, and 0.32% of added dose,
respectively, demonstrating that SenC binds to senescent cells at a
higher rate than to the other cell types (P=0.001). In the presence
of 20 .mu.M unlabeled SenC, labeled SenC bound to prostate
epithelium, normal fibroblasts, and senescent cells at an average
of 0.06%, 0.09%, and 0.25% of added dose, respectively. There was
no significant difference between binding rates in the presence or
absence of unlabeled SenC in the case of prostate epithelium and
normal fibroblasts (P=0.5 and 0.4 respectively), indicating that
the binding to these cell types is nonspecific. Labeled SenC did
bind at significantly different rates to senescent cells in the
absence vs. presence of unlabeled SenC (P=0.04), indicative of
specific binding (see FIG. 4).
Example 7
Isolation and Identification of Senescent Cell-Specific
Antigens
[0152] Fibroblasts (CCD-1070Sk) were cultured as outlined above and
divided into three groups: replicatively senescent, stress induced
prematurely senescent, and non-senescent. Cell surface proteins of
each group of cells were conjugated to biotin using the Pierce Cell
Surface Protein Isolation Kit (Thermo Scientific, product number
89881) following the instructions of the manufacturer. Membrane
proteins were captured using neutravidin following membrane
dissolution and sent for 2D gel electrophoresis. Spots from each
gel were analyzed to look for differences in protein expression
among the three groups of fibroblasts. Protein spots occurring in
the gels corresponding to senescent (replicative or stress-induced)
cells but not in gels corresponding to non-senescent fibroblasts
were identified as senescence-specific antigens and identified
using MALDI-TOF mass spectrometry.
[0153] Mutant beta-actin (SEQ ID NO:11; GI: 28336) and ACTB protein
(SEQ ID NO:12; GI: 15277503) were identified as cell-surface,
senescence specific antigens occurring in both replicatively
senescent and stress-induced prematurely senescent cells. Drug
resistance-related protein LRP (SEQ ID NO:13; GI: 1097308) and
major vault protein (SEQ ID NO:14; GI: 19913410) were identified as
senescence-specific surface proteins in the replicatively senescent
cells only. Senescence specific antigens that were identified in
stress induced prematurely senescent cells included thyroid hormone
binding protein precursor (SEQ ID NO:15; GI: 339647); unnamed
protein product (SEQ ID NO:20; GI: 35655); prolyl 4-hydroxylase,
beta subunit precursor (SEQ ID NO:16; GI: 20070125); chain A, human
protein disulfide isomerase (SEQ ID NO:17; GI: 159162689);
electron-transfer-flavoprotein, beta polypeptide (SEQ ID NO:18; GI:
4503609); unnamed protein product (SEQ ID NO:21; GI: 158257194);
unnamed protein product (SEQ ID NO:22; GI: 158259937); ATP
synthase, H.sup.+ transporting, mitochondrial F1 complex, alpha
subunit precursor (SEQ ID NO:19; GI: 4757810), and cathepsin B (SEQ
ID NO:23).
Methods
Induction of Replicative Senescence
[0154] CCD-1070Sk (fibroblasts) was cultured as described herein.
Cells were cultured until they underwent 68 population doublings at
which time they displayed typical senescent morphology and
underwent minimal further cell growth in response to mitogens.
Isolation of Cell Surface Proteins
[0155] Cell surface proteins were extracted from three groups of
cells (replicatively senescent fibroblasts, stress-induced
prematurely senescent fibroblasts, and non-senescent fibroblasts).
Each group of cells contained 10.sup.9 cells. Cell surface proteins
were isolated using the Pierce Cell Surface Protein Isolation Kit
(Thermo Scientific, product number 89881), following the
instructions of the manufacturer. Protein isolates were analyzed
using spectrophotometry which showed absorbances at 280 nm of 1.25,
1.375, and 1.347 for replicatively senescent cells, stress induced
prematurely senescent cells, and non-senescent cells respectively.
Total volume of each protein isolate was 500 .mu.L.
Identification of Cell Surface Proteins
[0156] Cell surface protein isolates were sent for analysis by the
proteomics core at the University of Massachusetts Medical School
(Worcester, Mass.). The analysis was carried out by performing a
buffer exchange for each protein isolate sample followed by 2D gel
electrophoresis. Each gel was compared to find protein spots that
occurred in the gels corresponding to the senescent (replicative or
stress-induced) cells but not in the gels corresponding to the
non-senescent cells. Protein spots that occurred in the senescent
cell samples but not in the non-senescent samples were digested and
sent for mass spectrometry analysis for identification.
Example 8
Immunostaining for Cathepsin B Expression on the Surface of
Senescent Cells
[0157] Senescent fibroblasts and non-senescent fibroblasts were
grown on cover slips and immunostained for cell surface expression
of cathepsin B. Images appear in FIGS. 5A and 5B, which show
surface staining for cathepsin B on the senescent cells but not on
their non-senescent counterparts.
[0158] Fibroblasts (CCD1070Sk) were grown in culture as detailed
herein. Replicatively senescent cells were acquired by growing
cells for 50 population doublings followed by plating on cover
slips. Non-senescent cells were acquired by plating mid passage
fibroblasts on cover slips. Cells on cover slips were allowed to
attach overnight. Cells were fixed with methanol and washed. Rabbit
polyclonal antibody to cathepsin B was diluted 1:100 in PBS with
0.2% BSA. Cells were incubated with primary antibody to cathepsin B
for one hour at room temperature followed by washing three times
with cold PBS. Secondary antibody (goat anti rabbit IgG conjugated
to FITC) was diluted 1:100 in PBS with 0.2% BSA and used to
incubate cells for 30 minutes at room temperature. Cells were
washed three times with cold PBS.
Example 9
Materials and Methods
Cell Culture
[0159] All cells were obtained from American Type Culture
Collection (Manassas, Va.). Each cell culture was grown at
37.degree. C. in 5% CO.sub.2. CCD-1070Sk (fibroblasts) were grown
in minimum essential medium (Eagle) with 2 mM L-glutamine and
Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
non-essential amino acids, and 1.0 mM sodium pyruvate, and
supplemented with 10% fetal bovine serum. RWPE-1 (non-cancerous
prostate epithelial cells) were grown in keratinocyte-serum free
medium supplemented with 5 ng/mL human recombinant EGF and 0.05
mg/mL bovine pituitary extract.
Chemical Induction of Cellular Senescence
[0160] CCD-1070Sk (fibroblasts) was cultured as described above to
70% confluence. Cells were then treated with 200 .mu.M hydrogen
peroxide (i.e., H.sub.2O.sub.2) for 2 hours. Media was then
replaced with fresh media and cells were allowed to grow for 3
days. Cells were then harvested by trypsinization, split 1:3, and
again grown to 70% confluence. Cells were then retreated with 200
.mu.M hydrogen peroxide. Lower passage number fibroblasts
frequently required two treatments with hydrogen peroxide, but
higher passage fibroblasts occasionally required only a single
treatment.
Peptide Synthesis
[0161] Peptides were synthesized using standard FMOC protected
chemistry. For in vitro cell cytotoxicity studies, peptide was
synthesized with a C-terminal, cell lytic sequence according to the
following: GVYHFAPLTPTPGGGS(KFAKFAK).sub.2 (SEQ: ID NO:6; SenL).
The peptide sequence GVYHFAPLTPTPGGGC (SEQ ID NO:5; SenC) was
synthesized for subsequent conjugation to ricin-A subunit and for
radio-labeling with 99m-technetium.
Conjugation of FITC to Peptide
[0162] FITC was conjugated to the N terminus of SenC according to
the following: (1) A senescent cell binding peptide (SenC; SEQ ID
NO:5) was prepared at a concentration of 5 mM in 125 .mu.L of 0.5 M
NaHCO.sub.3 buffer, pH 9.5, and (2) FITC was added to the peptide
solution at a 1:5 molar ratio (peptide:FITC) and diluted to a final
volume of 200 .mu.L. The solution was incubated in the dark for 2
hours. Peptide-FITC conjugate was purified on a P4 column using
PBS, pH 7.2 as an eluent.
Cell Internalization
[0163] Premature senescence of fibroblasts (CCD-1070Sk) was induced
as outlined above. Non-senescent fibroblasts were grown in culture
as detailed above. Cells were harvested and added to
collagen-coated coverslips and grown in MEM plus 10% FBS overnight.
