U.S. patent application number 10/363204 was filed with the patent office on 2004-09-02 for human and mouse targeting peptides identified by phage display.
Invention is credited to Arap, Wadih, Pasqualini, Renata.
Application Number | 20040170955 10/363204 |
Document ID | / |
Family ID | 32911759 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170955 |
Kind Code |
A1 |
Arap, Wadih ; et
al. |
September 2, 2004 |
Human and mouse targeting peptides identified by phage display
Abstract
The present invention concerns methods and compositions for in
vivo and in vitro targeting. A large number of targeting peptides
directed towards human organs, tissues or cell types are disclosed.
The peptides are of use for targeted delivery of therapeutic
agents, including but not limited to gene therapy vectors. A novel
class of gene therapy vectors is disclosed. Certain of the
disclosed peptides have therapeutic use for inhibiting
angiogenesis, inhibiting tumor growth, inducing apoptosis,
inhibiting pregnancy or inducing weight loss. Methods of
identifying novel targeting peptides in humans, as well as
identifying endogenous receptor-ligand pairs are disclosed. Methods
of identifying novel infectious agents that are causal for human
disease states are also disclosed. A novel mechanism for inducing
apoptosis is further disclosed.
Inventors: |
Arap, Wadih; (Houston,
TX) ; Pasqualini, Renata; (Houston, TX) |
Correspondence
Address: |
Blakely Sokoloff Taylor & Zafman
Seventh Floor
12400 Wilshire Boulevard
Los Angeles
CA
90025-1030
US
|
Family ID: |
32911759 |
Appl. No.: |
10/363204 |
Filed: |
October 6, 2003 |
PCT Filed: |
September 7, 2001 |
PCT NO: |
PCT/US01/27692 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60231266 |
Sep 8, 2000 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/6.14 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
3/10 20180101; A61P 15/00 20180101; A61P 31/12 20180101; A61P 9/00
20180101; A61P 29/00 20180101; A61P 11/00 20180101; A61P 7/12
20180101; A61P 35/00 20180101; C40B 40/02 20130101; A61P 31/00
20180101; A61P 27/02 20180101; C07K 7/06 20130101; C12N 2810/40
20130101; C07K 14/001 20130101; A61P 9/10 20180101; A61P 1/00
20180101; C12N 15/1037 20130101; A61P 31/04 20180101; C07K 7/08
20130101; A61K 38/00 20130101; A61P 19/02 20180101; A61P 37/04
20180101 |
Class at
Publication: |
435/005 ;
435/006 |
International
Class: |
C12Q 001/70; C12Q
001/68 |
Goverment Interests
[0002] This invention was made with government support under grants
DAMD 17-98-1-8041 and 17-98-1-8581 from the U.S. Army and grants
1R01CA78512-01A1, 1R1CA90810-01 and 1R01CA82976-01 from the
National Institutes of Health. The government has certain rights in
this invention.
Claims
What is claimed is:
1. A method comprising a) injecting a subject with a phage display
library; b) obtaining samples of one or more organs or tissues; c)
producing thin sections of the samples; and d) recovering phage
from the thin sections.
2. The method of claim 1, further comprising selecting one or more
portions of a thin section by PALM (Positioning and Ablation with
Laser Microbeams).
3. The method of claim 2, wherein the selected portion contains a
specific cell type.
4. The method of claim 2, wherein the selected portion contains a
homogenous population of cells.
5. The method of claim 3, wherein the cells are cancer cells.
6. The method of claim 1, wherein the phage are recovered by
infecting bacteria with the phage.
7. The method of claim 1, wherein the phage are recovered by
amplifying phage inserts and ligating the amplified inserts to
phage DNA to produce new phage.
8. A method of preparing a phage display library comprising: a)
immunizing a host animal with a target organ, tissue or cell type;
b) obtaining mRNAs encoding antibodies from the host animal; c)
preparing cDNAs from the mRNAs encoding antibodies; and d)
preparing a phage display library from the cDNAs.
9. The method of claim 8, further comprising using antibody
specific primers to amplify cDNAs that encode antibodies.
10. The method of claim 8, wherein the target organ, tissue or cell
is diseased.
11. The method of claim 10, wherein the target comprises cancer
cells.
12. The method of claim 8, further comprising: (i) injecting the
phage display library into a subject; and (ii) recovering phage
from one or more organs, tissues or cell types.
13. The method of claim 8, further comprising screening said
library against a target protein or peptide.
14. A phage display library prepared by the method of claim 8.
15. A method of interfering with pregnancy comprising; a) obtaining
a peptide comprising at least three contiguous amino acids of a
sequence selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,
SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45; and b)
administering the peptide to a female subject.
16. The method of claim 15, wherein the subject is pregnant.
17. The method of claim 15, further comprising attaching an agent
to the peptide.
18. A method of delivering an agent to a fetus comprising: a)
obtaining a peptide comprising at least three contiguous amino
acids of a sequence selected from SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45;
b) attaching the peptide to an agent; and b) administering the
peptide to a pregnant subject.
19. The method of claims 17 or 18, wherein the agent is a drug, a
pro-apoptotic agent, an anti-angiogenic agent, an enzyme, a
hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide,
a protein, an antibiotic, an antibody, a Fab fragment of an
antibody, an imaging agent, an antigen, a survival factor, an
anti-apoptotic agent, a hormone antagonist, a virus, a
bacteriophage, a bacterium, a liposome, a microparticle, a
microdevice, a cell or an expression vector.
20. A method of targeting delivery to adipose tissue comprising: a)
obtaining a targeting peptide comprising an amino acid sequence of
at least three contiguous amino acids selected from SEQ ID NO:47,
SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54 or SEQ ID NO:55; b) attaching the
peptide to an agent to form a complex; and c) administering the
complex to a subject.
21. The method of claim 20, further comprising inducing weight loss
in said subject.
22. An isolated peptide of 100 amino acids or less in size,
comprising at least 3 contiguous amino acids of a sequence selected
from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through
SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID
NO:250.
23. The isolated peptide of claim 22, wherein said peptide is 50
amino acids or less in size.
24. The isolated peptide of claim 22, wherein said peptide is 25
amino acids or less in size.
25. The isolated peptide of claim 22, wherein said peptide is 10
amino acids or less in size.
26. The isolated peptide of claim 22, wherein said peptide is 7
amino acids or less in size.
27. The isolated peptide of claim 22, wherein said peptide is 5
amino acids or less in size.
28. The isolated peptide of claim 22, wherein said peptide
comprises at least 5 contiguous amino acids of a sequence selected
from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through
SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID
NO:250.
29. The isolated peptide of claim 22, wherein said peptide is
attached to a molecule.
30. The isolated peptide of claim 29, wherein said molecule is a
drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis
agent, an anti-angiogenic agent, a hormone, a cytokine, a growth
factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an
antibody, a Fab fragment of an antibody, an imaging agent, survival
factor, an anti-apoptotic agent, a hormone antagonist or an
antigen.
31. The isolated peptide of claim 30, wherein said pro-aptoptosis
agent is selected from the group consisting of gramicidin,
magainin, mellitin, defensin, cecropin, (KLAKLAK).sub.2 (SEQ ID NO:
1), (KLAKKLA).sub.2 (SEQ ID NO:2), (KAAKKAA).sub.2 (SEQ ID NO:3)
and (KLGKKLG)3 (SEQ ID NO:4).
32. The isolated peptide of claim 30, wherein said anti-angiogenic
agent is selected from the group consisting of thrombospondin,
angiostatin5, pigment epithelium-drived factor, angiotensin,
laminin peptides, fibronectin peptides, plasminogen activator
inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, platelet factor 4, IP-10, Gro-.beta.,
thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,
angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP470, endostatin, paclitaxel,
Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor,
a signaling peptide, accutin, cidofovir, vincristine, bleomycin,
AGM-1470, platelet factor 4 and minocycline.
33. The isolated peptide of claim 30, wherein said cytokine is
selected from the group consisting of interleukin 1 (IL-l), IL-2,
IL-5, IL-10, IL-11, IL-12, IL-18, interferon-.gamma. (IF-.gamma.),
IF-.alpha., IF-.beta., tumor necrosis factor-.alpha. (INF-.alpha.),
or GM-CSF (granulocyte macrophage colony stimulating factor).
34. The isolated peptide of claim 22, wherein said peptide is
attached to a macromolecular complex.
35. The isolated peptide of claim 34, wherein said complex is a
virus, a bacteriophage, a bacterium, a liposome, a microparticle, a
magnetic bead, a yeast cell, a mammalian cell or a cell.
36. The isolated peptide of claim 34, wherein said peptide is
attached to a eukaryotic expression vector.
37. The isolated peptide of claim 36, wherein said vector is a gene
therapy vector.
38. The isolated peptide of claim 22, wherein said peptide is
attached to a solid support.
39. A composition comprising the isolated peptide of claim 22 in a
pharmaceutically acceptable carrier.
40. The composition of claim 39, wherein the isolated peptide is
attached to a molecule or a macromolecular complex.
41. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:5 through SEQ ID NO:19.
42. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:20 through SEQ ID NO:38.
43. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:210 through SEQ ID NO:234.
44. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:56 through SEQ ID NO:68.
45. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:69 through SEQ ID NO:88.
46. The isolated peptide of claim 22, wherein said sequence is
selected from any of SEQ ID NO:235 through SEQ ID NO:250.
47. The isolated peptide of claim 22, wherein said sequence is
selected from SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID
NO:92.
48. A kit comprising the isolated peptide of claim 22 and a control
peptide, each in a container.
49. An antibody that selectively binds to an isolated peptide, the
peptide comprising at least three contiguous amino acids selected
from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through
SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID
NO:250.
50. A method comprising; a) injecting a subject with a phage
display library; b) recovering at least one sample of at least one
organ, tissue or cell type; c) separating the sample into isolated
cells or clumps of cells; d) centrifuging the cells through an
organic phase to form a pellet; and e) recovering phage from the
pellet.
51. The method of claim 50, further comprising preselecting the
phage display library against a different organ, tissue or cell
type.
52. A gene therapy vector, wherein the vector expresses a targeting
peptide sequence as part of a surface protein, the targeting
peptide comprising at least three contiguous amino acids selected
from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through
SEQ ID NO:121, SEQ ID NO: 123 and SEQ ID NO: 125 through SEQ ID
NO:250.
53. A method of targeting delivery to an organ or tissue,
comprising: a) obtaining a peptide according to claim 22; b)
attaching the peptide to an agent; and c) administering the agent
to a subject.
54. The method of claim 53, wherein the subject is a human or a
mouse.
55. The method of claim 53, wherein the agent is a drug, a
chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an
anti-angiogenic agent, an enzyme, a hormone, a cytokine, a growth
factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an
antibody, a Fab fragment of an antibody, an imaging agent, an
antigen, a survival factor, an anti-apoptotic agent, a hormone
antagonist, a virus, a bacteriophage, a bacterium, a liposome, a
microparticle, a magnetic bead, a microdevice, a yeast cell, a
mammalian cell, a cell or an expression vector.
56. The method of claim 53, wherein the agent is an imaging
agent.
57. The method of claim 56, further comprising obtaining an image
of the subject.
58. The method of claim 57, wherein the image is diagnostic for a
disease.
59. The method of claim 58, wherein the disease is cancer,
arthritis, diabetes, inflammatory disease, atherosclerosis,
autoimmune disease, bacterial infection, viral infection,
cardiovascular disease or degenerative disease.
60. The method of claim 53, wherein the organ or tissue is bone
marrow, prostate, prostate cancer, ovary, ureter, placenta,
adipose, spleen, angiogenic tissue or ascites.
61. A method of targeting delivery to prostate cancer comprising:
a) obtaining a targeting peptide comprising at least three
contiguous amino acids selected from any of SEQ ID NO:20 through
SEQ ID NO:38; b) attaching the peptide to a therapeutic agent to
form a complex; and c) administering the complex to a subject with
prostate cancer.
62. A method of diagnosing prostate cancer comprising: a) obtaining
a targeting peptide comprising at least three contiguous amino
acids selected from any of SEQ ID NO:20 through SEQ ID NO:38; b)
administering the peptide to a subject suspected of having prostate
cancer; and c) detecting the peptide bound to prostate cancer
cells.
63. A method of identifying targeting peptides to angiogenic tissue
comprising: a) inducing hypoxia in a neonatal subject; b)
administering a phage display library to the subject; and c)
recovering phage from the retina of the subject.
64. A method of inducing apoptosis in a cell comprising: a)
obtaining a targeting peptide comprising at least three contiguous
amino acids selected from any of SEQ ID NO:93 through SEQ ID
NO:121; b) attaching the peptide to a permeabilizing agent to form
a complex; and c) administering the complex to the cell.
65. The method of claim 64, wherein the permeabilizing agent is
selected from a peptide with an amino acid sequence of SEQ ID
NO:122 or HIV Tat protein.
66. The method of claim 64, wherein the targeting peptide has the
amino acid sequence of SEQ ID NO:112.
67. A method of inducing apoptosis in a cell comprising: a)
attaching Annexin V to a permeabilizing agent to form a complex;
and b) administering the complex to the cell.
68. The method of claim 67, wherein the permeabilizing agent is
selected from a peptide with an amino acid sequence of SEQ ID NO:
122 or HIV Tat protein
69. A method of modulating angiogenesis comprising: a) obtaining a
peptide comprising at least three contiguous amino acids selected
from SEQ ID NO:93 through SEQ ID NO:11; and b) administering the
peptide to a subject.
70. The method of claim 69 wherein the subject has a tumor and the
peptide inhibits tumor growth or survival.
71. The method of claim 69, wherein the peptide is attached to an
agent.
72. The method of claim 71, wherein the agent is thrombospondin,
angiostatin5, pigment epithelium-drived factor, angiotensin,
laminin peptides, fibronectin peptides, plasminogen activator
inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, platelet factor 4, IP-10, Gro-.beta.,
thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, C M101, Marimastat, pentosan polysulphate,
angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet
factor 4 or minocycline.
73. The method of claim 69, wherein the peptide has anti-angiogenic
activity.
74. The method of claim 69, wherein the peptide has pro-angiogenic
activity.
75. The method of claim 73, further comprising administering the
peptide to a subject with ischemnia.
76. The method of claim 73, further comprising administering the
peptide to a subject with cardiovascular disease.
77. The method of claim 69, further comprising administering the
peptide to a subject with cancer, arthritis, diabetes,
cardiovascular disease, inflammation or macular degeneration.
78. A method of targeting delivery to an angiogenic tissue
comprising: a) obtaining a peptide comprising at least three
contiguous amino acids selected from SEQ ID NO:123, SEQ ID NO:125,
SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID
NO:130 or SEQ ID NO:131; b) attaching the peptide to a therapeutic
agent to form a complex; and b) administering the complex to a
subject.
79. The method of claim 78, wherein the peptide has an amino acid
sequence of SEQ ID NO:123.
80. The method of claim, 78, wherein the angiogenic tissue is from
a subject with cancer, arthritis, diabetes, cardiovascular disease,
inflammation or macular degeneration.
81. A method of detecting receptors for endostatin or angiostatin
comprising a) obtaining a sample from a tissue or organ; b)
incubating the sample with endostatin or angiostatin; and c)
detecting the presence of endostatin or angiostatin bound to the
sample.
82. The method of claim 81, wherein the sample is a thin section of
a tissue or organ.
83. The method of claim 82, further comprising assessing
specificity by inhibiting binding with a targeting peptide
selective for endostatin or angiostatin.
Description
[0001] This application claims priority from U.S. Provisional
Patent Application No. 60/231,266 filed Sep. 8, 2000, and U.S.
patent application Ser. No. 09/765,101, filed Jan. 17, 2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention concerns the fields of molecular
medicine and targeted delivery of therapeutic agents. More
specifically, the present invention relates to compositions and
methods for identification and use of peptides that selectively
target organs tissues or cell types in vivo or in vitro.
[0005] 2. Description of Related Art
[0006] Therapeutic treatment of many disease states is limited by
the systemic toxicity of the therapeutic agents used. Cancer
therapeutic agents in particular exhibit a very low therapeutic
index, with rapidly growing normal tissues such as skin and bone
marrow affected at concentrations of agent that are not much higher
than the concentrations used to kill tumor cells. Treatment of
cancer and other organ, tissue or cell type confined disease states
would be greatly facilitated by the development of compositions and
methods for targeted delivery to a desired organ, tissue or cell
type of a therapeutic agent.
[0007] Recently, an in vivo selection system was developed using
phage display libraries to identify organ, tissue or cell type
targeting peptides in a mouse model system. Phage display libraries
expressing transgenic peptides on the surface of bacteriophage were
initially developed to map epitope binding sites of immunoglobulins
(Smith and Scott, 1986, 1993). Such libraries can be generated by
inserting random oligonucleotides into cDNAs encoding a phage
surface protein, generating collections of phage particles
displaying unique peptides in as many as 10.sup.9 permutations.
(Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al
1998b).
[0008] Intravenous administration of phage display libraries to
mice was followed by the recovery of phage from individual organs
(Pasqualini and Ruoslahti, 1996). Phage were recovered that were
capable of selective homing to the vascular beds of different mouse
organs, tissues or cell types, based on the specific targeting
peptide sequences expressed on the outer surface of the phage
(Pasqualini and Ruoslahti, 1996). A variety of organ and
tumor-homing peptides have been identified by this method (Rajotte
et al., 1998, 1999; Koivunen et al., 1999; Burg et al., 1999;
Pasqualini, 1999). Each of those targeting peptides bound to
different receptors that were selectively expressed on the
vasculature of the mouse target tissue (Pasqualini, 1999;
Pasqualini et al., 2000; Folkman, 1995; Folkman 1997). Tumor-homing
peptides bound to receptors that were upregulated in the tumor
angiogenic vasculature of mice (Brooks et al., 1994; Pasqualini et
al., 2000). In addition to identifying individual targeting
peptides selective for an organ, tissue or cell type (Pasqualini
and Ruoslahti, 1996; Arap et al, 1998a; Koivunen et al., 1999),
this system has been used to identify endothelial cell surface
markers that are expressed in mice in vivo (Rajotte and Ruoslahti,
1999).
[0009] Attachment of therapeutic agents to targeting peptides
resulted in the selective delivery of the agent to a desired organ,
tissue or cell type in the mouse model system. Targeted delivery of
chemotherapeutic agents and proapoptotic peptides to receptors
located in tumor angiogenic vasculature resulted in a marked
increase in therapeutic efficacy and a decrease in systemic
toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b;
Ellerby et al., 1999).
[0010] In some cases, previous in vivo methods for phage display
screening resulted in relatively high backgrounds of non-specific
phage binding. This was particularly true for tissues belonging to
the reticuloendothelial system. A need exists for improved methods
of phage display that decrease non-specific phage binding, while
retaining specific interactions between targeting peptides and cell
receptors. A need also exists to target receptors for specific cell
populations within an organ, tissue or cell type. In many cases,
tissues or organs may contain highly heterologous populations of
different cell types. A need exists to be able to target phage
display screening to specific cell populations.
[0011] A need also exists to identify receptor-ligand pairs in
organs and tissues. Previous attempts to identify targeted
receptors and ligands binding to receptors have largely targeted a
single ligand at a time for investigation. Identification of
previously unknown receptors and previously uncharacterized ligands
has been a very slow and laborious process. Such novel receptors
and ligands may provide the basis for new therapies for a variety
of disease states, such as is diabetes mellitus, inflammatory
disease, arthritis, atherosclerosis, cancer, autoimmune disease,
bacterial infection, viral infection, cardiovascular disease or
degenerative disease.
SUMMARY OF THE INVENTION
[0012] The present invention solves a long-standing need in the art
by providing compositions and methods for the identifying and using
targeting peptides that are selective for organs, tissues or
specific cell types. In certain embodiments, the methods concern
Biopanning and Rapid Analysis of Selective Interactive Ligands
(BRASIL), a novel method for phage display that results in
decreased background of non-specific phage binding, while retaining
selective binding of phage to cell receptors. In preferred
embodiments, targeting peptides are identified by exposing a
subject to a phage display library, collecting samples of one or
more organs, tissues or cell types, separating the samples into
isolated cells or small clumps of cells suspended in an aqueous
phase, layering the aqueous phase over an organic phase,
centrifuging the two phases so that the cells are pelleted at the
bottom of a centrifuge tube and collecting phage from the pellet.
In an even more preferred embodiment, the organic phase is
dibutylphtalate.
[0013] In other embodiments, phage that bind to a target organ,
tissue or cell type, for example to placenta, may be pre-screened
or post-screened against a subject lacking that organ, tissue or
cell type. Phage that bind to the subject lacking the target organ,
tissue or cell type are removed from the library prior to screening
in subjects possessing the organ, tissue or cell type. In preferred
embodiments, the organ, tissue or cell type is placenta or adipose
tissue.
[0014] In preferred embodiments, targeting phage may be recovered
from specific cell types or sub-types present in an organ or tissue
after selection of the cell type by PALM (Positioning and Ablation
with Laser Microbeams). PALM allows specific cell types to be
selected from, for example, a thin section of an organ or tissue.
Phage may be recovered from the selected sample.
[0015] In another embodiment, a phage display library displaying
the antigen binding portions of antibodies from a subject is
prepared, the library is screened against one or more antigens and
phage that bind to the antigens are collected. In more preferred
embodiments, the antigen is a targeting peptide.
[0016] In certain embodiments, the methods and compositions may be
used to identify one or more receptors for a targeting peptide. In
alternative embodiments, the compositions and methods may be used
to identify naturally occurring ligands for known or newly
identified receptors.
[0017] In some embodiments, the methods may comprise contacting a
targeting peptide to an organ, tissue or cell containing a receptor
of interest, allowing the peptide to bind to the receptor, and
identifying the receptor by its binding to the peptide. In
preferred embodiments, the targeting peptide contains at least
three contiguous amino acids selected from any of SEQ ID NO:5
through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID
NO:123 and SEQ ID NO:125 through SEQ ID NO:251. In other preferred
embodiments, the targeting peptide comprises a portion of an
antibody against the receptor. In alternative embodiments, the
targeting peptide may contain a random amino acid sequence. The
skilled artisan will realize that the contacting step can utilize
intact organs, tissues or cells, or may alternatively utilize
homogenates or detergent extracts of the organs, tissues or cells.
In certain embodiments, the cells to be contacted may be
genetically engineered to express a suspected receptor for the
targeting peptide. In a preferred embodiment, the targeting peptide
is modified with a reactive moiety that allows its covalent
attachment to the receptor. In a more preferred embodiment, the
reactive moiety is a photoreactive group that becomes covalently
attached to the receptor when activated by light. In another
preferred embodiment, the peptide is attached to a solid support
and the receptor is purified by affinity chromatography. In other
preferred embodiments, the solid support comprises magnetic beads,
Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon
membrane, a column chromatography matrix, a high performance liquid
chromatography (HPLC) matrix or a fast performance liquid
chromatography (FPLC) matrix. In certain embodiments, the targeting
peptide inhibits the activity of the receptor upon binding to the
receptor. The skilled artisan will realize that receptor activity
can be assayed by a variety of methods known in the art, including
but not limited to catalytic activity and binding activity. In
another preferred embodiment, the receptor is an endostatin
receptor, a metalloprotease or an aminopeptidase.
[0018] In alternative embodiments, one or more ligands for a
receptor of interest may be identified by the disclosed methods and
compositions. One or more targeting peptides that mimic part or all
of a naturally occurring ligand may be identified by phage display
and biopanning in vivo or in vitro. A naturally occurring ligand
may be identified by homology with a single targeting peptide that
binds to the receptor, or a consensus motif of sequences that bind
to the receptor. In other alternative embodiments, an antibody may
be prepared against one or more targeting peptides that bind to a
receptor of interest. Such antibodies may be used for
identification or immunoaffinity purification of the native
ligand.
[0019] In certain embodiments, the targeting peptides of the
present invention are of use for the selective delivery of
therapeutic agents, including but not limited to gene therapy
vectors and fusion proteins, to specific organs, tissues or cell
types. The skilled artisan will realize that the scope of the
claimed methods of use include any disease state that can be
treated by targeted delivery of a therapeutic agent to a desired
organ, tissue or cell type. Although such disease states include
those where the diseased cells are confined to a specific organ,
tissue or cell type, such as non-metastatic cancer, other disease
states may be treated by an organ, tissue or cell type-targeting
approach.
[0020] One embodiment of the present invention concerns isolated
peptides of 100 amino acids or less in size, comprising at least 3
contiguous amino acids of a targeting peptide sequence, selected
from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through
SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID
NO:251.
[0021] In a preferred embodiment, the isolated peptide is 50 amino
acids or less, more preferably 30 amino acids or less, more
preferably 20 amino acids or less, more preferably 10 amino acids
or less, or even more preferably 5 amino acids or less in size. In
other preferred embodiments, the isolated peptide of claim 1
comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17,
18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a
targeting peptide sequence, selected from any of SEQ ID NO:5
through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID
NO:123 and SEQ ID NO:125 through SEQ ID NO:251.
[0022] In certain embodiments, the isolated peptide is attached to
a molecule. In preferred embodiments, the attachment is a covalent
attachment. In additional embodiments, the molecule is a drug, a
chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an
anti-angiogenic agent, a hormone, a cytokine, a growth factor, a
cytotoxic agent, a peptide, a protein, an antibiotic, an antibody,
a Fab fragment of an antibody, a survival factor, an anti-apoptotic
factor, a hormone antagonist, an imaging agent, a nucleic acid or
an antigen. Those molecules are representative only. Molecules
within the scope of the present invention include virtually any
molecule that may be attached to a targeting peptide and
administered to a subject. In preferred embodiments, the
pro-aptoptosis agent is granicidin, magainin, mellitin, defensin,
cecropin, (KLALAK).sub.2 (SEQ ID NO:1), (KLAKKLA).sub.2 (SEQ ID
NO:2), (KAAKKAA).sub.2 (SEQ ID NO:3) or (KLGKKLG).sub.3 (SEQ ID
NO:4). In other preferred embodiments, the anti-angiogenic agent is
angiostatin5, pigment epithelium-drived factor, angiotensin,
laminin peptides, fibronectin peptides, plasminogen activator
inhibitors, tissue metalloproteinase inhibitors, interferons,
interleukin 12, platelet factor 4, IP-10, Gro-.beta.,
thrombospondin, 2-methoxyoestradiol, proliferin-related protein,
carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,
angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP470, endostatin, paclitaxel,
docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor,
a signaling inhibitor (SU5416, SU6668, Sugen, South San Francisco,
Calif.), accutin, cidofovir, vincristine, bleomycin, AGM-1470,
platelet factor 4 or minocycline. In further preferred embodiments,
the cytokine is interleukin 1 (IL-1), IL-2, IL,5, IL-10, IL-11,
IL-12, IL-18, interferon-.gamma. (IF-.gamma.), IF-.alpha.,
IF-.beta., tumor necrosis factor-.alpha. (TNF-.alpha.), or GM-CSF
(granulocyte macrophage colony stimulating factor). Such examples
are representative only and are not intended to exclude other
pro-apoptosis agents, anti-angiogenic agents or cytokines known in
the art.
[0023] In other embodiments, the isolated peptide is attached to a
macromolecular complex. In preferred embodiments, the attachment is
a covalent attachment. In other preferred embodiments, the
macromolecular complex is a virus, a bacteriophage, a bacterium, a
liposome, a microparticle, a magnetic bead, a yeast cell, a
mammalian cell, a cell or a microdevice. These are representative
examples only. Macromolecular complexes within the scope of the
present invention include virtually any macromolecular complex that
may be attached to a targeting peptide and administered to a
subject. In other preferred embodiments, the isolated peptide is
attached to a eukaryotic expression vector, more preferably a gene
therapy vector.
[0024] In another embodiment, the isolated peptide is attached to a
solid support, preferably magnetic beads, Sepharose beads, agarose
beads, a nitrocellulose membrane, a nylon membrane, a column
chromatography matrix, a high performance liquid chromatography
(HPLC) matrix or a fast performance liquid chromatography (FPLC)
matrix.
[0025] Additional embodiments of the present invention concern
fusion proteins comprising at least 3 contiguous amino acids of a
sequence selected from any of SEQ ID NO:5 through SEQ ID NO:45, SEQ
ID NO:47 through SEQ ID NO:121, SEQ ID NO: 123 and SEQ ID NO:125
through SEQ ID NO:251.
[0026] Certain other embodiments concern compositions comprising
the claimed isolated peptides or fusion proteins in a
pharmaceutically acceptable carrier. Further embodiments concern
kits comprising the claimed isolated peptides or fusion proteins in
one or more containers.
[0027] Other embodiments concern methods of targeted delivery
comprising selecting a targeting peptide for a desired organ,
tissue or cell type, attaching said targeting peptide to a
molecule, macromolecular complex or gene therapy vector, and
providing said peptide attached to said molecule, complex or vector
to a subject. Preferably, the targeting peptide is selected to
include at least 3 contiguous amino acids from any of SEQ ID NO:5
through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID NO:121, SEQ ID
NO:123 and SEQ ID NO:125 through SEQ ID NO:251. In certain
preferred embodiments, the organ, tissue or cell type is bone
marrow, lymph node, prostate cancer or prostate cancer that has
metastasized to bone marrow. In other preferred embodiments, the
molecule attached to the targeting peptide is a chemotherapeutic
agent, an antigen or an imaging agent. The skilled artisan will
realize that within the scope of the present invention any organ,
tissue or cell type can be targeted for delivery, using targeting
peptides attached to any molecule, macromolecular complex or gene
therapy vector.
[0028] Other embodiments of the present invention concern isolated
nucleic acids of 300 nucleotides or less in size, encoding a
targeting peptide. In preferred embodiments, the isolated nucleic
acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or
even 10 nucleotides or less in size. In other preferred
embodiments, the isolated nucleic acid is incorporated into a
eukaryotic or a prokaryotic expression vector. In even more
preferred embodiments, the vector is a plasmid, a cosmid, a yeast
artificial chromosome (YAC), a bacterial artificial chromosome
(BAC), a virus or a bacteriophage. In other preferred embodiments,
the isolated nucleic acid is operatively linked to a leader
sequence that localizes the expressed peptide to the extracellular
surface of a host cell.
[0029] Additional embodiments of the present invention concern
methods of treating a disease state comprising selecting a
targeting peptide that targets cells associated with the disease
state, attaching one or more molecules effective to treat the
disease state to the peptide, and administering the peptide to a
subject with the disease state. Preferably, the targeting peptide
includes at least three contiguous amino acids selected from any of
SEQ ID NO:5 through SEQ ID NO:45, SEQ ID NO:47 through SEQ ID
NO:121, SEQ ID NO:123 and SEQ ID NO:125 through SEQ ID NO:251. In
preferred embodiments the disease state is diabetes mellitus,
inflammatory disease, arthritis, atherosclerosis, cancer,
autoimmune disease, bacterial infection, viral infection,
cardiovascular disease or degenerative disease.
[0030] Another embodiment of the present invention concerns
compositions and methods of use of tumor targeting peptides against
cancers. Tumor targeting peptides identified by the methods
disclosed in the instant application may be attached to therapeutic
agents, including but not limited to molecules or macromolecular
assemblages and administered to a subject with cancer, providing
for increased efficacy and decreased systemic toxicity of the
therapeutic agent. Therapeutic agents within the scope of the
present invention include but are not limited to chemotherapeutic
agents, radioisotopes, pro-apoptosis agents, cytotoxic agents,
cytostatic agents and gene therapy vectors. Targeted delivery of
such therapeutic agents to tumors provides a significant
improvement over the prior art for increasing the delivery of the
agent to the tumor, while decreasing the inadvertent delivery of
the agent to normal organs and tissues of the subject. In a
preferred embodiment, the tumor targeting peptide is incorporated
into the capsule of a phage gene therapy vector to target delivery
of the phage to angiogenic endothelial cells in tumor blood
vessels.
[0031] Certain embodiments concern methods of obtaining antibodies
against an antigen. In preferred embodiments, the antigen comprises
one or more targeting peptides. The targeting peptides are prepared
and immobilized on a solid support, serum containing antibodies is
added and antibodies that bind to the targeting peptides are
collected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0033] FIG. 1. Validation of placenta homing phage. Phage bearing
targeting peptides identified in Example 3 were injected into
pregnant mice and their recovery from placenta was compared to
control fd-tet phage without targeting sequences. The placenta
homing phage clones were: PA--TPKTSVT (SEQ ID NO:39), PC--RAPGGVR
(SEQ ID NO:41), PE--LGLRSVG (SEQ ID NO:44), PF--YIRPFIL (SEQ ID
NO:43).
[0034] FIG. 2. Validation of adipose homing peptides. Phage bearing
targeting peptides identified in Example 4 were injected into
pregnant mice and their recovery from adipose tissue was compared
to control fd-tet phage without targeting sequences.
[0035] FIG. 3. Spleen targeting in vitro using BRASIL. Binding of
Fab clones #2, #6, #10, #12 and control Fab clone NPC-3TT was
compared to binding of control Fd-tet phage.
[0036] FIG. 4. Spleen targeting in vitro using BRASIL. Binding of
Fab clones #2, #6, #10, #12 and control Fab clone NPC-3TT were
directly compared to each other.
[0037] FIG. 5. Spleen targeting in vivo using BRASIL. Binding of
Fab clones #2, #6, #10, #12 was compared to binding of Fd-tet
phage.
[0038] FIG. 6. Spleen, targeting in vivo using BRASIL. Binding of
Fab clone #10 to spleen tissue was compared to binding of Fab
control clone NPC-3TT and Fd-tet phage.
[0039] FIG. 7. Binding of Fab clone #10 to spleen versus bone
marrow in comparison to Fd-tet phage.
[0040] FIG. 8. Binding of Fab clones from an anti-Karposi's sarcoma
library to angiogenic retina.
[0041] FIG. 9. Binding of .beta.3 cytoplasmic domain-selected phage
to immobilized proteins. GST fusion proteins or GST alone were
coated on microtiter wells at 10 .mu.g/ml and used to bind phage
expressing endostatin targeting peptides. Each phage is identified
by the peptide sequence it displayed: GIDTYRGSP (SEQ ID NO:96);
YDWWYPWSW (SEQ ID NO:95); CLRQSYSYNC (SEQ ID NO:104); SDNRYIGSW
(SEQ ID NO:97); CEQRQTQEGC (SEQ ID NO:93); CFQNRC (SEQ ID NO:102).
The data represent the mean colony counts from triplicate wells,
with standard error of less than 10% of the mean.
[0042] FIG. 10. Binding of .beta.5 cytoplasmic domain-selected
phage to immobilized proteins. GST fusion proteins or GST alone
were coated on microfiter wells at 10 .mu.g/ml and used to bind
phage expressing endostatin binding peptides. Each phage is
identified by the peptide sequence it displayed: (A) DEEGYYMMR (SEQ
ID NO: 110); (B) KQFSYRYLL (SEQ ID NO:111); (C) CEPYWDGWFC (SEQ ID
NO:106); (D) VVISYSMPD (SEQ ID NO:112); and (E) CYIWPDSGLC (SEQ ID
NO:105). The data represent the mean colony counts from triplicate
wells, with standard error less than 10% of the mean.
[0043] FIG. 11. Binding of the cytoplasmic-domain binding phage to
.beta.3 immobilized protein and inhibition with the synthetic
peptide. Phage were incubated on wells coated with GST-.beta.3cyto
in the presence of increasing concentrations of the corresponding
synthetic peptide or a control peptide. The data represent the mean
colony counts from triplicate wells, with standard error less than
10% of the mean.
[0044] FIG. 12. Binding of the cytoplasmic-domain binding phage to
.beta.5 immobilized protein and inhibition with the synthetic
peptide. Phage were incubated on wells coated with GST-.beta.5cyto
in the presence of increasing concentrations of the corresponding
synthetic peptide or a control peptide. The data represent the mean
colony counts from triplicate wells, with standard error less than
10% of the mean.