Media was removed, and cells were washed once with PBS. Minimal
essential media without FBS was added to the cells. A senescent
cell binding peptide (SenC; SEQ ID NO:5) conjugated to FITC was
added to cells on coverslips at a concentration of 5 .mu.M and
incubated for 3 hours. Cells were than washed five times with PBS
followed by fixation with 1:1 methanol/acetone for 10 minutes at
-20.degree. C. Coverslips were air dried and mounted in fluorescent
mounting media with DAPI and visualized with an Olympus BX51
fluorescent microscope and DP70 digital camera with excitation and
emission wavelengths of 490 and 520 nm.
Cytotoxicity of Senescent Cell Binding Peptide Conjugated to a
Lytic Peptide Sequence
[0164] CCD-1070Sk and RWPE-1 were grown in culture as detailed
above. Premature senescence of fibroblasts was chemically induced
as described above. Cells were trypsinated and suspended in culture
media containing 10% FBS. Cells were centrifuged at 1,000 rpm for 5
minutes. Supernatant was removed and cells were resuspended in 1 mL
media without FBS. Cell suspensions were diluted to 20,000 cells/75
.mu.L. Twenty-five microliters of appropriately-diluted agent
solution (SenL; SEQ ID NO:6) was added to cell samples to give
various concentrations of 0, 0.1, 0.5, 1.0, 2.5, 5.0 or 10 .mu.M.
Each sample was prepared in triplicate. Cell suspensions were
transferred to a 96 well plate and incubated in the presence of
various concentrations of SenL for 2 hours at 37.degree. C. Six
samples of each cell type contained no agent. The assay plate was
removed from the incubator, and 2 .mu.L of lysis solution (Tris 25
mM, pH 7.5, 0.5% triton X-100) was added to three samples of each
cell type without agent to generate a positive control maximum LDH
release. LDH release was measured in each sample by adding 100
.mu.L of CytoTox-ONE Reagent (Roche Applied Science) to each well
and mixing on a plate shaker for 30 seconds. Samples were incubated
at 22.degree. C. for 10 minutes. The reaction was terminated by
adding 50 .mu.L of Stop Solution (per 100 .mu.l, of CytoTox-ONE.TM.
Reagent added) to each well. Fluorescence was measured in each well
using an excitation wavelength of 530 nm and an emission wavelength
of 620 nm (Cytofluor 4000). The CytoTox-ONE assay was shown to
yield a quantity of fluorescent product that is linearly
proportional to the number of cells killed (correlation
coefficient=0.99). The percentage of cells killed was calculated
using the following formula:
% cytotoxicity = 100 % .times. ( P - C ) ( M - C ) ##EQU00001##
where P=LDH release in wells of peptide incubated cells; C=LDH
release in wells of cells not incubated with peptide; and M=LDH
release in wells incubated in lysis solution. The formula is based
upon the assumptions that, in a linear relationship between
CytoTox-ONE product development and number of cells killed, C is
the y-intercept, and M is due to 100% cell killing.
Effect of SenL on Cell Proliferation
[0165] Cell proliferation was assayed using the Cell Proliferation
Reagent WST-1 (Roche, Mannheim, Germany) by following the
manufacturer's instructions.
In Vitro Cytotoxicity: Conjugation to Ricin-A
[0166] Peptide SenC (GVYHFAPLTPTPGGGC; SEQ ID NO:5) was conjugated
to ricin A subunit (Sigma-Aldrich) to form SenR. Ricin A was
obtained from manufacturer in solution. A buffer exchange was
performed with 0.1 M PBS/20% glycerol. Ricin A was conjugated with
NHS-PEO.sub.4-maleimide cross linker (Pierce, Rockford, Ill.) at a
1:10 molar ratio for 30 minutes at room temperature. Derivatized
ricin A was purified on a P4 column using 0.1 M PBS/20% glycerol as
an elution buffer. Derivatized ricin A was combined with P12S at a
1:1 molar ratio and reacted for 2 hours at room temperature.
[0167] CCD-1070Sk and RWPE-1 were grown in culture as detailed
above. Senescence was chemically-induced as described above. Cells
were trypsinized and suspended in culture media containing 10% FBS.
Cells were centrifuged at 1,000 rpm for 5 minutes. Supernatant was
removed and cells were resuspended in 1 mL media without FBS. Cell
suspensions were diluted to 20,000 cells/75 .mu.L. Twenty-five
microliters of appropriately-diluted peptide-ricin A conjugate was
added to each cell sample to give various concentrations. Each
sample was prepared in triplicate. Cell suspensions were
transferred to a 96 well plate and incubated in the presence of
peptide-ricin A conjugate for 2 hours at 37.degree. C. Percentage
of cells killed (FIG. 3) was determined as outlined above.
Radio-Labeling of Peptide Senescent Cell Binding Peptide
[0168] A senescent cell binding agent (SEQ ID NO:1) was conjugated
at its C terminus to the linker sequence GGGC (SEQ ID NO:9) by
synthesizing both as a single construct (i.e., SenC; SEQ ID NO:5).
The purpose of attaching GGGC was that it can be used to chelate
reduced 99m-Tc for radio-labeling. A 2 .mu.L, aliquot of conjugated
senescent cell binding agent (3 M) was mixed with 40 .mu.L, of 0.25
M ammonium acetate, 15 .mu.L, of tartrate buffer pH 8.7, 4 .mu.L,
of stannous chloride in 100 mM of sodium tartrate, and 30 .mu.L, of
99m-Tc pertechnetate. The mixture was heated for 25 minutes at
95.degree. C. Quality control was performed with Sep-Pak and was
always above 90% purity. A small aliquot was also injected on a
Waters 600 HPLC to check the radiological profile. Fractions were
collected and read on a gamma counter (Perkin-Elmer Wallac Wizard
1470).
Cell Binding Assay
[0169] Binding of radio-labeled SenC (SEQ ID NO:5) to senescent and
non-senescent cells was tested by competition with unlabeled SenC.
Peptide solutions were prepared to contain 0 or 20 .mu.M of
unlabeled SenC, 15 nM radio-labeled SenC, and 0.2% BSA.
Chemically-induced senescent fibroblasts, their non-senescent
counterparts, and prostate epithelial cells were prepared as above,
harvested, and centrifuged. The cell pellets were resuspended in
fresh media without FBS, and cells were counted. Peptide solution
containing 0 or 20 .mu.M unlabeled SenC and constant concentrations
of SenC and 10.sup.5 senescent cells were combined in a final
volume of 200 .mu.L, of PBS in Eppendorf tubes and incubated for 4
hours. Cells were pelleted by centrifuging at 2500 rpm for 2
minutes and washed twice with PBS and 0.2% BSA. Pellets were
suspended in 5 .mu.L, PBS and transferred to 12.times.75 mm tubes
for counting radioactivity using a gamma counter (Perkin-Elmer
Wallac Wizard 1470).
OTHER EMBODIMENTS
[0170] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0171] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth.
Sequence CWU 1
1
35112PRTArtificial SequenceSynthetic Construct 1Gly Val Tyr His Phe
Ala Pro Leu Thr Pro Thr Pro 1 5 10 212PRTArtificial
SequenceSynthetic Construct 2Ser Phe Gln Ser His Leu Ile Glu Phe
Ser Phe Glu 1 5 10 312PRTArtificial SequenceSynthetic Construct
3Ala Pro Ile Leu Lys Leu Ala Pro Leu Ile His Pro 1 5 10
414PRTArtificial SequenceSyntheitc Construct 4Lys Phe Ala Lys Phe
Ala Lys Lys Phe Ala Lys Phe Ala Lys 1 5 10 516PRTArtificial
SequenceSynthetic Construct 5Gly Val Tyr His Phe Ala Pro Leu Thr
Pro Thr Pro Gly Gly Gly Cys 1 5 10 15 629PRTArtificial
SequenceSynthetic Construct 6Gly Val Tyr His Phe Ala Pro Leu Thr
Pro Thr Pro Gly Gly Ser Lys 1 5 10 15 Phe Ala Lys Phe Ala Lys Lys
Phe Ala Lys Phe Ala Lys 20 25 716PRTArtificial SequenceSynthetic
Construct 7Gly Val Tyr His Phe Ala Pro Leu Thr Pro Thr Pro Gly Gly
Gly Cys 1 5 10 15 828PRTArtificial SequenceSynthetic Construct 8Gly
Val Tyr His Phe Ala Pro Leu Thr Pro Thr Pro Gly Gly Lys Phe 1 5 10
15 Ala Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Lys 20 25
94PRTArtificial SequenceSynthetic Construct 9Gly Gly Gly Cys 1
104PRTArtificial SequenceSynthetic Construct 10Gly Gly Gly Ser 1
11375PRTHomo sapiens 11Met Asp Asp Asp Ile Ala Ala Leu Val Val Asp
Asn Gly Ser Gly Met 1 5 10 15 Cys Lys Ala Gly Phe Ala Gly Asp Asp
Ala Pro Arg Ala Val Phe Pro 20 25 30 Ser Ile Val Gly Arg Pro Arg
His Gln Gly Val Met Val Gly Met Gly 35 40 45 Gln Lys Asp Ser Tyr
Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile 50 55 60 Leu Thr Leu
Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp 65 70 75 80 Asp
Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val 85 90
95 Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro
100 105 110 Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr
Phe Asn 115 120 125 Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Met Leu
Ser Leu Tyr Ala 130 135 140 Ser Gly Arg Thr Thr Gly Ile Val Met Asp
Ser Gly Asp Gly Val Thr 145 150 155 160 His Thr Val Pro Ile Tyr Glu
Gly Tyr Ala Leu Pro His Ala Ile Leu 165 170 175 Arg Leu Asp Leu Ala
Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 180 185 190 Leu Thr Glu
Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile 195 200 205 Val
Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu 210 215
220 Gln Glu Met Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys Ser Tyr
225 230 235 240 Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu
Arg Phe Arg 245 250 255 Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu
Gly Met Glu Ser Cys 260 265 270 Gly Ile His Glu Thr Thr Phe Asn Ser
Ile Met Lys Cys Asp Val Asp 275 280 285 Ile Arg Lys Asp Leu Tyr Asp
Asn Thr Val Leu Ser Gly Gly Thr Thr 290 295 300 Met Tyr Pro Gly Ile
Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu 305 310 315 320 Ala Pro
Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys 325 330 335
Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 340
345 350 Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro
Ser 355 360 365 Ile Val His Arg Lys Cys Phe 370 375 12360PRTHomo
Sapiens 12Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala
Val Phe 1 5 10 15 Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val
Met Val Gly Met 20 25 30 Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu
Ala Gln Ser Lys Arg Gly 35 40 45 Ile Leu Thr Leu Lys Tyr Pro Ile
Glu His Gly Ile Val Thr Asn Trp 50 55 60 Asp Asp Met Glu Lys Ile
Trp His His Thr Phe Tyr Asn Glu Leu Arg 65 70 75 80 Val Ala Pro Glu
Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 85 90 95 Pro Lys
Ala Asn Leu Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe 100 105 110
Asn Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr 115
120 125 Ala Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly
Val 130 135 140 Thr His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro
His Ala Ile 145 150 155 160 Leu Arg Leu Asp Leu Ala Gly Arg Asp Leu
Thr Asp Tyr Leu Met Lys 165 170 175 Ile Leu Thr Glu Arg Gly Tyr Ser
Phe Thr Thr Thr Ala Glu Arg Glu 180 185 190 Ile Val Arg Asp Ile Lys
Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe 195 200 205 Glu Gln Glu Met
Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys Ser 210 215 220 Tyr Glu
Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe 225 230 235
240 Arg Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser
245 250 255 Cys Gly Ile His Glu Thr Thr Phe Asn Ser Ile Met Lys Cys
Asp Val 260 265 270 Asp Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu
Ser Gly Gly Thr 275 280 285 Thr Met Tyr Pro Gly Ile Ala Asp Arg Met
Gln Lys Glu Ile Thr Ala 290 295 300 Leu Ala Pro Ser Thr Met Lys Ile
Lys Ile Ile Ala Pro Pro Glu Arg 305 310 315 320 Lys Tyr Ser Val Trp
Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr 325 330 335 Phe Gln Gln
Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro 340 345 350 Ser
Ile Val His Arg Lys Cys Phe 355 360 13896PRTHomo Sapiens 13Met Ala
Thr Glu Glu Phe Ile Ile Arg Ile Pro Pro Tyr His Tyr Ile 1 5 10 15
His Val Leu Asp Gln Asn Ser Asn Val Ser Arg Val Glu Val Gly Pro 20
25 30 Lys Thr Tyr Ile Arg Gln Asp Asn Glu Arg Val Leu Phe Ala Pro
Met 35 40 45 Arg Met Val Thr Val Pro Pro Arg His Tyr Cys Thr Val
Ala Asn Pro 50 55 60 Val Ser Arg Asp Ala Gln Gly Leu Val Leu Phe
Asp Val Thr Gly Gln 65 70 75 80 Val Arg Leu Arg His Ala Asp Leu Glu
Ile Arg Leu Ala Gln Asp Pro 85 90 95 Phe Pro Leu Tyr Pro Gly Glu
Val Leu Glu Lys Asp Ile Thr Pro Leu 100 105 110 Gln Val Val Leu Pro
Asn Thr Ala Leu His Leu Lys Ala Leu Leu Asp 115 120 125 Phe Glu Asp
Lys Asp Gly Asp Lys Val Val Ala Gly Asp Glu Trp Leu 130 135 140 Phe
Glu Gly Pro Gly Thr Tyr Ile Pro Arg Lys Glu Val Glu Val Val 145 150
155 160 Glu Ile Ile Gln Ala Thr Ile Ile Arg Gln Asn Gln Ala Leu Arg
Leu 165 170 175 Arg Ala Arg Lys Glu Cys Trp Asp Arg Asp Gly Lys Glu
Arg Val Thr 180 185 190 Gly Glu Glu Trp Leu Val Thr Thr Val Gly Ala
Tyr Leu Pro Ala Val 195 200 205 Phe Glu Glu Val Leu Asp Leu Val Asp
Ala Val Ile Leu Thr Glu Lys 210 215 220 Thr Ala Leu His Leu Arg Ala
Arg Arg Asn Phe Arg Asp Phe Arg Gly 225 230 235 240 Val Ser Arg Arg
Thr Gly Glu Glu Trp Leu Val Thr Val Gln Asp Thr 245 250 255 Glu Ala
His Val Pro Asp Val His Glu Glu Val Leu Gly Val Val Pro 260 265 270
Ile Thr Thr Leu Gly Pro His Asn Tyr Cys Val Ile Leu Asp Pro Val 275
280 285 Gly Pro Asp Gly Lys Asn Gln Leu Gly Gln Lys Arg Val Val Lys
Gly 290 295 300 Glu Lys Ser Phe Phe Leu Gln Pro Gly Glu Gln Leu Glu
Gln Gly Ile 305 310 315 320 Gln Asp Val Tyr Val Leu Ser Glu Gln Gln
Gly Leu Leu Leu Arg Ala 325 330 335 Leu Gln Pro Leu Glu Glu Gly Glu