[0045] FIG. 13. Binding of phage to immobilized .beta.3-GST and
.beta.5-GST after phosphorylation. Phage were phosphorylated with
Fyn kinase. Insertless phage were used as a control. Phage were
incubated on wells coated with GST-.beta.3cyto or GST-.beta.3cyto.
The data represent the mean colony counts from triplicate wells,
with standard error less than 10% of the mean.
[0046] FIG. 14. Binding of phage to immobilized GST fusion proteins
after phosphorylation. Phages were phosphorylated with Fyn kinase.
Insertless phage was used as a control. Phage were incubated on
wells coated with GST-cytoplasmic domains. The data represent the
mean of colony counts from triplicate wells, with standard error
less than 10% of the mean.
[0047] FIG. 15. Effect of integrin cytoplasmic domain binding
peptides on cell proliferation. Serum-deprived cells were cultured
for 24 h and the proliferation was determined by [.sup.3H]
thymidine (1 .mu.Ci/ml) uptake measurements. In a positive control,
VEGF was added back to serum-starved cells. Each experiment was
performed three times with triplicates, and the results were
expressed as the mean .+-.SD.
[0048] FIG. 16. Effect of penetratin peptide chimeras on
endothelial cell migration. Cell migration assay were performed in
a 48-well microchemotaxis chamber. Five random high-power fields
(magnitude 40.times.) were counted in each well. The results show
that both .beta.3-integrin cytoplasmic domain binding peptides
(Y-18 and TYR-11) increase cell migration while penetratin does not
affect the cells.
[0049] FIG. 17. Penetratin peptide chimera binding to the .beta.5
cytoplasmic domain induces programmed cell death. 10.sup.6 HUVEC
cells were harvested in complete media and 15 .mu.M penetratin
peptide chimeras were added to the cells. After four, eight and
twelve hours the cells were stained with Propidium Iodide (PI) and
induction of apoptosis was analyzed by cytometric analysis. a)
Profile obtained with starved cells after 24 h. b) Confluent cells
in complete media. c) 15 .mu.M of penetratin after four hours. d)
15 .mu.M of VISY-penetratin chimera after four hours. Cells
analyzed after eight and twelve hours showed similar profiles for
the percentage of G.sub.0/G.sub.1.
[0050] FIG. 18. Specificity of the antibodies raised against
.beta.3- or .beta.5-selected phage (ELISA). Increasing dilutions of
sera obtained after three immunizations with GLDTYRGSP (SEQ ID
NO:96) or SDNRYIGSW (SEQ ID NO:97) conjugated to KLH were incubated
on microtiter wells coated with 10 .mu.g of SDNRYIGSW (SEQ ID
NO:97, Y-18), GLDTYRGSP (SEQ ID NO:96, TYR-11) or control peptides.
Preimmunesera were used as controls. After incubation with HRPgoat
anti-rabbit, OD was measured at 405 nm. The data represent the
means from triplicate wells, with standard error less than 10%.
[0051] FIG. 19. Specificity of the antibodies raised against
.beta.3- or .beta.5-selected phage (ELISA). Sera obtained after
three immunizations with SDNRYIGSW (SEQ ID NO:97, Y-18) or
GLDTYRGSP (SEQ ID NO:96, TYR-11) conjugated to KLH were incubated
in microtiter wells coated with 10 .mu.g of TYR-11 or Y-18.
GLDTYRGSP (SEQ ID NO:96) or SDNRYIGSW (SEQ ID NO:97) and control
peptides were added in solution. After incubation with HRP goat
anti-rabbit, OD was measured at 405 nm. The data represent the
means from triplicate wells, with standard error less than 10%.
Peptides added in solution specifically block the reactivity with
the immobilized peptides.
[0052] FIG. 20A. Competitive binding of Annexin V to .beta.5
integrin with VISY peptide. Binding assays were performed by
ELISA.
[0053] FIG. 20B. Relative levels of binding of anti-Annexin V
antibody to purified Annexin V protein and VISY peptide.
[0054] FIG. 21. Chimeric peptide containing VISY peptide linked to
penetratin (antennapedia) induces apoptosis. VISY induced apoptosis
was inhibited by addition of a caspase inhibitor (zVAD).
[0055] FIG. 22. APA-binding phage specifically bind tumors. Equal
amounts of phage were injected into the tail veins of mice bearing
MDA-MB-435-derived tumors and phage were recovered after perfusion.
Mean values for phage recovered from the tumor or control tissue
(brain) and the standard error from triplicate platings are
shown.
[0056] FIG. 23. CPRECESIC (SEQ ID NO:123) is a specific inhibitor
of APA activity. APA enzyme activity was assayed in the presence of
increasing concentrations of either GACVRLSACGA (SEQ ID NO:124)
(control) or CPRECESIC (SEQ ID NO:123) peptide. The IC.sub.50 for
APA inhibition by CPRECESIC (SEQ ID NO: 123) was estimated at 800
.mu.M. Error bars are the standard error of the means of triplicate
wells. The experiment was repeated three times with similar
results.
[0057] FIG. 24. CPRECESIC (SEQ ID NO: 123) inhibits HUVEC
migration. HUVECs were stimulated with VEGF-A (10 ng/ml). The assay
was performed in a Boyden microchemotaxis chamber, and cells were
allowed to migrate through an 8-.mu.m pore filter for 5 h at
37.degree. C. GACVRLSACGA (SEQ ID NO:124) (control) and CPRECESIC
(SEQ ID NO:123) peptides were tested at 1 mM concentration.
Migrated cells were stained and five high-power fields (magnitude
100.times.) for each microwell were counted. Error bars are the
standard error of the means of triplicate microwells.
[0058] FIG. 25. CPRECESIC (SEQ ID NO: 123) inhibits HUVEC
proliferation. Cells were stimulated with VEGF-A (10 ng/ml), and
growth was evaluated at the indicated times by a colorimetric assay
based on crystal violet staining. Error bars are the standard error
of the means of triplicate wells. Each experiment was repeated at
least twice with similar results.
[0059] FIG. 26. Protocol for in vivo biopanning for phage targeted
in mouse pancreas, kidneys, liver, lungs and adrenal gland.
[0060] FIG. 27. Protocol for recovery of phage by infection of E.
coli or recovery of phage DNA by amplification and subcloning.
[0061] FIG. 28. Pancreatic islet targeting peptides and homologous
proteins. Candidate endogenous proteins mimicked by the pancreatic
islet targeting peptides CVSNPRWKC (SEQ ID NO:197), CVPRRWDVC (SEQ
ID NO:194), CQHTSGRGC (SEQ ID NO:195) and CRARGWULLC (SEQ ID
NO:196), identified by standard homology searches.
[0062] FIG. 29. Pancreatic islet targeting peptides and homologous
proteins. Candidate endogenous proteins mimicked by the pancreatic
islet targeting peptides CGGVHALRC (SEQ ID NO:175), CFNRTWIGC (SEQ
ID NO:198) and CWSRQGGC (SEQ ID NO:200, identified by standard
homology searches.
[0063] FIG. 30. Pancreatic islet targeting peptides and homologous
proteins. Candidate endogenous proteins mimicked by the pancreatic
islet targeting peptides CLASGMDAC (SEQ ID NO:204), CHDERTGRC (SEQ
ID NO:205), CAEBALMEC (SEQ ID NO:206) and CMQGARTSC (SEQ ID
NO:208), identified by standard homology searches.
[0064] FIG. 31. Pancreatic islet targeting peptides and homologous
proteins. Candidate endogenous proteins mimicked by the pancreatic
islet targeting peptides CHVLWSTRC (SEQ ID NO:201), CMSSPGVAC (SEQ
ID NO:203) and CLGLUMAGC (SEQ ID NO:202), identified by standard
homology searches.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0065] As used herein in the specification, "a" or "an" may mean
one or more. As used herein in the claim(s), in conjunction with
the word "comprising," the words "a" or "an" may mean one or more
than one. As used herein "another" may mean at least a second or
more of an item.
[0066] A "targeting peptide" is a peptide comprising a contiguous
sequence of amino acids, that is characterized by selective
localization to an organ, tissue or cell type. Selective
localization may be determined, for example, by methods disclosed
below, wherein the putative targeting peptide sequence is
incorporated into a protein that is displayed on the outer surface
of a phage. Administration to a subject of a library of such phage
that have been genetically engineered to express a multitude of
such targeting peptides of different amino acid sequence is
followed collection of one or more organs, tissues or cell types
from the subject and identification of phage found in that organ,
tissue or cell type. A phage expressing a targeting peptide
sequence is considered to be selectively locallized to a tissue or
organ if it exhibits greater binding in that tissue or organ
compared to a control tissue or organ. Preferably, selective
localization of a targeting peptide should result in a two-fold or
higher enrichment of the phage in the target organ, tissue or cell
type, compared to a control organ, tissue or cell type. Selective
localization resulting in at least a three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold or
higher enrichment in the target organ compared to a control organ,
tissue or cell type is more preferred. Alternatively, a phage
expressing a targeting peptide sequence that exhibits selective
localization preferably shows an increased enrichment in the target
organ compared to a control organ when phage recovered from the
target organ are reinjected into a second host for another round of
screening. Further enrichment may be exhibited following a third
round of screening. Another alternative means to determine
selective localization is that phage expressing the putative target
peptide preferably exhibit a two-fold, more preferably a three-fold
or higher enrichment in the target organ compared to control phage
that express a non-specific peptide or that have not been
genetically engineered to express any putative target peptides.
Another means to determine selective localization is that
locallization to the target organ of phage expressing the target
peptide is at least partially blocked by the co-administration of a
synthetic peptide containing the target peptide sequence.
"Targeting peptide" and "homing peptide" are used synonymously
herein.
[0067] A "phage display library" means a collection of phage that
have been genetically engineered to express a set of putative
targeting peptides on their outer surface. In preferred
embodiments, DNA sequences encoding the putative targeting peptides
are inserted in frame into a gene encoding a phage capsule protein.
In other preferred embodiments, the putative targeting peptide
sequences are in part random mixtures of all twenty amino acids and
in part non-random. In certain preferred embodiments the putative
targeting peptides of the phage display library exhibit one or more
cysteine residues at fixed locations within the targeting peptide
sequence.
[0068] A "macromolecular complex" refers to a collection of
molecules that may be random, ordered or partially ordered in their
arrangement. The term encompasses biological organisms such as
bacteriophage, viruses, bacteria, unicellular pathogenic organisms,
multicellular pathogenic organisms and prokaryotic or eukaryotic
cells. The term also encompasses non-living assemblages of
molecules, such as liposomes, microcapsules, microparticles,
magnetic beads and microdevices. The only requirement is that the
complex contains more than one molecule. The molecules may be
identical, or may differ from each other.
[0069] A "receptor" for a targeting peptide includes but is not
limited to any molecule or complex of molecules that binds to a
targeting peptide. Non-limiting examples of receptors include
peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids,
carbohydrates, multi-molecular structures, a specific conformation
of one or more molecules and a morphoanatomic entity. In preferred
embodiments, a "receptor" is a naturally occurring molecule or
complex of molecules that is present on the lumenal surface of
cells forming blood vessels within a target organ, tissue or cell
type.
[0070] A "subject" refers generally to a mammal. In certain
preferred embodiments, the subject is a mouse or rabbit. In even
more preferred embodiments, the subject is a human.
[0071] Phage Display
[0072] The methods described herein for identification of targeting
peptides involve the in vivo administration of phage display
libraries. Various methods of phage display and methods for
producing diverse populations of peptides are well known in the
art. For example, U.S. Pat. Nos. 5,223,409; 5,622,699 and
6,068,829, each of which is incorporated herein by reference,
disclose methods for preparing a phage library. The phage display
technique involves genetically manipulating bacteriophage so that
small peptides can be expressed on their surface (Smith et al.,
1985, 1993). The potential range of applications for this technique
is quite broad, and the past decade has seen considerable progress
in the construction of phage-displayed peptide libraries and in the
development of screening methods in which the libraries are used to
isolate peptide ligands. For example, the use of peptide libraries
has made it possible to characterize interacting sites and
receptor-ligand binding motifs within many proteins, such as
antibodies involved in inflammatory reactions or integrins that
mediate cellular adherence. This method has also been used to
identify novel peptide ligands that serve as leads to the
development of peptidomimetic drugs or imaging agents (Arap et al.,
1998a). In addition to peptides, larger protein domains such as
single-chain antibodies can also be displayed on the surface of
phage particles (Arap et al., 1998a).
[0073] Targeting amino acid sequences selective for a given organ,
tissue or cell type can be isolated by "biopanning" (Pasqualini and
Ruoslahti, 1996; Pasqualini, 1999). In brief, a library of phage
containing putative targeting peptides is administered to an animal
or human and samples of organs, tissues or cell types containing
phage are collected. In preferred embodiments utilizing filamentous
phage, the phage may be propagated in vitro between rounds of
biopanning in pilus-positive bacteria. The bacteria are not lysed
by the phage but rather secrete multiple copies of phage that
display a particular insert. Phage that bind to a target molecule
can be eluted from the target organ, tissue or cell type and then
amplified by growing them in host bacteria. If desired, the
amplified phage can be administered to a host and samples of
organs, tissues or cell types again collected. Multiple rounds of
biopanning can be performed until a population of selective binders
is obtained. The amino acid sequence of the peptides is determined
by sequencing the DNA corresponding to the targeting peptide insert
in the phage genome. The identified targeting peptide can then be
produced as a synthetic peptide by standard protein chemistry
techniques (Arap et al., 1998a, Smith et al., 1985). This approach
allows circulating targeting peptides to be detected in an unbiased
functional assay, without any preconceived notions about the nature
of their target. Once a candidate target is identified as the
receptor of a targeting peptide, it can be isolated, purified and
cloned by using standard biochemical methods (Pasqualini, 1999;
Rajotte and Ruoslahti, 1999).
[0074] In certain embodiments, a subtraction protocol is used with
to further reduce background phage binding. The purpose of
subtraction is to remove phage from the library that bind to cells
other than the cell of interest, or that bind to inactivated cells.
In alternative embodiments, the phage library may be prescreened
against a subject who does not possess the targeted cell, tissue or
organ. For example, placenta binding peptides may be identified
after prescreening a library against a male or non-pregnant female
subject After subtraction the library may be screened against the
cell, tissue or organ of interest. In another alternative
embodiment, an unstimulated, quiescent cell type, tissue or organ
may be screened against the library and binding phage removed. The
cell line, tissue or organ is then activated, for example by
administration of a hormone, growth factor, cytokine or chemokine
and the activated cell type, tissue or organ screened against the
subtracted phage library.
[0075] Other methods of subtraction protocols are known and may be
used in the practice of the present invention, for example as
disclosed in U.S. Pat. Nos. 5,840,841, 5,705,610, 5,670,312 and
5,492,807, incorporated herein by reference.
[0076] Choice of Phage Display System.
[0077] Previous in vivo selection studies performed in mice
preferentially employed libraries of random peptides expressed as
fusion proteins with the gene III capsule protein in the fUSE5
vector (Pasqualini and Ruoslahti, 1996). The number and diversity
of individual clones present in a given library is a significant
factor for the success of in vivo selection. It is preferred to use
primary libraries, which are less likely to have an
over-representation of defective phage clones (Koivunen et al.,
1999). The preparation of a library should be optimized to between
10.sup.8-10.sup.9 transducing units (T.U.)/ml. In certain
embodiments, a bulk amplification strategy is applied between each
round of selection.
[0078] Phage libraries displaying linear, cyclic, or double cyclic
peptides may be used within the scope of the present invention.
However, phage libraries displaying 3 to 10 random residues in a
cyclic insert (CX.sub.3-10C) are preferred, since single cyclic
peptides tend to have a higher affinity for the target organ than
linear peptides. Libraries displaying double-cyclic peptides (such
as CX.sub.3C X.sub.3CX.sub.3C; Rojotte et al., 1998) have been
successfully used. However, the production of the cognate synthetic
peptides, although possible, can be complex due to the multiple
conformers with different dissulfide bridge arrangements .
[0079] Identification of homing peptides and receptors by in vivo
phage display in mice.
[0080] In vivo selection of peptides from phage-display peptide
libraries administered to mice has been used to identify targeting
peptides selective for normal mouse brain, kidney, lung, skin,
pancreas, retina, intestine, uterus, prostate, and adrenal gland
(Pasqualini and Ruoslahti, 1996; Pasqualini, 1999; Rajotte et al.,
1998). These results show that the vascular endothelium of normal
organs is sufficiently heterogenous to allow differential targeting
with peptide probes (Pasqualini and Ruoslahti, 1996; Rajotte et
al., 1998). A means of identifying peptides that home to the
angiogenic vasculature of tumors has been devised, as described
below. A panel of peptide motifs that target the blood vessels of
tumor xenografts in nude mice has been assembled (Arap et al.,
1998a; reviewed in Pasqualini, 1999). These motifs include the
sequences RGD4C, NGR, and GSL. The RGD-4C peptide has previously
been identified as selectively binding .alpha.v integrins and has
been shown to home to the vasculature of tumor xenografts in nude
mice (Arap et al., 1998a, 1998b; Pasqualini et al., 1997).
[0081] The receptors for the tumor homing RGD4C targeting peptide
has been identified as .alpha.v integrins (Pasqualini et al.,
1997). The .alpha.v integrins play an important role in
angiogenesis. The .alpha.v.beta.3 and .alpha.v.beta.5 integrins are
absent or expressed at low levels in normal endothelial cells but
are induced in angiogenic vasculature of tumors (rooks et al.,
1994; Hammes et al., 1996). Aminopeptidase N/CD13 has recently been
identified as an angiogenic receptor for the NGR motif (Burg et
al., 1999). Aminopeptidase N/CD13 is strongly expressed not only in
the angiogenic blood vessels of prostate cancer in TRAMP mice but
also in the normal epithelial prostate tissue.