Asp Glu Glu Lys Val Ser His Gln 340 345 350 Ala Gly Asp His Trp Leu
Ile Arg Gly Pro Leu Glu Tyr Val Pro Ser 355 360 365 Ala Lys Val Glu
Val Val Glu Glu Arg Gln Ala Ile Pro Leu Asp Glu 370 375 380 Asn Glu
Gly Ile Tyr Val Gln Asp Val Lys Thr Gly Lys Val Arg Ala 385 390 395
400 Val Ile Gly Ser Thr Tyr Met Leu Thr Gln Asp Glu Val Leu Trp Glu
405 410 415 Lys Glu Leu Pro Pro Gly Val Glu Glu Leu Leu Asn Lys Gly
Gln Asp 420 425 430 Pro Leu Ala Asp Arg Gly Glu Lys Asp Thr Ala Lys
Ser Leu Gln Pro 435 440 445 Leu Ala Pro Arg Asn Lys Thr Arg Val Val
Ser Tyr Arg Val Pro His 450 455 460 Asn Ala Ala Val Gln Val Tyr Asp
Tyr Arg Glu Lys Arg Ala Arg Val 465 470 475 480 Val Phe Gly Pro Glu
Leu Val Ser Leu Gly Pro Glu Glu Gln Phe Thr 485 490 495 Val Leu Ser
Leu Ser Ala Gly Arg Pro Lys Arg Pro His Ala Arg Arg 500 505 510 Ala
Leu Cys Leu Leu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile Thr 515 520
525 Ile Glu Thr Ala Asp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr Asn
530 535 540 Trp His Phe Glu Val Asn Asp Arg Lys Asp Pro Gln Glu Thr
Ala Lys 545 550 555 560 Leu Phe Ser Val Pro Asp Phe Val Gly Asp Ala
Cys Lys Ala Ile Ala 565 570 575 Ser Arg Val Arg Gly Ala Val Ala Ser
Val Thr Phe Asp Asp Phe His 580 585 590 Lys Asn Ser Ala Arg Ile Ile
Arg Thr Ala Val Phe Gly Phe Glu Thr 595 600 605 Ser Glu Ala Lys Gly
Pro Asp Gly Met Ala Leu Pro Arg Pro Arg Asp 610 615 620 Gln Ala Val
Phe Pro Gln Asn Gly Leu Val Val Ser Ser Val Asp Val 625 630 635 640
Gln Ser Val Glu Pro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln Arg 645
650 655 Ser Val Gln Leu Ala Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala
Ala 660 665 670 Ala Lys His Glu Ala Gln Arg Leu Glu Gln Glu Ala Arg
Gly Arg Leu 675 680 685 Glu Arg Gln Lys Ile Leu Asp Gln Ser Glu Ala
Glu Lys Ala Arg Lys 690 695 700 Glu Leu Leu Glu Leu Glu Ala Leu Ser
Met Ala Val Glu Ser Thr Gly 705 710 715 720 Thr Ala Lys Ala Glu Ala
Glu Ser Arg Ala Glu Ala Ala Arg Ile Glu 725 730 735 Gly Glu Gly Ser
Val Leu Gln Ala Lys Leu Lys Ala Gln Ala Leu Ala 740 745 750 Ile Glu
Thr Glu Ala Glu Leu Gln Arg Val Gln Lys Val Arg Glu Leu 755 760 765
Glu Leu Val Tyr Ala Arg Ala Gln Leu Glu Leu Glu Val Ser Lys Ala 770
775 780 Gln Gln Leu Ala Glu Val Glu Val Lys Lys Phe Lys Gln Met Thr
Glu 785 790 795 800 Ala Ile Gly Pro Ser Thr Ile Arg Asp Leu Ala Val
Ala Gly Pro Glu 805 810 815 Met Gln Val Lys Leu Leu Gln Ser Leu Gly
Leu Lys Ser Thr Leu Ile 820 825 830 Thr Asp Gly Ser Thr Pro Ile Asn
Leu Phe Asn Thr Ala Phe Gly Leu 835 840 845 Leu Gly Met Gly Pro Glu
Gly Gln Pro Leu Gly Arg Arg Val Pro Val 850 855 860 Ala Gln Pro Trp
Gly Gly Asp Ile Pro Pro Val Cys Ser Gly Pro Ser 865 870 875 880 Ser
Ser Trp Arg Gln Pro Arg Gly Ala Cys Thr Ala Leu Thr Pro Asp 885 890
895 14893PRTHomo Sapiens 14Met Ala Thr Glu Glu Phe Ile Ile Arg Ile
Pro Pro Tyr His Tyr Ile 1 5 10 15 His Val Leu Asp Gln Asn Ser Asn
Val Ser Arg Val Glu Val Gly Pro 20 25 30 Lys Thr Tyr Ile Arg Gln
Asp Asn Glu Arg Val Leu Phe Ala Pro Met 35 40 45 Arg Met Val Thr
Val Pro Pro Arg His Tyr Cys Thr Val Ala Asn Pro 50 55 60 Val Ser
Arg Asp Ala Gln Gly Leu Val Leu Phe Asp Val Thr Gly Gln 65 70 75 80
Val Arg Leu Arg His Ala Asp Leu Glu Ile Arg Leu Ala Gln Asp Pro 85
90 95 Phe Pro Leu Tyr Pro Gly Glu Val Leu Glu Lys Asp Ile Thr Pro
Leu 100 105 110 Gln Val Val Leu Pro Asn Thr Ala Leu His Leu Lys Ala
Leu Leu Asp 115 120 125 Phe Glu Asp Lys Asp Gly Asp Lys Val Val Ala
Gly Asp Glu Trp Leu 130 135 140 Phe Glu Gly Pro Gly Thr Tyr Ile Pro
Arg Lys Glu Val Glu Val Val 145 150 155 160 Glu Ile Ile Gln Ala Thr
Ile Ile Arg Gln Asn Gln Ala Leu Arg Leu 165 170 175 Arg Ala Arg Lys
Glu Cys Trp Asp Arg Asp Gly Lys Glu Arg Val Thr 180 185 190 Gly Glu
Glu Trp Leu Val Thr Thr Val Gly Ala Tyr Leu Pro Ala Val 195 200 205
Phe Glu Glu Val Leu Asp Leu Val Asp Ala Val Ile Leu Thr Glu Lys 210
215 220 Thr Ala Leu His Leu Arg Ala Arg Arg Asn Phe Arg Asp Phe Arg
Gly 225 230 235 240 Val Ser Arg Arg Thr Gly Glu Glu Trp Leu Val Thr
Val Gln Asp Thr 245 250 255 Glu Ala His Val Pro Asp Val His Glu Glu
Val Leu Gly Val Val Pro 260 265 270 Ile Thr Thr Leu Gly Pro His Asn
Tyr Cys Val Ile Leu Asp Pro Val 275 280 285 Gly Pro Asp Gly Lys Asn
Gln Leu Gly Gln Lys Arg Val Val Lys Gly 290 295 300 Glu Lys Ser Phe
Phe Leu Gln Pro Gly Glu Gln Leu Glu Gln Gly Ile 305 310 315 320 Gln
Asp Val Tyr Val Leu Ser Glu Gln Gln Gly Leu Leu Leu Arg Ala 325 330
335 Leu Gln Pro Leu Glu Glu Gly Glu Asp Glu Glu Lys Val Ser His Gln
340 345 350 Ala Gly Asp His Trp Leu Ile Arg Gly Pro Leu Glu Tyr Val
Pro Ser 355 360 365 Ala Lys Val Glu Val Val Glu Glu Arg Gln Ala Ile
Pro Leu Asp Glu 370 375 380 Asn Glu Gly Ile Tyr Val Gln Asp Val Lys
Thr Gly Lys Val Arg Ala 385 390 395 400 Val Ile Gly Ser Thr Tyr Met
Leu Thr Gln Asp Glu Val Leu Trp Glu 405 410 415 Lys Glu Leu Pro Pro
Gly Val Glu Glu Leu Leu Asn Lys Gly Gln Asp 420 425 430 Pro Leu Ala
Asp Arg Gly Glu Lys Asp Thr Ala Lys Ser Leu Gln Pro 435 440 445 Leu
Ala Pro Arg Asn Lys Thr Arg Val Val Ser Tyr Arg
Val Pro His 450 455 460 Asn Ala Ala Val Gln Val Tyr Asp Tyr Arg Glu
Lys Arg Ala Arg Val 465 470 475 480 Val Phe Gly Pro Glu Leu Val Ser
Leu Gly Pro Glu Glu Gln Phe Thr 485 490 495 Val Leu Ser Leu Ser Ala
Gly Arg Pro Lys Arg Pro His Ala Arg Arg 500 505 510 Ala Leu Cys Leu
Leu Leu Gly Pro Asp Phe Phe Thr Asp Val Ile Thr 515 520 525 Ile Glu
Thr Ala Asp His Ala Arg Leu Gln Leu Gln Leu Ala Tyr Asn 530 535 540
Trp His Phe Glu Val Asn Asp Arg Lys Asp Pro Gln Glu Thr Ala Lys 545
550 555 560 Leu Phe Ser Val Pro Asp Phe Val Gly Asp Ala Cys Lys Ala
Ile Ala 565 570 575 Ser Arg Val Arg Gly Ala Val Ala Ser Val Thr Phe
Asp Asp Phe His 580 585 590 Lys Asn Ser Ala Arg Ile Ile Arg Thr Ala
Val Phe Gly Phe Glu Thr 595 600 605 Ser Glu Ala Lys Gly Pro Asp Gly
Met Ala Leu Pro Arg Pro Arg Asp 610 615 620 Gln Ala Val Phe Pro Gln
Asn Gly Leu Val Val Ser Ser Val Asp Val 625 630 635 640 Gln Ser Val
Glu Pro Val Asp Gln Arg Thr Arg Asp Ala Leu Gln Arg 645 650 655 Ser
Val Gln Leu Ala Ile Glu Ile Thr Thr Asn Ser Gln Glu Ala Ala 660 665
670 Ala Lys His Glu Ala Gln Arg Leu Glu Gln Glu Ala Arg Gly Arg Leu
675 680 685 Glu Arg Gln Lys Ile Leu Asp Gln Ser Glu Ala Glu Lys Ala
Arg Lys 690 695 700 Glu Leu Leu Glu Leu Glu Ala Leu Ser Met Ala Val
Glu Ser Thr Gly 705 710 715 720 Thr Ala Lys Ala Glu Ala Glu Ser Arg
Ala Glu Ala Ala Arg Ile Glu 725 730 735 Gly Glu Gly Ser Val Leu Gln
Ala Lys Leu Lys Ala Gln Ala Leu Ala 740 745 750 Ile Glu Thr Glu Ala
Glu Leu Gln Arg Val Gln Lys Val Arg Glu Leu 755 760 765 Glu Leu Val
Tyr Ala Arg Ala Gln Leu Glu Leu Glu Val Ser Lys Ala 770 775 780 Gln
Gln Leu Ala Glu Val Glu Val Lys Lys Phe Lys Gln Met Thr Glu 785 790
795 800 Ala Ile Gly Pro Ser Thr Ile Arg Asp Leu Ala Val Ala Gly Pro
Glu 805 810 815 Met Gln Val Lys Leu Leu Gln Ser Leu Gly Leu Lys Ser
Thr Leu Ile 820 825 830 Thr Asp Gly Ser Thr Pro Ile Asn Leu Phe Asn
Thr Ala Phe Gly Leu 835 840 845 Leu Gly Met Gly Pro Glu Gly Gln Pro
Leu Gly Arg Arg Val Ala Ser 850 855 860 Gly Pro Ser Pro Gly Glu Gly
Ile Ser Pro Gln Ser Ala Gln Ala Pro 865 870 875 880 Gln Ala Pro Gly
Asp Asn His Val Val Pro Val Leu Arg 885 890 15508PRTHomo Sapiens
15Met Leu Arg Arg Ala Leu Leu Cys Leu Ala Val Ala Ala Leu Val Arg 1
5 10 15 Ala Asp Ala Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys
Ser 20 25 30 Asn Phe Ala Glu Ala Leu Ala Ala His Lys Tyr Leu Leu
Val Glu Phe 35 40 45 Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu
Ala Pro Glu Tyr Ala 50 55 60 Lys Ala Ala Gly Lys Leu Lys Ala Glu
Gly Ser Glu Ile Arg Leu Ala 65 70 75 80 Lys Val Asp Ala Thr Glu Glu
Ser Asp Leu Ala Gln Gln Tyr Gly Val 85 90 95 Arg Gly Tyr Pro Thr
Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser 100 105 110 Pro Lys Glu
Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp 115 120 125 Leu
Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Arg Asp Gly Ala 130 135
140 Ala Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe
145 150 155 160 Phe Lys Asp Val Glu Ser Asp Ser Ala Lys Gln Phe Leu
Gln Ala Ala 165 170 175 Glu Ala Ile Asp Asp Ile Pro Phe Gly Ile Thr
Ser Asn Ser Asp Val 180 185 190 Phe Ser Lys Tyr Gln Leu Asp Lys Asp
Gly Val Val Leu Phe Lys Lys 195 200 205 Phe Asp Glu Gly Arg Asn Asn
Phe Glu Gly Glu Val Thr Lys Glu Asn 210 215 220 Leu Leu Asp Phe Ile
Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe 225 230 235 240 Thr Glu
Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His 245 250 255
Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu 260
265 270 Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu
Phe 275 280 285 Ile Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile
Leu Glu Phe 290 295 300 Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val
Arg Leu Ile Thr Leu 305 310 315 320 Glu Glu Glu Met Thr Lys Tyr Lys
Pro Glu Ser Glu Glu Leu Thr Ala 325 330 335 Glu Arg Ile Thr Glu Phe
Cys His Arg Phe Leu Glu Gly Lys Ile Lys 340 345 350 Pro His Leu Met
Ser Gln Glu Arg Ala Gly Asp Trp Asp Lys Gln Pro 355 360 365 Val Lys
Val Pro Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu 370 375 380
Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys 385
390 395 400 Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr
Lys Asp 405 410 415 His Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr
Ala Asn Glu Val 420 425 430 Glu Ala Val Lys Val His Ser Phe Pro Thr
Leu Lys Phe Phe Pro Ala 435 440 445 Ser Ala Asp Arg Thr Val Ile Asp
Tyr Asn Gly Glu Arg Thr Leu Asp 450 455 460 Gly Phe Lys Lys Phe Leu
Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp 465 470 475 480 Asp Asp Asp
Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu 485 490 495 Glu
Asp Asp Asp Gln Lys Ala Val Lys Asp Glu Leu 500 505 16508PRTHomo
sapiens 16Met Leu Arg Arg Ala Leu Leu Cys Leu Ala Val Ala Ala Leu
Val Arg 1 5 10 15 Ala Asp Ala Pro Glu Glu Glu Asp His Val Leu Val
Leu Arg Lys Ser 20 25 30 Asn Phe Ala Glu Ala Leu Ala Ala His Lys
Tyr Leu Leu Val Glu Phe 35 40 45 Tyr Ala Pro Trp Cys Gly His Cys
Lys Ala Leu Ala Pro Glu Tyr Ala 50 55 60 Lys Ala Ala Gly Lys Leu
Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala 65 70 75 80 Lys Val Asp Ala
Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val 85 90 95 Arg Gly
Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser 100 105 110
Pro Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp 115
120 125 Leu Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly
Ala 130 135 140 Ala Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala Val
Ile Gly Phe 145 150 155 160 Phe Lys Asp Val Glu Ser Asp Ser Ala Lys
Gln Phe Leu Gln Ala Ala 165 170 175 Glu Ala Ile Asp Asp Ile Pro Phe
Gly Ile Thr Ser Asn Ser Asp Val 180 185 190 Phe Ser Lys Tyr Gln Leu
Asp Lys Asp Gly Val Val Leu Phe Lys Lys 195 200 205 Phe Asp Glu Gly
Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn 210 215 220 Leu Leu
Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe 225 230 235
240 Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His
245 250 255 Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly
Lys Leu 260 265 270 Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly
Lys Ile Leu Phe 275 280 285 Ile Phe Ile Asp Ser Asp His Thr Asp Asn
Gln Arg Ile Leu Glu Phe 290 295 300 Phe Gly Leu Lys Lys Glu Glu Cys
Pro Ala Val Arg Leu Ile Thr Leu 305 310 315 320 Glu Glu Glu Met Thr
Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala 325 330 335 Glu Arg Ile
Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys 340 345 350 Pro
His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro 355 360
365 Val Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu
370 375 380 Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly
His Cys 385 390 395 400 Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly
Glu Thr Tyr Lys Asp 405 410 415 His Glu Asn Ile Val Ile Ala Lys Met
Asp Ser Thr Ala Asn Glu Val 420 425 430 Glu Ala Val Lys Val His Ser
Phe Pro Thr Leu Lys Phe Phe Pro Ala 435 440 445 Ser Ala Asp Arg Thr
Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp 450 455 460 Gly Phe Lys
Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp 465 470 475 480
Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu 485
490 495 Glu Asp Asp Asp Gln Lys Ala Val Lys Asp Glu Leu 500 505
17120PRTHomo Sapiens 17Asp Ala Pro Glu Glu Glu Asp His Val Leu Val
Leu Arg Lys Ser Asn 1 5 10 15 Phe Ala Glu Ala Leu Ala Ala His Lys
Tyr Leu Leu Val Glu Phe Tyr 20 25 30 Ala Pro Trp Cys Gly His Cys
Lys Ala Leu Ala Pro Glu Tyr Ala Lys 35 40 45 Ala Ala Gly Lys Leu
Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys 50 55 60 Val Asp Ala
Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg 65 70 75 80 Gly
Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro 85 90
95 Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu
100 105 110 Lys Lys Arg Thr Gly Pro Ala Ala 115 120 18255PRTHomo
Sapiens 18Met Ala Glu Leu Arg Val Leu Val Ala Val Lys Arg Val Ile
Asp Tyr 1 5 10 15 Ala Val Lys Ile Arg Val Lys Pro Asp Arg Thr Gly
Val Val Thr Asp 20 25 30 Gly Val Lys His Ser Met Asn Pro Phe Cys
Glu Ile Ala Val Glu Glu 35 40 45 Ala Val Arg Leu Lys Glu Lys Lys
Leu Val Lys Glu Val Ile Ala Val 50 55 60 Ser Cys Gly Pro Ala Gln
Cys Gln Glu Thr Ile Arg Thr Ala Leu Ala 65 70 75 80 Met Gly Ala Asp