[0082] Tumor-homing phage co-localize with their receptors in the
angiogenic vasculature of tumors but not in non-angiogenic blood
vessels in normal tissues (Arap et al., 1998b). Immunohistochemical
evidence shows that vascular targeting phage bind to human tumor
blood vessels in tissue sections (Pasqualini et al., 2000) but not
to normal blood vessels. A negative control phage with no insert
(fd phage) did not bind to normal or tumor tissue sections. The
expression of the angiogenic receptors was evaluated in cell lines,
in non-proliferating blood vessels and in activated blood vessels
of tumors and other angiogenic tissues such as corpus luteum. Flow
cytometry and immunohistochemistry showed that these receptors are
expressed in a number of tumor cells and in activated HUVECs (data
not shown). The angiogenic receptors were not detected in the
vasculature of normal organs of mouse or human tissues.
[0083] The distribution of these receptors was analyzed by
immunohistochemistry in tumor cells, tumor vasculature, and normal
vasculature. Alpha v integrins, CD13, aminopeptidase A, NG2, and
MMP-2/MMP-9--the known receptors in tumor blood vessels--are
specifically expressed in angiogenic endothelial cells and
pericytes of both human and murine origin. Angiogenic
neovasculature expresses markers that are either expressed at very
low levels or not at all in non-proliferating endothelial cells
(not shown).
[0084] The markers of angiogenic endothelium include receptors for
vascular growth factors, such as specific subtypes of VEGF and
basic FGF receptors, and .alpha.v integrins, among many others
(Mustonen and Alitalo, 1995). Thus far, identification and
isolation of novel molecules characteristic of angiogenic
vasculature has been slow, mainly because endothelial cells undergo
dramatic phenotypic changes when grown in culture (Watson et al.,
1995).
[0085] Many of these tumor vascular markers are proteases and some
of the markers also serve as viral receptors. Alpha v integrins are
receptors for adenoviruses (Wickham et al., 1997c) and CD13 is a
receptor for coronaviruses (Look et al., 1989). MMP-2 and MMP-9 are
receptors for echoviruses (Koivunen et al., 1999). Aminopeptidase A
also appears to be a viral receptor. Bacteriophage may use the same
cellular receptors as eukaryotic viruses. These findings suggest
that receptors isolated by in vivo phage display will have cell
internalization capability, a key feature for utilizing the
identified peptide motifs as targeted gene therapy carriers.
[0086] Targeted Delivery
[0087] Peptides that home to tumor vasculature have been coupled to
cytotoxic drugs or proapoptotic peptides to yield compounds that
were more effective and less toxic than the parental compounds in
experimental models of mice bearing tumor xenografts (Arap et al.,
1998a; Ellerby et al, 1999). As described below, the insertion of
the RGD-4C peptide into a surface protein of an adenovirus has
produced an adenoviral vector that may be used for tumor targeted
gene therapy (Arap et al., 1998b).
[0088] BRASIL
[0089] In preferred embodiments, separation of phage bound to the
cells of a target organ, tissue or cell type from unbound phage is
achieved using the BRASIL technique (Provisional Patent Application
No. 60/231,266 filed Sep. 8, 2000; U.S. patent application
entitled, "Biopanning and Rapid Analysis of Selective Interactive
Ligands (BRASIL)" by Arap, Pasqualini and Giordano, filed
concurrently herewith, incorporated herein by reference in its
entirety). In BRASIL (Biopanning and Rapid Analysis of Soluble
Interactive Ligands), an organ, tissue or cell type is gently
separated into cells or small clumps of cells that are suspended in
an aqueous phase. The aqueous phase is layered over an organic
phase of appropriate density and centrifuged. Cells attached to
bound phage are pelleted at the bottom of the centrifuge tube,
while unbound phage remain in the aqueous phase. This allows a more
efficient separation of bound from unbound phage, while maintaining
the binding interaction between phage and cell. BRASIL may be
performed in an in vivo protocol, in which organs, tissues or cell
types are exposed to a phage display library by intravenous
administration, or by an ex vivo protocol, where the cells are
exposed to the phage library in the aqueous phase before
centrifugation.
[0090] Proteins and Peptides
[0091] In certain embodiments, the present invention concerns novel
compositions comprising at least one protein or peptide. As used
herein, a protein or peptide generally refers, but is not limited
to, a protein of greater than about 200 amino acids, up to a full
length sequence translated from a gene; a polypeptide of greater
than about 100 amino acids; and/or a peptide of from about 3 to
about 100 amino acids. For convenience, the terms "protein,"
"polypeptide" and "peptide are used interchangeably herein.
[0092] In certain embodiments the size of the at least one protein
or peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, aboout
130, about 140, about 150, about 160, about 170, about 180, about
190, about 200, about 210, about 220, about 230, about 240, about
250, about 275, about 300, about 325, about 350, about 375, about
400, about 425, about 450, about 475, about 500, about 525, about
550, about 575, about 600, about 625, about 650, about 675, about
700, about 725, about 750, about 775, about 800, about 825, about
850, about 875, about 900, about 925, about 950, about 975, about
1000, about 1100, about 1200, about 1300, about 1400, about 1500,
about 1750, about 2000, about 2250, about 2500 or greater amino
acid residues.
[0093] As used herein, an "amino acid residue" refers to any
naturally occuring amino acid, any amino acid derivitive or any
amino acid mimic known in the art. In certain embodiments, the
residues of the protein or peptide are sequential, without any
non-amino acid interrupting the sequence of amino acid residues. In
other embodiments, the sequence may comprise one or more non-amino
acid moieties. In particular embodiments, the sequence of residues
of the protein or peptide may be interrupted by one or more
non-amino acid moieties.
[0094] Accordingly, the term "protein or peptide" encompasses amino
acid sequences comprising at least one of the 20 common amino acids
found in naturally occurring proteins, or at least one modified or
unusual amino acid, including but not limited to those shown on
Table 1 below.
1TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad
2-Aminoadipic acid Baad 3-Aminoadipic acid Bala .beta.-alanine,
.beta.-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu
4-Aminobutyric acid, piperidinic acid Acp 6-Aminocaproic acid Ahe
2-Aminoheptanoic acid Aib 2-Aminoisobutyric acid Baib
3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu
2,4-Diaminobutyric acid Des Desmosine Dpm 2,2'-Diaminopimelic acid
Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn
N-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp
3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIle
allo-Isoleucine MeGly N-Methylglycine, sarcosine MeIle
N-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methyllvaline Nva
Norvaline Me Norleucine Orn Ornithine
[0095] Proteins or peptides may be made by any technique known to
those of skill in the art, including the expression of proteins,
polypeptides or peptides through standard molecular biological
techniques, the isolation of proteins or peptides from natural
sources, or the chemical synthesis of proteins or peptides. The
nucleotide and protein, polypeptide and peptide sequences
corresponding to various genes have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases
(http://www.ncbi.nlm.nih.gov/). The coding regions for known genes
may be amplified and/or expressed using the techniques disclosed
herein or as would be know to those of ordinary skill in the art.
Alternatively, various commercial preparations of proteins,
polypeptides and peptides are known to those of skill in the
art.
[0096] Peptide Mimetics
[0097] Another embodiment for the preparation of polypeptides
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules that mimic elements of protein
secondary structure. See, for example, Johnson et al., "Peptide
Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds.,
Chapman and Hall, New York (1993), incorporated herein by
reference. The underlying rationale behind the use of peptide
mimetics is that the peptide backbone of proteins exists chiefly to
orient amino acid side chains in such a way as to facilitate
molecular interactions, such as those of antibody and antigen. A
peptide mimetic is expected to permit molecular interactions
similar to the natural molecule. These principles may be used to
engineer second generation molecules having many of the natural
properties of the targeting peptides disclosed herein, but with
altered and even improved characteristics.
[0098] Fusion Proteins
[0099] Other embodiments of the present invention concern fusion
proteins. These molecules generally have all or a substantial
portion of a targeting peptide, linked at the N- or C-terminus, to
all or a portion of a second polypeptide or proteion. For example,
fusions may employ leader sequences from other species to permit
the recombinant expression of a protein in a heterologous host.
Another useful fusion includes the addition of an immunologically
active domain, such as an antibody epitope, to facilitate
purification of the fusion protein. Inclusion of a cleavage site at
or near the fusion junction will facilitate removal of the
extraneous polypeptide after purification. Other useful fusions
include linking of functional domains, such as active sites from
enzymes, glycosylation domains, cellular targeting signals or
transmembrane regions. In preferred embodiments, the fusion
proteins of the instant invention comprise a targeting peptide
linked to a therapeutic protein or peptide. Examples of proteins or
peptides that may be incorporated into a fusion protein include
cytostatic proteins, cytocidal proteins, pro-apoptosis agents,
anti-angiogenic agents, hormones, cytokines, growth factors,
peptide drugs, antibodies, Fab fragments antibodies, antigens,
receptor proteins, enzymes, lectins, MHC proteins, cell adhesion
proteins and binding proteins. These examples are not meant to be
limiting and it is contemplated that within the scope of the
present invention virtually and protein or peptide could be
incorporated into a fusion protein comprising a targeting peptide.
Methods of generating fusion proteins are well known to those of
skill in the art. Such proteins can be produced, for example, by
chemical attachment using bifunctional cross-linking reagents, by
de novo synthesis of the complete fusion protein, or by attachment
of a DNA sequence encoding the targeting peptide to a DNA sequence
encoding the second peptide or protein, followed by expression of
the intact fusion protein.
[0100] Protein Purification
[0101] In certain embodiments a protein or peptide may be isolated
or purified. Protein purification techniques are well known to
those of skill in the art. These techniques involve, at one level,
the homogenization and crude fractionation of the cells, tissue or
organ to polypeptide and non-polypeptide fractions. The protein or
polypeptide of interest may be further purified using
chromatographic and electrophoretic techniques to achieve partial
or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, gel exclusion
chromatography, polyacrylamide gel electrophoresis, affinity
chromatography, immunoaffinity chromatography and isoelectric
focusing. An example of receptor protein purification by affinity
chromatography is disclosed in U.S. Pat. No. 5,206,347, the entire
text of which is incorporated herein by reference. A particularly
efficient method of purifying peptides is fast protein liquid
chromatography (FPLC) or even HPLC.
[0102] A purified protein or peptide is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. An isolated or purified protein or
peptide, therefore, also refers to a protein or peptide free from
the environment in which it may naturally occur. Generally,
"purified" will refer to a protein or peptide composition that has
been subjected to fractionation to remove various other components,
and which composition substantially retains its expressed
biological activity. Where the term "substantially purified" is
used, this designation will refer to a composition in which the
protein or peptide forms the major component of the composition,
such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or more of the proteins in the
composition.
[0103] Various methods for quantifying the degree of purification
of the protein or peptide are known to those of skill in the art in
light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity therein, assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification, and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0104] Various techniques suitable for use in protein purification
are well known to those of skill in the art. These include, for
example, precipitation with ammonium sulphate, PEG, antibodies and
the like, or by heat denaturation, followed by: centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of these and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0105] There is no general requirement that the protein or peptide
always be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an TPLC apparatus will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0106] Affinity chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule to which it can specifically bind to. This is a
receptor-ligand type of interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(e.g., altered pH, ionic strength, temperature, etc.). The matrix
should be a substance that itself does not adsorb molecules to any
significant extent and that has a broad range of chemical, physical
and thermal stability. The ligand should be coupled in such a way
as to not affect its binding properties. The ligand should also
provide relatively tight binding. And it should be possible to
elute the substance without destroying the sample or the
ligand.
[0107] Synthetic Peptides
[0108] Because of their relatively small size, the targeting
peptides of the invention can be synthesized in solution or on a
solid support in accordance with conventional techniques. Various
automatic synthesizers are commercially available and can be used
in accordance with known protocols. See, for example, Stewart and
Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany
and Merrifield (1979), each incorporated herein by reference. Short
peptide sequences, usually from about 6 up to about 35 to 50 amino
acids, can be readily synthesized by such methods. Alternatively,
recombinant DNA technology may be employed wherein a nucleotide
sequence which encodes a peptide of the invention is inserted into
an expression vector, transformed or transfected into an
appropriate host cell, and cultivated under conditions suitable for
expression.
[0109] Antibodies
[0110] In certain embodiments, it may be desirable to make
antibodies against the identified targeting peptides or their
receptors. The appropriate targeting peptide or receptor, or
portions thereof, may be coupled, bonded, bound, conjugated, or
chemically-linked to one or more agents via linkers, polylinkers,
or derivatized amino acids. This may be performed such that a
bispecific or multivalent composition or vaccine is produced. It is
further envisioned that the methods used in the preparation of
these compositions are familiar to those of skill in the art and
should be suitable for administration to humans, i.e.,
pharmaceutically acceptable. Preferred agents are the carriers are
keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
[0111] The term "antibody" is used to refer to any antibody-like
molecule that has an antigen binding region, and includes antibody
fragments such as Fab', Fab, F(ab').sub.2, single domain antibodies
(DABs), Fv, scFv (single chain Fv), and the like. Techniques for
preparing and using various antibody-based constructs and fragments
are well known in the art. Means for preparing and characterizing
antibodies are also well known in the art (See, e.g., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference).
[0112] Cytokines and Chemokines
[0113] In certain embodiments, it may be desirable to couple
specific bioactive agents to one or more targeting peptides for
targeted delivery to an organ, tissue or cell type. Such agents
include, but are not limited to, cytokines, chemikines,
pro-apoptosis factors and anti-angiogenic factors. The term
"cytokine" is a generic term for proteins released by one cell
population which act on another cell as intercellular mediators.
Examples of such cytokines are lymphokines, monokines, growth
factors and traditional polypeptide hormones. Included among the
cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and -.beta.; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-.beta.; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -..beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M- CSF, EPO,
kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor
necrosis factor and LT. As used herein, the term cytokine includes
proteins from natural sources or from recombinant cell culture and
biologically active equivalents of the native sequence
cytokines.
[0114] Chemokines generally act as chemoattractants to recruit
immune effector cells to the site of chemokine expression. It may
be advantageous to express a particular chemokine gene in
combination with, for example, a cytokine gene, to enhance the
recruitment of other immune system components to the site of
treatment. Chemokines include, but are not limited to, RANTES,
MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilled artisan will
recognize that certain cytokines are also known to have
chemoattractant effects and could also be classified under the term
chemokines.
[0115] Imaging Agents and Radioisotopes
[0116] In certain embodiments, the claimed peptides or proteins of
the present invention may be attached to imaging agents of use for
imaging and diagnosis of various diseased organs, tissues or cell
types. Many appropriate imaging agents are known in the art, as are
methods for their attachment to proteins or peptides (see, e.g.,
U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein by
reference). Certain attachment methods involve the use of a metal
chelate complex employing, for example, an organic chelating agent
such a DTPA attached to the protein or peptide (U.S. Pat. No.
4,472,509). Proteins or peptides also may be reacted with an enzyme
in the presence of a coupling agent such as glutaraldehyde or
periodate. Conjugates with fluorescein markers are prepared in the
presence of these coupling agents or by reaction with an
isothiocyanate.
[0117] Non-limiting examples of paramagnetic ions of potential use
as imaging agents include chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium
(III), samarium (III), ytterbium (III), gadolinium (III), vanadium
(II), terbium (III), dysprosium (III), holmium (III) and erbium
(III), with gadolinium being particularly preferred. Ions useful in
other contexts, such as X-ray imaging, include but are not limited
to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0118] Radioisotopes of potential use as imaging or therapeutic
agents include astatine.sup.211, .sup.14carbon, .sup.51chromium,
.sup.36chlorine, .sup.57cobalt, .sup.58cobalt, copper.sup.67,
.sup.152Eu, gallium.sup.67, .sup.3hydrogen, iodine.sup.123,
iodine125, iodine.sup.131, indium.sup.111, .sup.59iron,
.sup.32phosphorus, rhenium.sup.186, rhenium.sup.188,
.sup.75selenium, .sup.35sulphur, technicium.sup.99m and
yttrium.sup.90. .sup.125I is often being preferred for use in
certain embodiments, and technicium.sup.99m and indium.sup.111 are
also often preferred due to their low energy and suitability for
long range detection.
[0119] Radioactively labeled proteins or peptides of the present
invention may be produced according to well-known methods in the
art. For instance, they can be iodinated by contact with sodium or
potassium iodide and a chemical oxidizing agent such as sodium
hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Proteins or peptides according to the invention
may be labeled with technetium-.sup.99m by ligand exchange process,
for example, by reducing pertechnate with stannous solution,
chelating the reduced technetium onto a Sephadex column and
applying the peptide to this column or by direct labeling
techniques, e.g., by incubating pertechnate, a reducing agent such
as SNCl.sub.2, a buffer solution such as sodium-potassium phthalate
solution, and the peptide. Intermediary functional groups which are
often used to bind radioisotopes which exist as metallic ions to
peptides are diethylenetriaminepentaacetic acid ODTPA) and ethylene
diaminetetracetic acid (EDTA). Also contemplated for use are
fluorescent labels, including rhodarmine, fluorescein
isothiocyanate and renographin.
[0120] In certain embodiments, the claimed proteins or peptides may
be linked to a secondary binding ligand or to an enzyme (an enzyme
tag) that will generate a colored product upon contact with a
chromogenic substrate. Examples of suitable enzymes include urease,
alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose
oxidase. Preferred secondary binding ligands are biotin and avidin
or streptavidin compounds. The use of such labels is well known to
those of skill in the art in light and is described, for example,
in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241; each incorporated herein by
reference.
[0121] Cross-Linkers
[0122] Bifunctional cross-linking reagents have been extensively
used for a variety of purposes including preparation of affinity
matrices, modification and stabilization of diverse structures,
identification of ligand and receptor binding sites, and structural
studies. Homobifunctional reagents that carry two identical
functional groups proved to be highly efficient in inducing
cross-linking between identical and different macromolecules or
subunits of a macromolecule, and linking of polypeptide ligands to
their specific binding sites. Heterobifunctional reagents contain
two different functional groups. By taking advantage of the
differential reactivities of the two different functional groups,
cross-linking can be controlled both selectively and sequentially.
The bifunctional cross-linking reagents can be divided according to
the specificity of their functional groups, e.g., amino,
sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,
reagents directed to free amino groups have become especially
popular because of their commercial availability, ease of synthesis
and the mild reaction conditions under which they can be applied. A
majority of heterobifunctional cross-linking reagents contains a
primary amine-reactive group and a thiol-reactive group.
[0123] Exemplary methods for cross-linking ligands to liposomes are
described in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511,
each specifically incorporated herein by reference in its
entirety). Various ligands can be covalently bound to liposomal
surfaces through the cross-linking of amine residues, liposomes, in
particular, multilamellar vesicles (MLV) or unilamellar vesicles
such as microemulsified liposomes (MEL) and large unilamellar
liposomes (LUVET), each containing phosphatidylethanolamine (PE),
have been prepared by established procedures. The inclusion of PE
in the liposome provides an active functional residue, a primary
amine, on the liposomal surface for cross-linking purposes. Ligands
such as epidermal growth factor (EGF) have been successfully linked
with PE-liposomes. Ligands are bound covalently to discrete sites
on the liposome surfaces. The number and surface density of these
sites are dictated by the liposome formulation and the liposome
type. The liposomal surfaces may also have sites for non-covalent
association. To form covalent conjugates of ligands and liposomes,
cross-linking reagents have been studied for effectiveness and
biocompatibility. Cross-linking reagents include glutaraldehyde
(GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether
(EGDE), and a water soluble carbodiimide, preferably
1-ethyl-3-(3-dimethylaminopropyl- ) carbodiimide (EDC). Through the
complex chemistry of cross-linking, linkage of the amine residues
of the recognizing substance and liposomes is established.
[0124] In another example, heterobifunctional cross-linking
reagents and methods of using the cross-linking reagents are
described (U.S. Pat. No. 5,889,155, specifically incorporated
herein by reference in its entirety). The cross-linking reagents
combine a nucleophilic hydrazide residue with an electrophilic
maleimide residue, allowing coupling in one example, of aldehydes
to free thiols. The cross-linking reagent can be modified to
cross-link various functional groups.
[0125] Nucleic Acids
[0126] Nucleic acids according to the present invention may encode
a targeting peptide, a receptor protein or a fusion protein. The
nucleic acid may be derived from genomic DNA, complementary DNA
(cDNA) or synthetic DNA. Where incorporation into an expression
vector is desired, the nucleic acid may also comprise a natural
intron or an intron derived from another gene. Such engineered
molecules are sometime referred to as "mini-genes."
[0127] A "nucleic acid" as used herein includes single-stranded and
double-stranded molecules, as well as DNA, RNA, chemically modified
nucleic acids and nucleic acid analogs. It is contemplated that a
nucleic acid within the scope of the present invention may be of 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120,
aboout 130, about 140, about 150, about 160, about 170, about 180,
about 190, about 200, about 210, about 220, about 230, about 240,
about 250, about 275, about 300, about 325, about 350, about 375,
about 400, about 425, about 450, about 475, about 500, about 525,
about 550, about 575, about 600, about 625, about 650, about 675,
about 700, about 725, about 750, about 775, about 800, about 825,
about 850, about 875, about 900, about 925, about 950, about 975,
about 1000, about 1100, about 1200, about 1300, about 1400, about
1500, about 1750, about 2000, about 2250, about 2500 or greater
nucleotide residues in length.
[0128] It is contemplated that targeting peptides, fusion proteins
and receptors may be encoded by any nucleic acid sequence that
encodes the appropriate amino acid sequence. The design and
production of nucleic acids encoding a desired amino acid sequence
is well known to those of skill in the art, using standardized
codon tables (see Table 2 below). In preferred embodiments, the
codons selected for encoding each amino acid may be modified to
optimize expression of the nucleic acid in the host cell of
interest. Codon preferences for various species of host cell are
well known in the art.
2 TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine
Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA
GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K
AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0129] In addition to nucleic acids encoding the desired targeting
peptide, fusion protein or receptor amino acid sequence, the
present invention encompasses complementary nucleic acids that
hybridize under high stringency conditions with such coding nucleic
acid sequences. High stringency conditions for nucleic acid
hybridization are well known in the art. For example, conditions
may comprise low salt and/or high temperature conditions, such as
provided by about 0.02 M to about 0.15 M NaCl at temperatures of
about 50.degree. C. to about 70.degree. C. It is understood that
the temperature and ionic strength of a desired stringency are
determined in part by the length of the particular nucleic acid(s),
the length and nucleotide content of the target sequence(s), the
charge composition of the nucleic acid(s), and to the presence or
concentration of formamide, tetramethylammonium chloride or other
solvent(s) in a hybridization mixture.
[0130] Vectors for Cloning, Gene Transfer and Expression
[0131] In certain embodiments expression vectors are employed to
express the targeting peptide or fusion protein, which can then be
purified and used. In other embodiments, the expression vectors are
used in gene therapy. Expression requires that appropriate signals
be provided in the vectors, and which include various regulatory
elements, such as enhancers/promoters from both viral and mammalian
sources that drive expression of the genes of interest in host
cells. Elements designed to optimize messenger RNA stability and
translatability in host cells also are known.
[0132] Regulatory Elements
[0133] The terms "expression construct" or "expression vector" are
meant to include any type of genetic construct containing a nucleic
acid coding for a gene product in which part or all of the nucleic
acid coding sequence is capable of being transcribed. In preferred
embodiments, the nucleic acid encoding a gene product is under
transcriptional control of a promoter. A "promoter" refers to a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a gene. The phrase "under transcriptional control"
means that the promoter is in the correct location and orientation
in relation to the nucleic acid to control RNA polymerase
initiation and expression of the gene.
[0134] The particular promoter employed to control the expression
of a nucleic acid sequence of interest is not believed to be
important, so long as it is capable of directing the expression of
the nucleic acid in the targeted cell. Thus, where a human cell is
targeted, it is preferable to position the nucleic acid coding
region adjacent and under the control of a promoter that is capable
of being expressed in a human cell. Generally speaking, such a
promoter might include either a human or viral promoter.
[0135] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, rat insulin promoter, and
glyceraldehyde-3-phosphate dehydrogenase promoter can be used to
obtain high-level expression of the coding sequence of interest.
The use of other viral or mammalian cellular or bacterial phage
promoters which are well-known in the art to achieve expression of
a coding sequence of interest is contemplated as well, provided
that the levels of expression are sufficient for a given
purpose.
[0136] Where a cDNA insert is employed, typically one will
typically include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed, such as human growth hormone and SV40 polyadenylation
signals. Also contemplated as an element of the expression
construct is a terminator. These elements can serve to enhance
message levels and to minimize read through from the construct into
other sequences.
[0137] Selectable Markers
[0138] In certain embodiments of the invention, the cells
containing nucleic acid constructs of the present invention may be
identified in vitro or in vivo by including a marker in the
expression construct. Such markers would confer an identifiable
change to the cell permitting easy identification of cells
containing the expression construct. Usually the inclusion of a
drug selection marker aids in cloning and in the selection of
transformants. For example, genes that confer resistance to
neomycin, puromycin, hygromycin, DBFR, GPT, zeocin, and histidinol
are useful selectable markers. Alternatively, enzymes such as
herpes simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be employed. Immunologic markers also
can be employed. The selectable marker employed is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable markers are well known to one of
skill in the art.
[0139] Delivery of Expression Vectors
[0140] There are a number of ways in which expression vectors may
introduced into cells. In certain embodiments of the invention, the
expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to
enter cells via receptor-mediated endocytosis, to integrate into
host cell genome, and express viral genes stably and efficiently
have made them attractive candidates for the transfer of foreign
genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein,
1988; Baichwal and Sugden, 1986; Temin, 1986). Preferred gene
therapy vectors are generally viral vectors.
[0141] Although some viruses that can accept foreign genetic
material are limited in the number of nucleotides they can
accommodate and in the range of cells they infect, these viruses
have been demonstrated to successfully effect gene expression.
However, adenoviruses do not integrate their genetic material into
the host genome and therefore do not require host replication for
gene expression making them ideally suited for rapid, efficient,
heterologous gene expression. Techniques for preeparing replication
infective viruses are well known in the art.
[0142] In using viral delivery systems, one will desire to purify
the virion sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering viral,
particles or endotoxins and other pyrogens such that it will not
cause any untoward reactions in the cell, animal or individual
receiving the vector construct. A preferred means of purifying the
vector involves the use of buoyant density gradients, such as
cesium chloride gradient centrifugation.
[0143] DNA viruses used as gene vectors include the papovaviruses
(e.g., simian virus 40, bovine papilloma virus, and polyoma)
(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses
(Ridgeway, 1988; Baichwal and Sugden, 1986).
[0144] One of the preferred methods for in vivo delivery involves
the use of an adenovirus expression vector. Although adenovirus
vectors are known to have a low capacity for integration into
genomic DNA, this feature is counterbalanced by the high efficiency
of gene transfer afforded by these vectors. "Adenovirus expression
vector" is meant to include, but is not limited to, constructs
containing adenovirus sequences sufficient to (a) support packaging
of the construct and (b) tq express an antisense or a sense
polynucleotide that has been cloned therein.
[0145] The expression vector comprises a genetically engineered
form of adenovirus. Knowledge of the genetic organization of
adenovirus, a 36 kb, linear, double-stranded DNA virus, allows
substitution of large pieces of adenoviral DNA with foreign
sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to
retroviral infection, the adenoviral infection of host cells does
not result in chromosomal integration because adenoviral DNA can
replicate in an episomal manner without potential genotoxicity.
Also, adenoviruses are structurally stable, and no genome
rearrangement has been detected after extensive amplification.
Adenovirus can infect virtually all epithelial cells regardless of
their cell cycle stage. So far, adenoviral infection appears to be
linked only to mild disease such as acute respiratory disease in
humans.
[0146] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target cell range and high
infectivity. Both ends of the viral genome contain 100-200 base
pair inverted repeats (ITRs), which are cis elements necessary for
viral DNA replication and packaging. The early (E) and late (L)
regions of the genome contain different transcription units that
are divided by the onset of viral DNA replication. The E1 region
(E1A and E1B) encodes proteins responsible for the regulation of
transcription of the viral genome and a few cellular genes. The
expression of the E2 region (E2A and E2B) results in the synthesis
of the proteins for viral DNA replication. These proteins are
involved in DNA replication, late gene expression and host cell
shut-off (Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by the
major late promoter (MLP). The MLP, (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the mRNAs issued. from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNAs for
translation.
[0147] In currently used systems, recombinant adenovirus is
generated from homologous recombination between shuttle vector and
provirus vector. Due to the possible recombination between two
proviral vectors, wild-type adenovirus may be generated from this
process. Therefore, it is critical to isolate a single clone of
virus from an individual plaque and examine its genomic
structure.
[0148] Generation and propagation of adenovirus vectors which are
replication deficient depend on a unique helper cell line,
designated 293, which is transformed from human embryonic kidney
cells by Ad5 DNA fragments and constitutively expresses E1 proteins
(Graham et al., 1977). Since the E3 region is dispensable from the
adenovirus genome (Jones and Shenk, 1978), the current adenovirus
vectors, with the help of 293 cells, carry foreign DNA in either
the E1, the E3, or both regions (Graham and Prevec, 1991). In
nature, adenovirus can package approximately 105% of the wild-type
genome (Ghosh-Choudhury et al., 1987), providing capacity for about
2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA
that is replaceable in the E1 and E3 regions, the maximum capacity
of the current adenovirus vector is under 7.5 kb, or about 15% of
the total length of the vector. More than 80% of the adenovirus
viral genome remains in the vector backbone and is the source of
vector-borne cytotoxicity. Also, the replication deficiency of the
E1-deleted virus is incomplete. For example, leakage of viral gene
expression has been observed with the currently available vectors
at high multiplicities of infection (MOI) (Mulligan, 1993).
[0149] Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells or
other human embryonic mesenchymal or epithelial cells.
Alternatively, the helper cells may be derived from the cells of
other mammalian species that are permissive for human adenovirus.
Such cells include, e.g., Vero cells or other monkey embryonic
mesenchymal or epithelial cells. As discussed, the preferred helper
cell line is 293.
[0150] Racher et al., (1995) disclosed improved methods for
culturing 293 cells and propagating adenovirus. In one format,
natural cell aggregates are grown by inoculating individual cells
into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the
cell viability is estimated with trypan blue. In another format,
Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) are
employed as follows. A cell innoculum, resuspended in 5 ml of
medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer
flask and left stationary, with occasional agitation, for 1 to 4 h.
The medium is then replaced with 50 ml of fresh medium and shaking
is initiated. For virus production, cells are allowed to grow to
about 80% confluence, after which time the medium is replaced (to
25% of the final volume) and adenovirus added at an MOI of 0.05.
Cultures are left stationary overnight, following which the volume
is increased to 100% and shaking is commenced for another 72
hr.
[0151] Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be of
any of the 42 different known serotypes or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the present invention. This is because Adenovirus
type 5 is a human adenovirus about which a great deal of
biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector.
[0152] A typical vector applicable to practicing the present
invention is replication defective and will not have an adenovirus
E1 region. Thus, it are most convenient to introduce the
polynucleotide encoding the gene at the position from which the
E1-coding sequences have been removed. However, the position of
insertion of the construct within the adenovirus sequences is not
critical. The polynucleotide encoding the gene of interest may also
be inserted in lieu of the deleted E3 region in E3 replacement
vectors as described by Karlsson et al., (1986) or in the E4 region
where a helper cell line or helper virus complements the E4
defect.
[0153] Alkylating Agents
[0154] Alkylating agents are drugs that directly interact with
genomic DNA to prevent the cancer cell from proliferating. This
category of chemotherapeutic drugs represents agents that affect
all phases of the cell cycle, that is, they are not phase-specific.
An alkylating agent, may include, but is not limited to, a nitrogen
mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a
nitrosourea or a triazines. They include but are not limited to:
busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),
dacarbazine, ifosfamide, mechlorethamine (mustargen), and
melphalan.
[0155] Antimetabolites
[0156] Antimetabolites disrupt DNA and RNA synthesis. Unlike
alkylating agents, they specifically influence the cell cycle
during S phase. Antimetabolites can be differentiated into various
categories, such as folic acid analogs, pyrimidine analogs and
purine analogs and related inhibitory compounds. Antimetaboiltes
include but are not limited to, 5-fluorouracil (5-FU), cytarabine
(Ara-C), fludarabine, gemcitabine, and methotrexate.
[0157] Natural Products
[0158] Natural products generally refer to compounds originally
isolated from a natural source, and identified has having a
pharmacological activity. Such compounds, analogs and derivatives
thereof may be, isolated from a natural source, chemically
synthesized or recombinantly produced by any technique known to
those of skill in the art. Natural products include such categories
as mitotic inhibitors, antitumor antibiotics, enzymes and
biological response modifiers.
[0159] Mitotic inhibitors include plant alkaloids and other natural
agents that can inhibit either protein synthesis required for cell
division or mitosis. They operate during a specific phase during
the cell cycle. Mitotic inhibitors include, for example, docetaxel,
etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine,
vincristine, and vinorelbine.
[0160] Taxoids are a class of related compounds isolated from the
bark of the ash tree, Taxus brevifolia. Taxoids include but are not
limited to compounds such as docetaxel and paclitaxel. Paclitaxel
binds to tubulin (at a site distinct from that used by the vinca
alkaloids) and promotes the assembly of microtubules.
[0161] Vinca alkaloids are a type of plant alkaloid identified to
have pharmaceutical activity. They include such compounds as
vinblastine (VLB) and vincristine.
[0162] Antitumor Antibiotics
[0163] Antitumor antibiotics have both antimicrobial and cytotoxic
activity. These drugs also interfere with DNA by chemically
inhibiting enzymes and mitosis or altering cellular membranes.
These agents are not phase specific so they work in all phases of
the cell cycle. Examples of antitumor antibiotics include, but are
not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin
(Adriamycin), plicarnycin (mithramycin) and idarubicin.
[0164] Hormones
[0165] Corticosteroid hormones are considered chemotherapy drugs
when they are implemented to kill or slow the growth of cancer
cells. Corticosteroid hormones can increase the effectiveness of
other chemotherapy agents, and consequently, they are frequently
used in combination treatments. Prednisone and dexamethasone are
examples of corticosteroid hormones.
[0166] Progestins such as hydroxyprogesterone caproate,
medroxyprogesterone acetate, and megestrol acetate have been used
in cancers of the endometrium and breast. Estrogens such as
diethylstilbestrol and ethinyl estradiol have been used in cancers
such as breast and prostate. Antiestrogens such as tamoxifen have
been used in cancers such as breast. Androgens such as testosterone
propionate and fluoxymesterone have also been used in treating
breast cancer. Antiandrogens such as flutamide have been used in
the treatment of prostate cancer. Gonadotropin-releasing hormone
analogs such as leuprolide have been used in treating prostate
cancer.
[0167] Miscellaneous Agents
[0168] Some chemotherapy agents do not fall into the previous
categories based on their activities. They include, but are not
limited to, platinum coordination complexes, anthracenedione,
substituted urea, methyl hydrazine derivative, adrenalcortical
suppressant, amsacrine, L-asparaginase, and tretinoin. It is
contemplated that they may be used within the compositions and
methods of the present invention.
[0169] Platinum coordination complexes include such compounds as
carboplatin and cisplatin (cis-DDP).
[0170] An anthracenedione such as mitoxantrone has been used for
treating acute granulocytic leukemia and breast cancer. A
substituted urea such as hydroxyurea has been used in treating
chronic granulocytic leukemia, polycythemia vera, essental
thrombocytosis and malignant melanoma. A methyl hydrazine
derivative such as procarbazine (N-methylhydrazine, MIH) has been
used in the treatment of Hodgkin's disease. An adrenocortical
suppressant such as mitotane has been used to treat adrenal cortex
cancer, while aminoglutethimide has been used to treat Hodgkin's
disease.
[0171] Regulators of Programmed Cell Death
[0172] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
folicular lymphoma, plays a prominent role in controlling apoptosis
and enhancing cell survival in response to diverse apoptotic
stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et
al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The
evolutionarily conserved Bcl-2 protein now is recognized to be a
member of a family of related proteins, which can be categorized as
death agonists or death antagonists.