Arg Gly Ile His Val Glu Val Pro Pro Ala Glu Ala 85 90 95 Glu Arg
Leu Gly Pro Leu Gln Val Ala Arg Val Leu Ala Lys Leu Ala 100 105 110
Glu Lys Glu Lys Val Asp Leu Val Leu Leu Gly Lys Gln Ala Ile Asp 115
120 125 Asp Asp Cys Asn Gln Thr Gly Gln Met Thr Ala Gly Phe Leu Asp
Trp 130 135 140 Pro Gln Gly Thr Phe Ala Ser Gln Val Thr Leu Glu Gly
Asp Lys Leu 145 150 155 160 Lys Val Glu Arg Glu Ile Asp Gly Gly Leu
Glu Thr Leu Arg Leu Lys 165 170 175 Leu Pro Ala Val Val Thr Ala Asp
Leu Arg Leu Asn Glu Pro Arg Tyr 180 185 190 Ala Thr Leu Pro Asn Ile
Met Lys Ala Lys Lys Lys Lys Ile Glu Val 195 200 205 Ile Lys Pro Gly
Asp Leu Gly Val Asp Leu Thr Ser Lys Leu Ser Val 210 215 220 Ile Ser
Val Glu Asp Pro Pro Gln Arg Thr Ala Gly Val Lys Val Glu 225 230 235
240 Thr Thr Glu Asp Leu Val Ala Lys Leu Lys Glu Ile Gly Arg Ile 245
250 255 19553PRTHomo sapiens 19 Met Leu Ser Val Arg Val Ala Ala Ala
Val Val Arg Ala Leu Pro Arg 1 5 10 15 Arg Ala Gly Leu Val Ser Arg
Asn Ala Leu Gly Ser Ser Phe Ile Ala 20 25 30 Ala Arg Asn Phe His
Ala Ser Asn Thr His Leu Gln Lys Thr Gly Thr 35 40 45 Ala Glu Met
Ser Ser Ile Leu Glu Glu Arg Ile Leu Gly Ala Asp Thr 50 55 60 Ser
Val Asp Leu Glu Glu Thr Gly Arg Val Leu Ser Ile Gly Asp Gly 65 70
75 80 Ile Ala Arg Val His Gly Leu Arg Asn Val Gln Ala Glu Glu Met
Val 85 90 95 Glu Phe Ser Ser Gly Leu Lys Gly Met Ser Leu Asn Leu
Glu Pro Asp 100 105 110 Asn Val Gly Val Val Val Phe Gly Asn Asp Lys
Leu Ile Lys Glu Gly 115 120 125 Asp Ile Val Lys Arg Thr Gly Ala Ile
Val Asp Val Pro Val Gly Glu 130 135 140 Glu Leu Leu Gly Arg Val Val
Asp Ala Leu Gly Asn Ala Ile Asp Gly 145 150 155 160 Lys Gly Pro Ile
Gly Ser Lys Thr Arg Arg Arg Val Gly Leu Lys Ala 165 170 175 Pro Gly
Ile Ile Pro Arg Ile Ser Val Arg Glu Pro Met Gln Thr Gly 180 185 190
Ile Lys Ala Val Asp Ser Leu Val Pro Ile Gly Arg Gly Gln Arg Glu 195
200 205 Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys Thr Ser Ile Ala Ile
Asp 210 215 220 Thr Ile Ile Asn Gln Lys Arg Phe Asn Asp Gly Ser Asp
Glu Lys Lys 225 230 235 240 Lys Leu Tyr Cys Ile Tyr Val Ala Ile Gly
Gln Lys Arg Ser Thr Val 245 250 255 Ala Gln Leu Val Lys Arg Leu Thr
Asp Ala Asp Ala Met Lys Tyr Thr 260 265 270 Ile Val Val Ser Ala Thr
Ala Ser Asp Ala Ala Pro Leu Gln Tyr Leu 275 280 285 Ala Pro Tyr Ser
Gly Cys Ser Met Gly Glu Tyr Phe Arg Asp Asn Gly 290 295 300 Lys His
Ala Leu Ile Ile Tyr Asp Asp Leu Ser Lys Gln Ala Val Ala 305 310 315
320 Tyr Arg Gln Met Ser Leu Leu Leu Arg Arg Pro Pro Gly Arg Glu Ala
325 330 335 Tyr Pro Gly Asp Val Phe Tyr Leu His Ser Arg Leu Leu Glu
Arg Ala 340 345 350 Ala Lys Met Asn Asp Ala Phe Gly Gly Gly Ser Leu
Thr Ala Leu Pro 355 360 365 Val Ile Glu Thr Gln Ala Gly Asp Val Ser
Ala Tyr Ile Pro Thr Asn 370 375 380 Val Ile Ser Ile Thr Asp Gly Gln
Ile Phe Leu Glu Thr Glu Leu Phe 385 390 395 400 Tyr Lys Gly Ile Arg
Pro Ala Ile Asn Val Gly Leu Ser Val Ser Arg 405 410 415 Val Gly Ser
Ala Ala Gln Thr Arg Ala Met Lys Gln Val Ala Gly Thr 420 425 430 Met
Lys Leu Glu Leu Ala Gln Tyr Arg Glu Val Ala Ala Phe Ala Gln 435 440
445 Phe Gly Ser Asp Leu Asp Ala Ala Thr Gln Gln Leu Leu Ser Arg Gly
450 455
460 Val Arg Leu Thr Glu Leu Leu Lys Gln Gly Gln Tyr Ser Pro Met Ala
465 470 475 480 Ile Glu Glu Gln Val Ala Val Ile Tyr Ala Gly Val Arg
Gly Tyr Leu 485 490 495 Asp Lys Leu Glu Pro Ser Lys Ile Thr Lys Phe
Glu Asn Ala Phe Leu 500 505 510 Ser His Val Val Ser Gln His Gln Ala
Leu Leu Gly Thr Ile Arg Ala 515 520 525 Asp Gly Lys Ile Ser Glu Gln
Ser Asp Ala Lys Leu Lys Glu Ile Val 530 535 540 Thr Asn Phe Leu Ala
Gly Phe Glu Ala 545 550 20508PRTHomo
sapiensmisc_feature(12)..(12)Xaa can be any naturally occurring
amino acid 20Met Leu Arg Arg Ala Leu Leu Cys Leu Pro Trp Xaa Ala
Leu Val Arg 1 5 10 15 Ala Asp Ala Pro Glu Glu Glu Asp His Val Leu
Val Leu Arg Lys Ser 20 25 30 Asn Phe Ala Glu Ala Leu Ala Ala His
Lys Tyr Pro Pro Val Glu Phe 35 40 45 His Ala Pro Trp Cys Gly His
Cys Lys Ala Leu Ala Pro Glu Tyr Ala 50 55 60 Lys Ala Ala Gly Lys
Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala 65 70 75 80 Lys Val Asp
Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val 85 90 95 Arg
Gly Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser 100 105
110 Pro Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp
115 120 125 Leu Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp
Gly Ala 130 135 140 Ala Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala
Val Ile Gly Phe 145 150 155 160 Phe Lys Asp Val Glu Ser Asp Ser Ala
Lys Gln Phe Leu Gln Ala Ala 165 170 175 Glu Ala Ile Asp Asp Ile Pro
Phe Gly Ile Thr Ser Asn Ser Asp Val 180 185 190 Phe Ser Lys Tyr Gln
Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys 195 200 205 Phe Asp Glu
Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn 210 215 220 Leu
Leu Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe 225 230
235 240 Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr
His 245 250 255 Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp
Gly Lys Leu 260 265 270 Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys
Gly Lys Ile Leu Phe 275 280 285 Ile Phe Ile Asp Ser Asp His Thr Asp
Asn Gln Arg Ile Leu Glu Phe 290 295 300 Phe Gly Leu Lys Lys Glu Glu
Cys Pro Ala Val Arg Leu Ile Thr Leu 305 310 315 320 Glu Glu Glu Met
Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala 325 330 335 Glu Arg
Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys 340 345 350
Pro His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro 355
360 365 Val Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp
Glu 370 375 380 Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys
Gly His Cys 385 390 395 400 Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu
Gly Glu Thr Tyr Lys Asp 405 410 415 His Glu Asn Ile Val Ile Ala Lys
Met Asp Ser Thr Ala Asn Glu Val 420 425 430 Glu Ala Val Lys Val His
Gly Phe Pro Thr Leu Gly Phe Phe Pro Ala 435 440 445 Ser Ala Asp Arg
Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp 450 455 460 Gly Phe
Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp 465 470 475
480 Val Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu
485 490 495 Glu Asp Asp Asp Gln Lys Ala Val Lys Asp Glu Leu 500 505
21255PRTHomo sapiens 21Met Ala Glu Leu Arg Val Leu Val Ala Val Lys
Arg Val Ile Asp Tyr 1 5 10 15 Ala Val Lys Ile Arg Val Lys Pro Asp
Arg Thr Gly Val Val Thr Asp 20 25 30 Gly Val Lys His Ser Met Asn
Pro Phe Cys Glu Ile Ala Val Glu Glu 35 40 45 Ala Val Arg Leu Lys
Glu Lys Lys Leu Val Lys Glu Val Ile Ala Val 50 55 60 Ser Cys Gly
Pro Ala Gln Cys Gln Glu Thr Ile Arg Thr Ala Leu Ala 65 70 75 80 Met
Gly Ala Asp Arg Gly Ile His Val Glu Val Pro Pro Ala Glu Ala 85 90