[0173] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, Bcl.sub.S,
Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0174] Non-limiting examples of pro-apoptosis agents contemplated
within the scope of the present invention include gramicidin,
magainin, mellitin, defensin, cecropin, (KLAKLAK).sub.2 (SEQ ID
NO:1), (KLAKKLA).sub.2 (SEQ ID NO:2), (KAAKKAA).sub.2 (SEQ ID NO:3)
or (KLGKKLG).sub.3 (SEQ ID NO:4).
[0175] Angiogenic Inhibitors
[0176] In certain embodiments the present invention may concern
administration of targeting peptides attached to anti-angiogenic
agents, such as angiotensin, laminin peptides, fibronectin
peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet
factor 4, IP-10, Gro-.beta., thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CM101,
Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,
Linomide, thalidomide, pentoxifylline, genistein, TNP-470,
endostatin, paclitaxel, accutin, angiostatin, cidofovir,
vincristine, bleomycin, AGM-1470, platelet factor 4 or
minocycline.
[0177] Dosages
[0178] The skilled artisan is directed to "Remington's
Pharmaceutical Sciences" 15th Edition, chapter 33, and in
particular to pages 624-652. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet
sterility, pyrogenicity, and general safety and purity standards as
required by the FDA Office of Biologics standards.
EXAMPLES
[0179] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Bone Marrow Targeting Peptides
[0180] A non-limiting example of an organ of specific interest for
targeting petides is bone marrow. Bone is the preferred site of
metastasis in the large majority of patients with prostate cancer
(Fidler, 1999). This striking selectivity has been viewed as an
example of site-specific interactions that were essential to cancer
progression (Rak, 1995; Zetter, 1998). Despite the clinical
relevance, little is known about the mechanisms that control
prostate cancer spread to bone. In addition, there were no
effective strategies for targeting therapeutic agents for the
treatment of metastatic prostate cancer (Brodt et. al, 1996).
[0181] A subset of peptides capable of selective homing to bone
marrow through the circulation is likely to simulate the behavior
of prostate cancer cells during bone metastasis formation. The
vascular markers targeted by using phage display might also be
utilized by tumor cells to metastasize. This concept has already
been proven to be true for lung-homing peptides. Peptides that home
to lung blood vessels inhibit experimental metastasis. These
results fit a "modified seed and soil" model, in which the basis
for site-specific metastasis is the presence of homing receptors in
blood vessels of certain tissues to which metastasis preferentially
occurs. Such selective vascular markers are exposed to tumor cells
during adhesion, the first step of the metastastic cascade.
Isolation of bone marrow-homing peptides is of utility for
identifying those vascular markers that mediate prostate cancer
cell homing during the metastatic process, and for potential
therapeutic intervention in preventing metastases to bone, or in
selectively imaging and/or treating cancer that has already
metastasized to bone.
[0182] Methods
[0183] In vivo screening of phage libraries was used to isolate
peptides that bind to bone marrow in mice. The bone marrow
targeting peptides were characterized with regard to their ability
to inhibit metastasis in prostate cancer mouse models. Affinity
chromatography and molecular cloning were used to identify the
receptors for the bone-marrow binding peptides. The compositions
and methods disclosed herein were of use to develop new
anti-prostate cancer therapeutic strategies that focus on the
prevention and treatment of bone metastasis.
[0184] In vivo screenings to isolate peptides that home to bone
marrow in mice.
[0185] Phage libraries were injected intravenously. The libraries
were prepared according to the protocol of Smith and Scott (1985)
with improvements, discussed below. The phage in these libraries
displayed inserts ranging from 5 to if 11 residues. Tissue samples
were processed for phage rescue by transfer to 1 ml DMEM-PI in a
glass tube and homogenized with a grinder. Bone marrow does not
require homogenization, whereas other organs that were used as
controls needed to be minced before they could be efficiently
homogenized. Samples were transferred to autoclaved 2 ml Eppendorf
tubes. The tissues were washed with ice cold DMEM-PI containing 1%
BSA. After 3 washes, the pellets were resuspended and brought to
37.degree. C. before adding bacteria. Incubation of the washed
tissue samples with 1.5 ml of competent K91-kan bacteria
(OD.sub.600 0.2 in 1:10 dil.) for one hour at room temperature was
used for recovering the phage particles.
[0186] Multiple aliquots were plated in LB tet/kan plates or dishes
containing 40 .mu.g/ml of tetracycline and 100 .mu.g/ml kanamycin.
Platings were performed at several concentrations, covering a large
range of sample, i.e. 3 ml, 1 ml, 300 .mu.l, 100 .mu.l, 30 .mu.l.
The beads that were used for plating were passed on to two
subsequent 10 cm LB tet/kan plates so as to recover every
potentially phage infected bacterial clone trapped on the bead
surface. The dishes were incubated overnight at 37.degree. C. The
remaining 2 -3 ml of infected culture (including the homogenized
tissue) was transferred into 10 ml of LB medium containing 40
.mu.g/ml tetracycline and 100 .mu.g/ml kanamycin (LB tet/kan) and
placed in the shaker at 37.degree. C. for 2 h. The 12 ml cultures
were expanded to 1 liter LB tettkan and grown overnight in the
37.degree. C. shaker. Phage were rescued from the bulk amplified
bacterial culture after 12-16 h, according to standard protocols
and saved for subsequent rounds of selection. From the
plates/dishes in the incubator, well separated colonies from bone
marrow were sequenced. The colonies were transferred to 96 well
plates containing 20 .mu.l PBS/well for sequencing
[0187] Immunohistochemical staining with an anti-M13 antibody was
used to examine phage targeting in various tissues (Pasqualiri and
Ruoslahti, 1996; Arap et al, 1998). The phage were injected IV and
allowed to circulate for 5 min or 24 h. Mice in the 5 min
experiment were perfused with DMEM after the phage injection to
remove unbound phage from the circulation. There was little
circulating phage after 24 h (Arap et al, 1998a, 1998b). The
animals were sacrificed, their tissues collected, fixed with
Bouin's solution, sectioned and stained with antibodies against the
phage.
[0188] Results
[0189] Murine Bone Marrow Targeting In Vivo in Mice
[0190] Bone marrow targeting sequence motifs were identified by
intravenously injecting phage libraries into mice and recovering
phage from bone marrow. Phage were injected intravenously,
recovered from the bone marrow, repeatedly amplified in vitro and
re-injected to obtain sufficient enrichment. After three rounds of
selection, phage preparations that homed to mouse bone marrow were
obtained. The individual phage exhibited similar organ specificity
as the pooled phage after intravenous injection. Several peptide
motifs were identified and characterized. The most promising motifs
that show specificity in vivo are shown in Table 3
3TABLE 3 Sequences in phage that target murine bone marrow in vivo.
CX.sub.3CX.sub.3CX.sub.3C peptide library C V M T C A P R C F E H C
(SEQ ID NO:5) C D G V C A P R C G E R C (SEQ ID NO:6) C T G G C V V
D C L S I C (SEQ ID NO:7) C G V P C R P A C R G L C (SEQ ID NO:8) C
A G F C V P G C H S K C (SEQ ID NO:9) C A G A C P V G C G T G C
(SEQ ID NO:10) X6 peptide library A E R L W R S (SEQ ID NO:11) S Q
H V V S G (SEQ ID NO:12) I A W R L E H (SEQ ID NO:13) W Y T V M S W
(SEQ ID NO:14) R L T Y K L Q (SEQ ID NO:15) W Q R L Y A W (SEQ ID
NO:16) E F R L G S K (SEQ ID NO:17) L G S N S K A (SEQ ID NO:18) C
G V V K F A (SEQ ID NO:19)
[0191] The skilled artisan will realize that the bone marrow
targeting peptide sequences identified herein will be of use for
numerous applications within the scope of the present invention,
including but not limited to targeted delivery of therapeutic
agents or gene therapy, in vivo imaging of normal or diseased
organs, tissues or cell types, identification of receptors and
receptor ligands in organs, tissues or cell types, and therapeutic
treatment of a number of human diseases, particularly metastatic
prostate cancer.
Example 2
Prostate and Prostate Cancer Targeting Peptides
[0192] Another non-limiting organ of particular interest for
targeting is the prostate. Prostate is an unusual organ because it
continues to growth throughout adult life. As a result, benign
prostate hypertrophy (BPH) affects most elderly men to some degree.
Even more serious, the prostate is a frequent site of malignant
tumors. One out of eleven men will develop prostate cancer during
their lifetime. Because serum markers for prostate cancer were
available, many of these malignant tumors were currently detected
early in the course of the disease. In the absence of reliable ways
of predicting which ones will progress clinically, many were
aggressively treated with surgery or radiotherapy, often with
devastating side-effects such as incontinence and impotence (Lane
and Shah, 1999; Mikolajczyk et al., 2000). There is a clear need
for improved methods for detection, prognosis, and treatment of
human prostate cancer.
[0193] Many interesting genes in the prostate may be expressed in
restricted--but perhaps highly specific or accessible--cellular
locations such as the prostate vasculature. Thus, potential targets
for intervention may easily be overlooked by high-throughput
sequencing or gene array approaches that do not account for the
molecular heterogeneity intrinsic to microanatomic or physiological
contexts.
[0194] The methods of the present invention allow the
identification of peptides that home to specific target sites in
vivo (Pasqualini et al., 1996, 1997, Koivunen et al., 1999;
Pasqualini, 1999). In vivo selection of phage peptide libraries
yields peptides that are capable of homing to specific receptors in
target tissues through the circulation. These studies have revealed
a surprising degree of specialization in various normal tissues
(Pasqualini, 1999; Rajotte et al., 1998, 1999). The present example
concerns compositions and methods of use of prostate targeting
peptides.
[0195] Methods
[0196] In Vivo Phage Targeting of the Prostate
[0197] Phage display libraries were injected intravenously. Samples
were kept on ice at all times. Prostate tissue samples were
processed as follows. The first sample was stored at -80.degree. C.
as a backup. The second sample was processed for
histology/pathology and HE or anti-M13 phage immunostaining. The
third sample was divided under clean conditions to obtain three
fragments with the same weight.
[0198] The triplicates from the third prostate sample were
processed for host bacterial infection and phage recovery. The
prostate sample was transferred to 1 ml DMEM-protease inhibitors
(PI) in a glass tissue grinder, homogenized and transfered cell
suspension to an autoclaved 2 ml eppendorf tube. Next, the prostate
tissue samples were washed three times with ice cold DMEM-PI
containing 1% BSA. The tissue was mixed with DMEM-PI and vortexed
for 30 seconds after each wash. After spinnig at 4,000 rpm for 3
min and the supernatant was carefully discarded (the tissue pellet
should remain undisturbed). Next, 1.5 ml DMEM-PI/BSA was added.
After the third wash, the pellet was briefly vortexed to re-suspend
the dissolved pellet warmed briefly to 37.degree. before adding the
host bacteria. Then, the admixture was incubated with 1.5 ml of
competent K91-kan bacteria (OD.sub.600=2) for one hour at room
temperature.
[0199] The admixture was transferred to Falcon tubes containing 10
ml of LB medium plus 0.2 .mu.g/ml of tetracycline at RT for 20
minute. Multiple aliquots were plated in LB tet/kan plates or
dishes containing 40 .mu.g/ml of tetracycline and 100 .mu.g/ml
kanamycin. Finally, dishes were incubated at 37.degree. C. and the
phage transducing unit count determined after an overnight
incubation.
[0200] Prostate Cancer Targeting
[0201] Tumor blood vessels are known to be leaky. Longer term
exposure of the mouse subject to a phage display library may result
in migration of phage and binding to prostate cancer cell markers.
Mice were incubated with a phage library for 24 hrs to target
cancer cell markers.
[0202] Male nude mice (4 mice) were injected with 10.sup.6 DU-145
cells in PBS. After tumor growth, the mice were injected with
either a CX10C phage display library. After allowing the library to
circulate for 24 hours, the mice were sacrificed and tissue samples
were collected. Samples were homogenized in Dounce homogenizer and
K91 bacteria were added to recover phage. Bacteria were plated on
kan/tet LB plates in triplicate at a series of dilution. The
remaing tumor homogenate with bacteria was incubated for 1 hr at RT
with 10 ml of LB/tet/kan. Another 10 ml of LK/tet/kan and incubated
at 37.degree. C. in a rotator to provide bulk phage stock. 216
colonies were picked from the plates to make a combined stock for
second round screening. After the clones wer amplified, they were
pooled and phage were precipitated with PEG/NaCl. Nude mice bearing
prostate tumors were subjected to a second round of selection as
described above, using the pooled phage recovered from round 1. A
third round of screening was perfromed as described.
[0203] Results
[0204] Normal Mouse Prostate
[0205] Mouse prostate targeting peptide motifs obtained by the
methods disclosed above are shown in Table 4.
4TABLE 4 Mouse Prostate Targeting Peptides Obtained by In vivo
Phage Display RVGTWGR SEQ ID NO:20 GRGRWGS SEQ ID NO:21 VQGIGRL SEQ
ID NO:22 VGSGRLS SEQ ID NO:23 GWTVRDG SEQ ID NO:24 GSRIRTP SEQ ID
NO:25 GGGSRIS SEQ ID NO:26 VMGGVVS SEQ ID NO:27 YGNDRRN SEQ ID
NO:28 SGKDRRS SEQ ID NO:29 YICPGPC SEQ ID NO:30 SYQSPGP SEQ ID
NO:31 AAAGSKH SEQ ID NO:32 GSRIRTP SEQ ID NO:33 SWGSRIR SEQ ID
NO:34 GGGSRIS SEQ ID NO:35 RVVGSRS SEQ ID NO:36 DGSTNLS SEQ ID
NO:37 VGSGRLS SEQ ID NO:38
[0206] Prostate Cancer
[0207] By the third round of in vivo screening, phage were obtained
that exhibited high selectivity for tumor localization compared to
control normla kidney tissue (not shown). Prostate cancer targeting
sequences identified by DNA sequencing the phage inserts are listed
in Table 5.
5TABLE 5 Prostate cancer targeting peptides LSRLVTGDVIC (SEQ ID
NO:210) CGNMGGSLYYVC (SEQ ID NO:211) CLHWEATFNPQC (SEQ ID NO:212)
CRTEVWRSNQRC (SEQ ID NO:213) CHVRDEHHEQGC (SEQ ID NO:214)
CPMQATRNLWHC (SEQ ID NO:215) CRDDAKVMRYNC (SEQ ID NO:216)
CNNWGELLGFNC (SEQ ID NO:217) CEGGYENLVLKC (SEQ ID NO:218)
CRNAWNKHGSRC (SEQ ID NO:219) CKERMYREQRRC (SEQ ID NO:220)
CRTIDIENNELC (SEQ ID NO:221) CHRGINRSTTDC (SEQ ID NO:222)
CETGREIDRSDC (SEQ ID NO:223) CCGRKTRGVAIC (SEQ ID NO:224)
CLASMLNMSTLC (SEQ ID NO:225) CGQGFAPRNLVC (SEQ ID NO:226)
CLGKWKSSRGTC (SEQ ID NO:227) CGEGFGSEWPPC (SEQ ID NO:228)
CKPDYMDSNKMC (SEQ ID NO:229) CTRNITKSRMMC (SEQ ID NO:230)
CVRNVDQNTNTC (SEQ ID NO:231) CFWTRENRGWTC (SEQ ID NO:232)
CRIRGIQLRPAC (SEQ ID NO:233) CEVGLSAAMAYCC (SEQ ID NO:234)
[0208] The skilled artisan will realize that the prostate targeting
peptide sequences identified herein will be of use for numerous
applications within the scope of the present invention, including
but not limited to targeted delivery of therapeutic agents or gene
therapy, in vivo imaging of normal or diseased organs, tissues or
cell types, identification of receptors and receptor ligands in
organs, tissues or cell types, and therapeutic treatment of a
number of diseases, particularly prostate cancer.
Example 3
Identification of Mouse Placenta, Adipose, Ovary and Ureter
Targeting Peptides
[0209] Identification of Placenta Homing Peptides
[0210] Peptides homing to the mouse placenta were identified by a
post-clearing protocol using a phage display library. A first round
of biopanning was performed on pregnant mice. Samples of placenta
were removed and phage rescued according to the standard protocols
described above, with one modification. In the typical bipanning
protocol, thousands of phage may be recovered from a single organ,
tissue or cell type. Typically, between 200 and 300 individual
colonies were selected from plated phage and these were amplified
and pooled to form the phage display library for the second or
third rounds of biopanning. In this example, all phage rescued from
the first round of biopanning were amplified in bulk on solid
medium and then pooled to form the phage display library for the
second round of biopanning. That is, there was no restriction of
the rescued phage from the first round of biopanning. This in vivo
biopanning without restriction was performed for three rounds
(rounds I-III), then a post-clearing procedure was used.
[0211] In the post-clearing protocol (round IV), phage were
administered to a non-pregnant mouse. Phage that bound to tissues
other than placenta were absorbed from the circulation. Remaining
phage were recovered from the plasma of the non-pregnant mouse.
This protocol was designed to isolate phage that bound to placenta
but not to other mouse organs, tissues or cell types. The following
placenta targeting peptides were identified, along with their
frequencies. A search of the GenBank database disclosed that none
of the SEQ ID NO's listed below was 100% homologous with any known
peptide sequence.
6 TPKTSVT (SEQ ID NO:39) 7.4% in round III, 8.5% in round IV
RMDGPVR (SEQ ID NO:40) 3.1% in round III, 8.5% in round IV RAPGGVR
(SEQ ID NO:41) <1% in round III, 8.5% in round IV VGLHARA (SEQ
ID NO:42) 4.2% in round III, 7.4% in round IV YIRPFTL (SEQ ID
NO:43) 2.1% in round III, 5.3% in round IV LGLRSVG (SEQ ID NO:44)
<1% in round III, 5.3% in round IV PSERSPS (SEQ ID NO:45) (data
not available)
[0212] As can be seen, the use of a post-clearing procedure
resulted in a substantial enrichment of phage bearing placenta
targeting peptides. Although this procedure was used for placenta,
the skilled artisan will realize that post-clearance can be
performed on for any organ, tissue or cell type where a phage
library can be administered to a subject lacking that organ, tissue
or cell type. For example, a post-clearing procedure for prostate
or testicle targeting peptides could be performed in a female
subject, and for ovary, vagina or uterus in a male subject.
[0213] A homology search identified several candidate proteins as
endogenous analogs of the placental targeting peptides, including
TCR gamma-1 (TPKTSVT, SEQ ID NO:39), tenascin (RMDGPVR, SEQ ID
NO:40 and RAPGGVR, SEQ ID NO:41), MHC Class II (LGLRSVG, SEQ ID
NO:44), angiotensin I (YIRPFIL, SEQ ID NO:43) and MFC H2-D-q alpha
chain (VGLIARA, SEQ ID NO:42).
[0214] Validation of Placenta Homing Peptides and Inhibition of
Pregnancy
[0215] The placenta homing peptides were validated in vivo by
injection into pregnant mice and recovery from the placenta. FIG. 1
shows the results of the validation studies for selected placenta
homing phage. The phage clones are identified as: PA--TPKTSVT (SEQ
ID NO:39), PC--RAPGGVR (SEQ ID NO:41), PE--LGLRSVG (SEQ ID NO:44),
PF--YIRPFIL (SEQ ID NO:43). It can be seen that the PA clone
exhibited placental homing more than an order of magnitude greater
than observed with control fd-tet phage. The PC clone also showed
substantially higher placental localization, while the PE and PF
clones were not substantially enriched in placenta compared to
control phage.
[0216] Despite the absence of apparent enrichment of the PF clone
in placental tissue, both the PA and PF peptides showed
anti-placental activity. Table 6 shows the effects of the PA and PF
placental targeting peptides injected into pregnant mice, attached
to FITC (fluorescein isothiocyanate), GST (glutathion
S-transferase) or to phage. At lower dosages (450 .mu.g total),
FITC conjugated PA and PF showed a slight effect on pregnancy
(Table 6). At higher dosages (800 to 1000 .mu.g protein or
4.5.times.10.sup.10 phage), both protein and phage conjugated PA
and PF peptides substantially interfered with fetal development
(Table 6), apparently resulting in death of the fetuses in most
cases. The CARAC peptide (SEQ ID NO:46), an adipose targeting
peptide (FE, TREVHRS, SEQ ID NO:47) or fd-tet phage were used as
non-placental targeting controls.
7TABLE 6 Effect of placental targeting peptides on fetal
development Peptide Injected Pregnancy Outcome Peptide Effect on
Embryo Inhibition with FITC conjugates -I 1 mouse injected iv
(predominantly) or ip .about.every other day, day 1-day 18, 9
times, Total 450 mM (.about.450 .mu.g) CARAC-FITC Delivery: 18 d,
No effect (-control) 5 normal pups PA-FITC Delivery: 19 d, No
effect (placenta homer) 8 normal pups PF-FITC Delivery: 21 d,
Development (placenta homer) 1 dead pup delay, toxicity Inhibition
with FITC conjugates -II 1 mouse injected sc (predominantly) or iv
.about.every other day, day 4-day 17, 10 times, Total 1 M (.about.1
mg) CARAC-FITC Delivery: 20 d, Slight (-control) 5 pups, 1-dead
toxicity? PA-FITC No fetuses inside Pregnancy (placenta homer)
after 21 d termination PF-FITC No fetuses inside Pregnancy
(placenta homer) after 21 d termination Inhibition with phage
conjugates -I 1 mouse injected iv (predominantly) or ip
.about.every other day, day 1-day 18, 9 times, Total 4.5 .times.
10.sup.10 TU Fd-Tet Avertin OD=>death. ? (-control) fetuses-OK
PA-phage Delivery: 24 d, Development delay, (placenta homer) 4
pups, 1-dead toxicity PF-phage Delivery: 25 d, Development delay,
(placenta homer) 8 pups, all dead toxicity Inhibition with GST
conjugates -I 1 mouse injected sc (predominantly) or iv
.about.every other day, day 4-day 17, 10 times, Total 800 .mu.g
GST-FE Delivery: 20 d, No effect (-control) 2 pups, OK GST-PA No
delivery or Pregnancy (placenta homer) fetuses after 21 d
termination GST-PF Day 15: no fetuses Pregnancy (placenta homer)
inside, uterus necrotic termination
[0217] These results validate the placental targeting peptide
sequences identified above. They further demonstrate that even in
the absence of substantial enrichment of phage bearing the
targeting sequence to the target organ (e.g. peptide PF, FIG. 1),
the targeting peptide may nevertheless provide for targeted
delivery of therapeutic agents to the target organ. In this study,
it appeared that at lower dosages the PF peptide was more effective
than the PA peptide at interfering with pregnancy, despite the
observation that the PA peptide produced a many-fold higher level
of phage localization to placenta.
[0218] The skilled artisan will realize that the disclosed methods
and peptides may be of use for targeted delivery of therapeutic
agents to the fetus through the placenta, as well as for novel
approaches to terminating pregnancy.
[0219] Adipose Targeting Peptides
[0220] A similar procotol was used to isolate fat targeting
peptides from a genetically obese mouse (Zhang et al., 1994;
Pelleymounter et al., 1995), with post-clearing performed in a
normal mouse. The fat-targeting peptides isolated included TRNTGNI
(SEQ ID NO:48), FDGQDRS (SEQ ID NO:49); WGPKRL (SEQ ID NO:50);
WGESRL (SEQ ID NO:51); VMGSVTG (SEQ ID NO:52), KGGRAKD (SEQ ID
NO:53), RGEVLWS (SEQ ID NO:54), TREVHRS (SEQ ID NO:47) and HGQGVRP
(SEQ ID NO:55).
[0221] Homology searches identified several candidate proteins as
the endogenous analogs of the fat targeting peptides, including
stem cell growth factor (SCGF) (KGGRAKD, SEQ ID NO:53), attractin
(mahogany) (RGEVLWS, SEQ ID NO:54), angiopoitin-related adipose
factor (FIAF) (TREVHRS, SEQ ID NO:47), adipophilin (ADRP) (VMGSVTG,
SEQ ID NO:52), Flt-1 or procollagen type XVII (TRNTGNI, SEQ ID
NO:48) and fibrillin 2 or transferrin-like protein p97 (HGQGVRP,
SEQ ID NO:55)
[0222] Validation of Adipose Targeting Peptides
[0223] The fat homing peptides were validated by in vivo homing, as
shown in FIG. 2. The fat homing clones selected were: FA--KGGRAKD
(SEQ ID NO:53), FC--RGEVLWS (SEQ ID NO:54), FE--TREVHRS (SEQ ID
NO:47) and FX--VMGSVTG (SEQ ID NO:52). As seen in FIG. 2, all of
these clones exhibited some elevation of homing to adipose tissue,
with clone FX showing several orders of magnitude higher adipose
localization than control fd-tet phage. Clone FX also exhibited
substantially higher localization than the other selected fat
homing clones. However, by analogy with the placental homing
peptides disclosed above, the skilled artisan will realize that fat
homing clones exhibiting lower levels of adipose tissue
localization may still be of use for targeted delivery of
therapeutic agents.
[0224] The skilled artisan will realize that targeting peptides
selective for angiogenic vasculature in adipose tissue could be of
use for weight reduction or for preventing weight gain. By
attaching anti-angiogenic or toxic moieties to an adipose targeting
peptide, the blood vessels supplying new fat tissue could be
selectively inhibited, preventing the growth of new deposits of fat
and potentially killing existing fat deposits.
[0225] Ovary and Ascites Targeting Peptides
[0226] Additional targeting peptide sequences have been identified
against mouse ovary and ascites fluid, listed below.
[0227] Mouse ovary targeting peptides include GLAKLIP (SEQ ID
NO:56), HIMDMS (SEQ ID NO:57), LQHWLLS (SEQ ID NO:58), ALVLQG (SEQ
ID NO:59). TGVALQS (SEQ ID NO:60), YVQSREG (SEQ ID NO:61), PLFWPYS
(SEQ ID NO:62), DGSG (SEQ ID NO:63), EGSG (SEQ ID NO:64), SSPRPGV
(SEQ ID NO:65), DGYPAIA (SEQ ID NO:66) GHAIE (SEQ ID NO:67) and
IWSTSER (SEQ ID NO:68).
[0228] Targeting peptides against mouse ascites include YRLRG (SEQ
ID NO:69), YRARG (SEQ ID NO:70), SQPLG (SEQ ID NO:71), SQPWG (SEQ
ID NO:72), QRLVTP (SEQ ID NO:73), QVLVTP (SEQ ID NO:74), QRLVHP
(SEQ ID NO:75), QVLVHP (SEQ ID NO:76), nRMTRYL (SEQ ID NO:77),
SLGGMSG (SEQ ID NO:78), SQLAAG (SEQ ID NO:79), SLLAAG (SEQ ID
NO:80), SQLVAG (SEQ ID NO:81), SLLAAG (SEQ ID NO:82), GLPSGLL (SEQ
ID NO:83), HGGSANP (SEQ ID NO:84), SLEAFFL (SEQ ID NO:85),
CVPELGHEC (SEQ ID NO:86), CELGFELGC (SEQ ID NO:87) AND CPFLRDWFC
(SEQ ID NO:88).
[0229] Ureter Targeting Peptides
[0230] Similar protocols were used to identify ureter targeting
peptides in C57B1 mice, disclosed in Table 7.
8TABLE 7 Ureter targeting peptides Motif Peptide LRXGN (SEQ ID
NO:235) GVMLRRG (SEQ ID NO:238) YSLRIGL (SEQ ID NO:239) LRDGNGE
(SEQ ID NO:240) CLRGGNLR (SEQ ID NO:241) RGAG (SEQ ID NO:236)
VRGLAAA (SEQ ID NO:242) ARGAGLA (SEQ ID NO:243) RGAGTGWT (SEQ ID
NO:244) ARGVNGA (SEQ ID NO:245) DLLR (SEQ ID NO:237) DLLRARW (SEQ
ID NO:246) DLLRTEW (SEQ ID NO:247) EFDLVRQ (SEQ ID NO:248) none
GCDEGGG (SEQ ID NO:249) none GDSPVES (SEQ ID NO:250)
Example 4
Screening an Alpha-Spleen Antibody Library In Vivo by BRASIL
[0231] Targeting peptides against spleen have not been previously
identified. As part of the reticulo-endothelial system, biopanning
against spleen tissue is complicated by the high background of
non-specific phage localization to spleen. The decreased background
observed in biopanning with the BRASIL method is advantageous for
identifying targeting peptides against tissues such as spleen.
[0232] This example demonstrates an illustrative embodiment of the
BRASIL method. A phage library based on immunoglobulins derived
against the target organ (mouse spleen) was developed and then
subjected to in vivo biopanning. To construct the immunoglobulin
library, mouse spleen was injected into a chicken. After boosting,
the chicken spleen was collected and immunoglobulin variable domain
sequences were obtained by PCR.TM. amplification of chicken spleen
mRNA. The amplified immunoglobulin variable sequences were inserted
into a phage display library (.alpha.-library) that was then used
for in vivo biopanning against mouse spleen. Thus, the spleen
targeting peptide sequences obtained from phage localized to mouse
spleen in vivo were derived from antibody fragments produced in the
chicken in response to mouse spleen antigens. The success of this
example further shows the broad utility of the BRASIL method. The
skilled artisan will realize that the present invention is not
limited to the embodiments disclosed herein and that many further
developments of the BRASIL methodology are included in the scope of
the present invention.
[0233] Materials and Methods
[0234] Library Construction
[0235] A white leghorn chicken was immunized with spleen homogenate
(about 150 mg per injection) from a perfused (10 ml MEM) Balb/c
mouse. The chicken received spleen homogenate boosters at 4 weeks
and 8 weeks after the initial immunization. Immune response to
mouse spleen by FACS analysis showed that the chicken immune serum
contained antibodies against a mouse cell-line (TRAMP-C1). The
chicken was sacrificed and its spleen was removed to TRI Reagent
(Molecular Research Center, Inc., Cincinatti, Ohio) 12 weeks after
the first immunization.
[0236] Total RNA was prepared from the chicken spleen using the
manufacturer's protocol for the TRI reagent. cDNA was prepared from
the total RNA using oligo(dT)-primers and Superscript enzyme (Life
Technologies). cDNAs encoding chicken spleen immunoglobulin
variable regions were amplified by CHybVH and ChybIgB (V heavy) or
by CSCVK and CHHybL-B (V.sub.kappa) primers according to standard
techniques. Light chain variable regions and constant regions were
PCR.TM. amplified together using CSC-F and lead-B primers and
V.sub.kappa and C.sub.kappa templates. Heavy chain variable regions
and constant regions were PCR.TM. amplified together using dp-seq
and lead-F primers and V.sub.heavy and C.sub.heavy templates.
Heavy- and light-chain fragments were PCR.TM. amplified together
with CSC-F and dp-Ex primers. PCR primers were purchased from
Genosys or GenBase, using primer sequences listed in the Cold
Spring Harbor laboratory course manual, "Phage Display of
Combinatorial Antibody Libraries" (Barbas et al., 2000), the text
of which is incorporated herein by reference.
[0237] After digestion with Sfi I, the amplification products were
ligated to SfiI-digested pComb3.times. for insertion into the phage
library. Ligated pComb3-123 plasmid was electroporated into ER2537
E.coli and phage production was started with subsequent VCM13
(helper phage) infection. The resulting library size was about
5.times.10.sup.6 cfu.
[0238] In Vivo Screening of .alpha.-Spleen Library Using BRASIL
[0239] Four rounds of in vivo screening in mice were performed
using the chicken .alpha.-spleen library. About 0.8 to
2.0.times.10.sup.10 TU were injected into a Balb/c mouse. The
library was allowed to circulate for 5 minutes. After sacrifice,
the mouse spleen was recovered and a single cell suspension was
prepared by pressing the spleen through a 70 .mu.m cell strainer
nylon mesh. The single cell suspension was centrifuged over oil
(9:1 dibutyl phtalate:cyclohexane) using the BRASIL technique and
200 .mu.l of log phase ER2537 E. coli were infected with the
pellet. Amplified phage recovered from the mouse spleen was used
for the subsequent round of screening. No obvious enrichment in the
screening rounds was seen in the number of phage homing to spleen
and brain compared with the conventional biopanning method, using a
piece of spleen obtained prior to BRASIL.
[0240] Phage localized in mouse spleen from the fourth round of
screening of the chicken Fab inserts were PCR.TM. amplified and the
PCR product was digested with Bst I. Half of the clones out of 90
analyzed produced a similar restriction pattern. Of those, 20
clones were sequenced from which only two had an identical
restriction pattern. Four of the antibody based phage clones
(numbers 2, 6, 10 and 12) were subjected to further analysis using
binding and localization assays.
[0241] Testing the Clones In Vitro Using BRASIL.
[0242] A singe cell suspension was prepared from two mouse spleens.
The suspension was divided into five tubes and. incubated on ice
with 3.times.10.sup.9 TU of Fab clones #2, #6, #10, #12 and
2.times.10.sup.9 TU tet-phage. Phage bound to mouse spleen cells
were recovered by BRASIL. 200 .mu.l of log phase ER2537 E.coli was
infected with the pellet and serial dilutions were plated on
LB/carbenicillin and LB/tetracyclin plates for assessment of phage
binding. Fd-tet was used as an internal control to normalize all
the phage homing experiments.
[0243] Testing Clones In Vivo With BRASIL
[0244] Phage (3.times.10.sup.9) of Fab clones #2, #6, #10, #12 and
2.times.10.sup.9 TU tet-phage were injected into the tail veins of
Balb/c mice and allowed to circulate for 5 minutes. The spleens
were recovered and single cell suspensions were prepared on ice
from whole spleens. Cell bound phage were recovered by BRASIL. 200
.mu.l of log phase ER2537 E.coli was infected with the pellet and
serial dilutions were plated on LB/carbenicillin and
LB/tetracycline plates for assessment of the phage recovery.
[0245] Testing Clone #10 Versus, Control Phage NPC-3TT In Vivo With
BRASIL
[0246] Phage (3.times.10.sup.9 TU) of Fab clone #10 and NPC-3TT
(control Fab phage) and 1.times.10.sup.9 TU of control Fd-tet-phage
were injected to mice (2 mice for NPC-3TT, 2 mice for clone #10)
and allowed to circulate for 5 minutes. Spleens were recovered and
single cell suspensions were prepared on ice. Cell-bound phage were
recovered by BRASIL. 200 .mu.l of log phase ER2537 E.coli was
infected with the pellet and serial dilutions were plated on
LB/carbenicillin and LB/tetracycline plates. The NPC-3TT phage is a
human anti-tetanus toxin Fab fragment displaying phage.
[0247] Homing of Fab Clone #10 to Spleen Versus Bone Marrow
[0248] Phage (3.times.10.sup.9 TU) of Fab clone #10 and NPC-3tt
control and 1.times.10.sup.9 TU of Fd-tet control phage were
injected into mice (2 mice for NPC-3TT, 2 mice for clone #10) and
allowed to circulate for 5 minutes. The spleens were recovered and
single cell suspensions were prepared. Bone marrow was recovered
from the same mice (both femurs) as a control for organ specific
homing. Cell-bound phage were recovered by BRASIL.
[0249] Fab-Fragment Production
[0250] The plasmid pComb3 containing the chicken Fab inserts was
electroporated into ER2537 bacteria. Serial dilutions were plated
onto LB/carbenicillin plates and incubated overnight at 37.degree.
C. Fab production culture (in super broth with 100 .mu.g/ml
carbenicillin) was started from a single plated colony. Fab
production was induced with 1 mM IPTG for 7 hours at 30.degree. C.
The Fab fragment was purified from the periplasmic fraction SN2 by
affinity purification after determination of the Fab concentration
in bacteria supernatant, periplasmic fractions SN1 and SN2 and in
the bacteria lysate by ELUSA. An .alpha.-Fab-Protein G-column was
coupled (2 mg/ml) with dimethylpimelimidate (DMP) using standard
protocols (Harlow and Lane, 1988).
[0251] For purifying Fab fragments the following method was used.