95 Glu Arg Leu Gly Pro Leu Gln Val Ala Arg Val Leu Ala Lys Leu Ala
100 105 110 Glu Lys Glu Lys Val Asp Leu Val Leu Leu Gly Lys Gln Ala
Ile Tyr 115 120 125 Asp Asp Cys Asn Gln Thr Gly Gln Met Thr Ala Gly
Phe Leu Asp Trp 130 135 140 Pro Gln Gly Thr Phe Ala Ser Gln Val Met
Leu Glu Gly Asp Lys Leu 145 150 155 160 Lys Val Glu Arg Glu Ile Asp
Gly Gly Leu Glu Thr Leu Arg Leu Lys 165 170 175 Leu Pro Ala Val Val
Thr Ala Asp Leu Arg Leu Asn Glu Pro Arg Tyr 180 185 190 Ala Thr Leu
Pro Asn Ile Met Lys Ala Lys Lys Lys Lys Ile Glu Val 195 200 205 Ile
Lys Pro Gly Asp Leu Gly Val Asp Leu Thr Ser Lys Leu Ser Val 210 215
220 Ile Ser Val Glu Asp Pro Pro Gln Arg Thr Ala Gly Val Lys Val Glu
225 230 235 240 Thr Thr Glu Asp Leu Val Ala Lys Leu Lys Glu Ile Gly
Arg Ile 245 250 255 22503PRTHomo sapiens 22 Met Ser Ser Ile Leu Glu
Glu Arg Ile Leu Gly Ala Asp Thr Ser Val 1 5 10 15 Asp Leu Glu Glu
Thr Gly Arg Val Leu Ser Ile Gly Asp Gly Ile Ala 20 25 30 Arg Val
His Gly Leu Arg Asn Val Gln Ala Glu Glu Met Val Glu Phe 35 40 45
Ser Ser Gly Leu Lys Gly Met Ser Leu Asn Leu Glu Pro Asp Asn Val 50
55 60 Gly Val Val Val Phe Gly Asn Asp Lys Leu Ile Lys Glu Gly Asp
Ile 65 70 75 80 Val Lys Arg Thr Gly Ala Ile Val Asp Val Pro Val Gly
Glu Glu Leu 85 90 95 Leu Gly Arg Val Val Asp Ala Leu Gly Asn Ala
Ile Asp Gly Lys Gly 100 105 110 Pro Ile Gly Ser Lys Thr Arg Arg Arg
Val Gly Leu Lys Ala Pro Gly 115 120 125 Ile Ile Pro Arg Ile Ser Val
Arg Glu Pro Met Gln Thr Gly Ile Lys 130 135 140 Ala Val Asp Ser Leu
Val Pro Ile Gly Arg Gly Gln Arg Glu Leu Ile 145 150 155 160 Ile Gly
Asp Arg Gln Thr Gly Lys Thr Ser Ile Ala Ile Asp Thr Ile 165 170 175
Ile Asn Gln Lys Arg Phe Asn Asp Gly Ser Asp Glu Lys Lys Lys Leu 180
185 190 Tyr Cys Ile Tyr Val Ala Ile Gly Gln Lys Arg Ser Thr Val Ala
Gln 195 200 205 Leu Val Lys Arg Leu Thr Asp Ala Asp Ala Met Lys Tyr
Thr Ile Val 210 215 220 Val Ser Ala Thr Ala Ser Asp Ala Ala Pro Leu
Gln Tyr Leu Ala Pro 225 230 235 240 Tyr Ser Gly Cys Ser Met Gly Glu
Tyr Phe Arg Asp Asn Gly Lys His 245 250 255 Ala Leu Ile Ile Tyr Asp
Asp Leu Ser Lys Gln Ala Val Ala Tyr Arg 260 265 270 Gln Met Ser Leu
Leu Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro 275 280 285 Gly Asp
Val Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys 290 295 300
Met Asn Asp Ala Phe Gly Gly Gly Ser Leu Thr Ala Leu Pro Val Ile 305
310 315 320 Glu Thr Gln Ala Gly Asp Val Ser Ala Tyr Ile Pro Thr Asn
Val Ile 325 330 335 Ser Ile Thr Asp Gly Gln Ile Phe Leu Glu Thr Glu
Leu Phe Tyr Lys 340 345 350 Gly Ile Arg Pro Ala Ile Asn Val Gly Leu
Ser Val Ser Arg Val Gly 355 360 365 Ser Ala Ala Gln Thr Arg Ala Met
Lys Gln Val Ala Gly Thr Met Lys 370 375 380 Leu Glu Leu Ala Gln Tyr
Arg Glu Val Ala Ala Phe Ala Gln Phe Gly 385 390 395 400 Ser Asp Leu
Asp Ala Ala Thr Gln Gln Leu Leu Ser Arg Gly Val Arg 405 410 415 Leu
Thr Glu Leu Leu Lys Gln Gly Gln Tyr Ser Pro Met Ala Ile Glu 420 425
430 Glu Gln Val Ala Val Ile Tyr Ala Gly Val Arg Gly Tyr Leu Asp Lys
435 440 445 Leu Glu Pro Ser Lys Ile Thr Lys Phe Glu Asn Ala Phe Leu
Ser His 450 455 460 Val Val Ser Gln His Gln Ala Leu Leu Gly Thr Ile
Arg Ala Asp Gly 465 470 475 480 Lys Ile Ser Glu Gln Ser Asp Ala Lys
Leu Lys Glu Ile Val Thr Asn 485 490 495 Phe Leu Ala Gly Phe Glu Ala
500 23339PRTHomo sapiens 23Met Trp Gln Leu Trp Ala Ser Leu Cys Cys
Leu Leu Val Leu Ala Asn 1 5 10 15 Ala Arg Ser Arg Pro Ser Phe His
Pro Leu Ser Asp Glu Leu Val Asn 20 25 30 Tyr Val Asn Lys Arg Asn
Thr Thr Trp Gln Ala Gly His Asn Phe Tyr 35 40 45 Asn Val Asp Met
Ser Tyr Leu Lys Arg Leu Cys Gly Thr Phe Leu Gly 50 55 60 Gly Pro
Lys Pro Pro Gln Arg Val Met Phe Thr Glu Asp Leu Lys Leu 65 70 75 80
Pro Ala Ser Phe Asp Ala Arg Glu Gln Trp Pro Gln Cys Pro Thr Ile 85
90 95 Lys Glu Ile Arg Asp Gln Gly Ser Cys Gly Ser Cys Trp Ala Phe
Gly 100 105 110 Ala Val Glu Ala Ile Ser Asp Arg Ile Cys Ile His Thr
Asn Ala His 115 120 125 Val Ser Val Glu Val Ser Ala Glu Asp Leu Leu
Thr Cys Cys Gly Ser 130 135 140 Met Cys Gly Asp Gly Cys Asn Gly Gly
Tyr Pro Ala Glu Ala Trp Asn 145 150 155 160 Phe Trp Thr Arg Lys Gly
Leu Val Ser Gly Gly Leu Tyr Glu Ser His 165 170 175 Val Gly Cys Arg
Pro Tyr Ser Ile Pro Pro Cys Glu His His Val Asn 180 185 190 Gly Ser
Arg Pro Pro Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Ser 195 200 205
Lys Ile Cys Glu Pro Gly Tyr Ser Pro Thr Tyr Lys Gln Asp Lys His 210
215 220 Tyr Gly Tyr Asn Ser Tyr Ser Val Ser Asn Ser Glu Lys Asp Ile
Met 225 230 235 240 Ala Glu Ile Tyr Lys Asn Gly Pro Val Glu Gly Ala
Phe Ser Val Tyr 245 250 255 Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val
Tyr Gln His Val Thr Gly 260 265 270 Glu Met Met Gly Gly His Ala Ile
Arg Ile Leu Gly Trp Gly Val Glu 275 280 285 Asn Gly Thr Pro Tyr Trp
Leu Val Ala Asn Ser Trp Asn Thr Asp Trp 290 295 300 Gly Asp Asn Gly
Phe Phe Lys Ile Leu Arg Gly Gln Asp His Cys Gly 305 310 315 320 Ile
Glu Ser Glu Val Val Ala Gly Ile Pro Arg Thr Asp Gln Tyr Trp 325 330
335 Glu Lys Ile 2410PRTArtificial sequenceSynthetic construct 24Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 259PRTArtificial
SequenceSynthetic construct 25Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1
5 266PRTArtificial SequenceSynthetic construct 26His His His His
His His 1 5 27238PRTAequorea victoria 27Met Ser Lys Gly Glu Glu Leu
Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55
60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr
Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val
Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185
190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu
Tyr Lys 225 230 235 28194PRTAliiyibrio fischeri 28 Met Phe Lys Gly
Ile Val Glu Gly Ile Gly Ile Ile Glu Lys Ile Asp 1 5 10 15 Ile Tyr
Thr Asp Leu Asp Lys Tyr Ala Ile Arg Phe Pro Glu Asn Met 20 25 30
Leu Asn Gly Ile Lys Lys Glu Ser Ser Ile Met Phe Asn Gly Cys Phe 35
40 45 Leu Thr Val Thr Ser Val Asn Ser Asn Ile Val Trp Phe Asp Ile
Phe 50 55 60 Glu Lys Glu Ala Arg Lys Leu Asp Thr Phe Arg Glu Tyr
Lys Val Gly 65 70 75 80 Asp Arg Val Asn Leu Gly Thr Phe Pro Lys Phe
Gly Ala Ala Ser Gly 85 90 95 Gly His Ile Leu Ser Ala Arg Ile Ser
Cys Val Ala Ser Ile Ile Glu 100 105 110 Ile Ile Glu Asn Glu Asp Tyr
Gln Gln Met Trp Ile Gln Ile Pro Glu 115 120 125 Asn Phe Thr Glu Phe
Leu Ile Asp Lys Asp Tyr Ile Ala Val Asp Gly 130 135 140 Ile Ser Leu
Thr Ile Asp Thr Ile Lys Asn Asn Gln Phe Phe Ile Ser 145 150 155 160
Leu Pro Leu Lys Ile Ala Gln Asn Thr Asn Met Lys Trp Arg Lys Lys 165
170 175 Gly Asp Lys Val Asn Val Glu Leu Ser Asn Lys Ile Asn Ala Asn
Gln 180 185 190 Cys Trp 29225PRTMontastraea cavernosa 29Met Ser Val
Ile Lys Ser Val Met Lys Ile Lys Leu Arg Met Asp Gly 1 5 10 15 Ile
Val Asn Gly His Lys Phe Met Ile Thr Gly Glu Gly Glu Gly Lys 20 25
30 Pro Phe Glu Gly Thr His Thr Ile Ile Leu Lys Val Lys Glu Gly Gly
35 40 45 Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Ala Phe Gln
Tyr Gly 50 55 60 Asn Arg Val Phe Thr Lys Tyr Pro Lys Asp Ile Pro
Asp Tyr Phe Lys 65 70 75 80 Gln Ser Phe Pro Glu Gly Tyr Ser Trp Glu
Arg Ser Met Thr Phe Glu 85 90 95 Asp Gln Gly Val
Cys Thr Val Thr Ser Asp Ile Lys Leu Glu Gly Asp 100 105 110 Cys Phe
Phe Tyr Glu Ile Arg Phe Tyr Gly Val Asn Phe Pro Ser Ser 115 120 125
Gly Pro Val Met Gln Lys Lys Thr Leu Lys Trp Glu Pro Ser Thr Glu 130
135 140 Asn Met Tyr Val Arg Asp Gly Val Leu Leu Gly Asp Val Ser Arg
Thr 145 150 155 160 Leu Leu Leu Glu Gly Asp Lys His His Arg Cys Asn
Phe Arg Ser Thr 165 170 175 Tyr Gly Ala Lys Lys Gly Val Val Leu Pro
Glu Tyr His Phe Val Asp 180 185 190 His Arg Ile Glu Ile Leu Ser His
Asp Lys Asp Tyr Asn Thr Val Glu 195 200 205 Val Tyr Glu Asn Ala Val
Ala Arg Pro Ser Met Leu Pro Val Lys Ala 210 215 220 Lys 225
30230PRTDiscosoma sp. 30 Met Ser Cys Ser Lys Asn Val Ile Lys Glu
Phe Met Arg Phe Lys Val 1 5 10 15 Arg Met Glu Gly Thr Val Asn Gly
His Glu Phe Glu Ile Lys Gly Glu 20 25 30 Gly Glu Gly Arg Pro Tyr
Glu Gly His Cys Ser Val Lys Leu Met Val 35 40 45 Thr Lys Gly Gly
Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser Pro Gln 50 55 60 Phe Gln
Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro 65 70 75 80
Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 85
90 95 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Ser Gln Asp Ser
Ser 100 105 110 Leu Lys Asp Gly Cys Phe Ile Tyr Glu Val Lys Phe Ile
Gly Val Asn 115 120 125 Phe Pro Ser Asp Gly Pro Val Met Gln Arg Arg
Thr Arg Gly Trp Glu 130 135 140 Ala Ser Ser Glu Arg Leu Tyr Pro Arg
Asp Gly Val Leu Lys Gly Asp 145 150 155 160 Ile His Met Ala Leu Arg
Leu Glu Gly Gly Gly His Tyr Leu Val Glu 165 170 175 Phe Lys Ser Ile
Tyr Met Val Lys Lys Pro Ser Val Gln Leu Pro Gly 180 185 190 Tyr Tyr
Tyr Val Asp Ser Lys Leu Asp Met Thr Ser His Asn Glu Asp 195 200 205
Tyr Thr Val Val Glu Gln Tyr Glu Lys Thr Gln Gly Arg His His Pro 210
215 220 Phe Ile Lys Pro Leu Gln 225 230 31550PRTPhotinus pyralis 31
Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5
10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys
Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala
His Ile Glu 35 40 45 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met
Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn
Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gln
Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala
Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu
Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser
Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 130 135
140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly
145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro
Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp
Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser
Thr Gly Ser Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala
Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn
Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro
Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255
Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260
265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu
Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile
Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly
Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala
Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu
Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp
Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu
Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380
Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385
390 395 400 Tyr Val Asn Asp Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys
Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu
Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile
Lys Tyr Lys Gly Cys Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser
Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala
Gly Leu Pro Gly Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala
Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu
Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505
510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly
515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala
Lys Lys 530 535 540 Gly Gly Lys Ser Lys Leu 545 550 32311PRTRenilla
reniformis 32Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg
Met Ile Thr 1 5 10 15 Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met
Asn Val Leu Asp Ser 20 25 30 Phe Ile Asn Tyr Tyr Asp Ser Glu Lys
His Ala Glu Asn Ala Val Ile 35 40 45 Phe Leu His Gly Asn Ala Ala
Ser Ser Tyr Leu Trp Arg His Val Val 50 55 60 Pro His Ile Glu Pro
Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly 65 70 75 80 Met Gly Lys
Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 85 90 95 His
Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 100 105
110 Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His
115 120 125 Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His
Ala Glu 130 135 140 Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp
Pro Asp Ile Glu 145 150 155 160 Glu Asp Ile Ala Leu Ile Lys Ser Glu
Glu Gly Glu Lys Met Val Leu 165 170 175 Glu Asn Asn Phe Phe Val Glu
Thr Met Leu Pro Ser Lys Ile Met Arg 180 185 190 Lys Leu Glu Pro Glu
Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 195 200 205 Lys Gly Glu
Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro 210 215 220 Leu
Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr 225 230
235 240 Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe Ile
Glu 245 250 255 Ser Asp Pro Gly Phe Phe Ser Asn Ala Ile Val Glu Gly
Ala Lys Lys 260 265 270 Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly
Leu His Phe Ser Gln 275 280 285 Glu Asp Ala Pro Asp Glu Met Gly Lys
Tyr Ile Lys Ser Phe Val Glu 290 295 300 Arg Val Leu Lys Asn Glu Gln
305 310 3321PRTArtificial SequenceSynthetic construct 33Lys Phe Ala
Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Lys Lys Phe1 5 10 15Ala Lys
Phe Ala Lys 203430PRTArtificial SequenceSynthetic construct 34Gly
Val Tyr His Phe Ala Pro Leu Thr Pro Thr Pro Gly Gly Gly Ser1 5 10
15Lys Phe Ala Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Lys 20 25
30355PRTArtificial SequenceSynthetic construct 35Gly Gly Gly Ser
Xaa1 5
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