The SN2 fraction was loaded into a 1 ml HiTrap-protein
G-.alpha.-Fab-column (Amersham Pharmacia Biotech, Piscataway, N.J.)
either over 2 hours (if using lower than 50 ml volume with
superloop) or overnight (with more than 50 ml volume using a
peristaltic pump). The column was washed with 10-20 ml of PBS
(phosphate buffered saline). The Fab fragments were eluted with 10
ml of 20 mM glycine buffer, pH 2.2, 150 mM NaCl and 1 ml fractions
were collected. Fractions are neutralized with 1 M Tris immediately
after elution. Protein concentrations were quantified by
A.sub.280.
[0252] Intravascular Staining
[0253] To determine in vivo distribution of the recovered Fab
fragments, 50 to 60 .mu.g of Fab fragment (Fab#10, NPC3-tt or R#16)
was injected into the tail vein of a Balb/c mouse and allowed to
circulate for 8 minutes. 50 .mu.g of L.esculentum lectin-FITC was
injected into the mouse and the mouse tissues were fixed by
perfusion with 25 to 30 ml of 4% paraformaldehyde/PBS after 2
minutes of lectin circulation. Tissues were removed and post-fixed
in 4% paraformaldehyde for 1 hour. Fixed tissues were incubated in
30% sucrose/PBS overnight at 4.degree. C., changing the solution at
least twice. The tissues were embedded in the freezing media and
frozen on dry ice.
[0254] Fixed tissue sections were stained for Fab as follows.
Frozen tissue sections (55 .mu.m) were cut on a microtome and
washed 3.times. with PBS. The thin sections were blocked with
PBS/0.3% TritonX-100/5% goat serum for 1 hr at room temperature.
Sections were incubated overnight at room temperature with 1:400
Cy3 conjugated .alpha.-human anti-Fab antibody. The conjugated
sections were washed 6.times. with PBS/0.3% Triton X-100, 3.times.
with PBS, and fixed with 4% paraformaldehyde for 15 minutes. After
fixation the sections were washed again 2.times. with PBS and
2.times. with distilled water, then mounted on slides using
VectorShield.
[0255] Results
[0256] The in vitro localization to mouse spleen cells of phage
clones expressing chicken Fab fragments was examined by BRASIL. As
shown in FIG. 3, the Fab phage clones isolated by BRASIL showed
differential binding to mouse spleen cells compared to Fd-tet
insertless control phage. Clone #6 showed the lowest degree of
differential binding, similar to the control phage NPC-3TT, which
contained a Fab fragment but was not isolated from mouse spleen.
Clones #2, #10 and #12 all showed selective binding to mouse spleen
cells compared to the Fd-tet control, with at least a two-fold
increased binding observed for clones #2 and #10. The amino acid
sequences determined for the clone inserts were:
9 Clone #2: CQPAMAAVTLDESGGGLQTPGGALSLVCKASGFTFNSYPMGWVRQA- PGKGLE
(SEQ ID NO:89) WVAVISSSGTTWYAPAVKGRATISRDNGQSTVRLQLSNLRAED Clone
#6: CQPAMAAVTLDESGGGLQTPGGTLSLVCKASGISIGYGMNWV- RQAPGKGLEY (SEQ ID
NO:90) VASISGDGNFAHYGAPVKGRATISRDDGQNTVTLQLNNLR Clone #10:
CQPAMAAVTLDESGGGLQTPGGTLSLVCKGSGFIFSRYD- MAWVRQAPGKGLE (SEQ ID
NO:91) WVAGIDDGGGYTTLYAPAVKGRATITSRDNGQSTVRLQ- LNNLR Clone #12:
ANQPWPPLTLDESGGGLQTPGGALSLVCKASGFT- MSSYDMFWVRQAPGKGLE (SEQ ID
NO:92) FVAGISSSGSSTEYGAAVKGRATISRDNGQSTV- RLQLNNLRAED
[0257] A direct comparison was made of in vitro phage binding for
the Fab clones compared to NPC-3TT. As shown in FIG. 4, clones #2
and #10 exhibited the highest levels of binding to mouse spleen
cells in vitro. Clones #6 and #12 showed levels of binding to mouse
spleen that was only slightly higher than the binding of phage
NPC-3TT.
[0258] The preferential binding of the chicken Fab phage clones was
confirmed by in vivo studies using BRASIL. As shown in FIG. 5,
selective localization to mouse spleen was even more dramatic in
vivo, with Fab clones #2, #6 and #10 showing many-fold increased
binding to spleen compared to Fd-tet phage. In contrast, Fab clone
#12 did not exhibit significantly elevated binding to mouse spleen
compared to Fd-tet phage. These results show that in vitro results
obtained with spleen targeting phage are confirmed in vivo.
[0259] Fab clone #10 was selected for additional characterization
by in vivo localization to mouse spleen. The results, shown in FIG.
6, confirm that Fab clone #10 exhibted 3 to 10 fold enrichment in
spleen compared to Fd-tet. This effect was not due to general Fab
binding, since the Fab control phage NPC-3TT did not exhibit
selective localization in spleen compared to Fd-tet insertless
phage.
[0260] Binding of Fab clone #10 was organ specific, as demonstrated
in FIG. 7. Phage from Fab clone #10 and NPC-3TT control were
recovered from spleen and bone marrow tissue from the same injected
mice. It can be seen in FIG. 7 that Fab clone #10 exhibited
selective localization to spleen but not to bone marrow tissue. The
control phage did not exhibit selective localization to bone marrow
(FIG. 7) or spleen (not shown).
[0261] These results show that Fab phage clone #10 selectively
targets mouse spleen tissue for binding both in vitro and in vivo.
These results were further validated by vascular staining for in
vivo phage distribution. Control phage used for this study were
clones NPC-3TT (Fab fragment) and clone R#16 (isolated from
angiogenic retina screening).
[0262] Fab clone #10 was observed to bind to mouse spleen tissue in
vivo by fluorescent staining (not shown). The control phage NPC-3TT
and R#16 did not stain spleen tissue under identical conditions.
The clone #10 and NPC-3TT phage were observed to intensively stain
kidneys of injected animals, perhaps due to glomerular filtration
(not shown). Other control organs (lung, brain, liver, heart and
skeletal muscle) did not show staining with clone #10 (not
shown).
[0263] These results demonstrate that spleen targeting phage
peptides can be identified by the BRASIL method. They further show
the feasibility of the phage display technique using antibody
fragments against a target organ, tissue or cell type to obtain a
starting phage library. The ability to obtain targeting peptides
against spleen, a tissue that has proven refractory to biopanning
using standard phage display protocols because of the high
non-specific background, shows the advantages of the BRASIL
method.
Example 5
In Vivo Screening of .alpha.-Kaposi's Sarcoma Library in Angiogenic
Retinas
[0264] An angiogenic retinal system has been developed as a model
for angiogenic tumor tissues. Hypoxia in neonatal mice causes an
angiogenic response in the retina. The angiogenic retinal tissue
receptors show similarities with angiogenic tumor tissues in phage
display binding.
[0265] Materials and Methods
[0266] Angiogenic Model System
[0267] One-week-old C57BL/6J mice were exposed to a 75% oxygen
atmosphere for 5 days and then kept in room air for another five
days. The proliferative neovascular response was quantified by
counting the nuclei of new vessels extending from the retina into
the vitreous region in 6 .mu.m cross-sections. This model was used
to assess binding to newly formed angiogenic vessels of a phage
display library injected intravenously into mice. The peak of
neovascularization was observed between postnatal days 17 to
21.
[0268] Phage Display
[0269] A Fab phage library (.alpha.-KS) was produced against
Karposi's sarcoma tumor tissue that had been immunized into a
rabbit, using the same methods disclosed above for spleen. Three
rounds of in vivo screening were performed using the .alpha.-KS
library in the angiogenic retinal model system. About 3 to
10.times.10.sup.10 TU of .alpha.-KS phage were injected into 2
C57BL/6 mice with hypoxia-induced retinal neaovascularization on
postnatal days 18 to 20. The library was allowed to circulate for 5
minutes. Eyes were enucleated and retinas separated from the rest
of the eye. A single cell suspension was prepared from the retinas
by crushing them between two glass slides. Single cell suspensions
were processed by BRASIL as described above and 200 .mu.l of log
phase ER2537 E.coli was infected with the pellet. Phage that had
been amplified overnight were recovered from the retinal tissue and
used for subsequent rounds of screening. The recovery after each
round of selection was between 3400-5000 TU.
[0270] After three rounds of selection, 90 selected clones were
tested for their ability to bind to HUVEC cells. Microtiter wells
were coated with HUVECs in complete media. Cells were fixed and
incubated overnight with supernatant from IPTG-induced cultures
from phage infected bacteria. Fab production was detected by
.alpha.-Fab EIUSA. Fab binding to HUVECs was detected by
.alpha.-Fab-AFOS ELISA.
[0271] FIG. 8 shows the results of Fab clone binding to HUVECs
using an .alpha.-KS phage library. Clone #16 appeared to bind well
to HUVECs in vitro.
[0272] These results confirm the utility of using Fab antibody
fragments for production of phage display libraries. Such libraries
should be enriched in peptide sequences targeted against the
specific organ, tissue or cell type used to immunize the host
animal, compared to the random sequence phage display libraries
that have been used in previous biopanning methods. The results
also confirm the utility of the BRASIL method for identifying
targeting peptide sequences.
Example 6
Identification of Receptor/Ligand Pairs: Targeting Peptides Against
Integrin Receptors
[0273] Certain embodiments of the present invention concern the
identification of receptor/ligand pairs for various applications.
Targeting peptides selective for organs, tissues or cell types bind
to receptors (as defined above), normally located on the lumenal
surface of blood vessels within the target. In certain embodiments,
targeting peptides may be used to identify or characterize such
receptors, either directly or indirectly. In addition to their use
as targets for delivery of gene therapy vectors, other therapeutic
agents or imaging agents for in vivo imaging, such naturally
occuring receptors are of use as potential targets for development
of new therapeutic agents directed against the receptor itself, for
development of vaccines directed against the receptor, and for
understanding the molecular mechanisms underlying various disease
states. Naturally, the targeting peptides themselves may serve as
the basis for new therapeutic agents directed against the
receptors.
[0274] Targeting peptides may frequently act as mimeotopes of
endogenous ligands that bind to the targeted receptor. In other
embodiments, the endogenous ligands may be identified and
characterized using the disclosed methods. Such ligands are also of
potential use as targets for development of new therapeutic agents,
etc.
[0275] The present example illustrates one embodiment related to
identification of receptor/ligand pairs, in this case, integrin
receptors. Non-limiting examples of applications of targeting
peptides directed against integrins include regulation of cell
proliferation and chemotaxis, pro-apoptosis and anti-angiogenesis.
In this embodiment, purified integrins attached to a solid
substrate were used to screen phage display libraries to identify
targeting peptides directed against integrins.
[0276] Background
[0277] Integrin function is regulated by cytokines and other
soluble factors in a variety of biological systems. Most commonly,
exposure to such factors leads to conformational alterations that
result in changes in the activation state of the receptors (i.e.,
increased or decreased affinity for a given ligand and/or receptor
clustering in the plasma membrane). Changes in integrin-dependent
adhesion ultimately activate various complex signal transduction
pathways. At the molecular level, the induced co-localization of
cytoskeleton proteins with integrin cytoplasmic domains controls
signal transduction.
[0278] Cytoplasmic domains are key regulators of integrin function
(reviewed in Hynes, 1992; Ruoslahti, 1996). Individual ax and B
subunit cytoplasmic domains are highly conserved among different
species (Hemler et al., 1994). Although the cytoplasmic domains of
various .beta. subunits share similar primary structures, they
differ in certain functional characteristics. Experiments with
chimeric integrins have shown that the cytoplasmic domains of
.beta. chains are responsible for regulating receptor distribution
and recruitment to focal adhesion sites (Pasqualini and Hemler,
1994). Thus, certain cytoplasmic domains are critical for
integrin-mediated signaling into the cell (outside-in signaling)
and activation of integrin-ligand binding activity (inside-out
signaling) (Heraler et al., 1994).
[0279] The integrins .alpha.v.beta.3 and .alpha.v.beta.5 are
selectively expressed in angiogenic vasculature but not in normal
vasculature (Brooks et al., 1994a, 1994b; Pasqualini et al., 1997;
Arap et al., 1998). Moreover, .alpha.v integrin antagonists have
been shown to block the growth of neovessels (Brooks et al., 1994a,
1994b, 1995; Hammes et al., 1996). In these experiments,
endothelial cell apoptosis was identified as the mechanism for the
inhibition of angiogenesis (Brooks et al., 1994a, 1994b, 1995).
Angiogenesis initiated by bFGF can be inhibited by an
anti-.alpha.v.beta.3 blocking antibody, whereas VEGF-mediated
angiogenesis can be prevented by a blocking antibody against
.alpha.v.beta.5. The integrins .alpha.v.beta.3 and .alpha.v.beta.5
have been reported to be preferentially displayed in different
types of ocular neovascular disease (Friedlander et al., 1995,
1996). Thus, distinct cytokine-induced pathways that lead to
angiogenesis seem to depend on specific .alpha.v integrins.
[0280] Although both .alpha.v.beta.3 and .alpha.v.beta.5 integrins
bind to vitronectin, they probably mediate different post-ligand
binding events. For instance, in the absence of exogenous soluble
factors, the integrin .alpha.v.beta.5 fails to promote cell
adhesion, spreading, migration, and angiogenesis. On the other
hand, the .alpha.v.beta.3 integrin can induce such events without
additional stimulation by cytokines (Klemke et al., 1994; Lewis et
al., 1996; Friedlander et al., 1995).
[0281] Experiments designed to study the molecular basis for
cytokine regulation of .alpha.v.beta.5 function have shown that
upon binding to immobilized vitronectin, inactivated
.alpha.v.beta.5 is barely detectable in association with actin,
.alpha.-actinin, talin, tensin, p130.sup.cas, and vinculin. In
contrast, .alpha.v.beta.3 induces the localized accumulation of
such molecules. Upon activation of protein kinase C (PKC),
.alpha.v.beta.5 behaves similarly to .alpha.v.beta.3, but cannot
recruit talin (Lewis et al., 1996). Furthermore, calphostin C, an
inhibitor of PKC, seems to block angiogenesis mediated by
.alpha.v.beta.5 but not by .alpha.v.beta.3 (Friedlander et al.,
1995). These observations suggest that PKC activation probably
affects the conformation or phosphorylation state of the .beta.5
cytoplasmic domain. Similar changes may occur in cytoplasmic
proteins as well (Kolanus and Seed, 1997). The cytokine regulation
of .alpha.v.beta.5 integrin is unusual because ligand binding is
unchanged, but the events that follow ligand binding differ (Lewis
et al., 1996). Therefore, cellular events mediated by
.alpha.v.beta.3 or .alpha.v.beta.5 are clearly controlled by
different mechanisms (Filardo and Cheresh, 1994b).
[0282] The search for .alpha.v integrin-associated molecules has
been hampered by technical difficulties. First, the physical
associations involved are likely to rely on an assembly of
multimeric ligands that no longer occurs when cells are not intact.
Second, their association to integrins is usually of low affinity.
Finally, changes in the conformation and phosphorylation states of
the associating proteins may add a further level of complexity in
these transiently modulated interactions. Because of these
problems, only a limited number of proteins that bind to integrin
cytoplasmic domains have been identified. These proteins, such as
paxillin and ICAP-1, mainly associate with the .beta.1 chain
(Shattil and Ginsberg, 1997). Cytohesin-1 and filamin associate
with the cytoplasmic domain of .beta.2.
[0283] Several other proteins reportedly interact with .beta.
integrin cytoplasmic domains in general: talin, filamin,
.alpha.-actinin, focal adhesion kinase, the serine/threonine kinase
ILK, and skelemin. Talin, .alpha.-actinin, and focal adhesion
kinase no longer co-localize with .beta.1 integrins after deletion
of their putative binding sites in the .beta.1 cytoplasmic domain.
Similar approaches have shown that other cytoskeleton-associated
proteins and signaling molecules co-localize with integrins.
[0284] Integrins associate with molecules that are involved in
growth factor signaling. In addition to the 190-kDa protein and
IRS-1, which can be found in association with .alpha.v.beta.3
(Vuori and Ruoslahti, 1994), analysis of the association of the
.alpha.v.beta.3 integrin with molecules related to the insulin and
PDGF signaling pathways revealed that both the insulin and
PDGF.beta. receptors co-immunoprecipitate with .alpha.v.beta.3. The
receptor molecules associated with the integrin represent a highly
phosphorylated and highly activated subfraction of such molecules.
These results are important because they reinforce the notion that
integrin-mediated cell attachment coordinates cellular responses to
growth factors. Integrin-dependent signaling processes synergize
with proliferation signals (Frisch and Ruoslahti, 1997; Clark and
Brugge, 1995; Longhurst and Jennings, 1998).
[0285] Protein phosphorylation is one of the earliest events
detected in response to integrin stimulation. The ability of
tyrosine kinase inhibitors to obstruct the formation of focal
adhesions suggests a role for tyrosine phosphorylation in the
signaling pathways linked to integrin receptors (Defilippi et al.,
1994). Serine/threonine kinase families, such as protein kinase C
(PKC) and mitogen-activated protein (MAP) kinase, are also
activated upon integrin stimulation, and inhibitors of PKC block
cell attachment and spreading in certain cell systems (Vuori and
Ruoslahti, 1993). Integrins seem to affect cell survival by
regulating programmed cell death, a response that also depends on
tyrosine phosphorylation. Several proteins that associate with
integrin protein complexes contain modular domains, termed Src
homology 2 (SH2) and 3 (SH3), that specifically mediate
protein-protein coupling. SH2 domains bind to proteins through
interactions with specific peptide motifs containing
phosphotyrosine, whereas SH3 domains bind to short proline-rich
peptide motifs on their protein targets (Clark and Brugge, 1995).
Integrin-mediated cell adhesion causes activation of MAP kinases
and increased tyrosine phosphorylation of focal adhesion kinase
(FAK). Autophosphorylation of FAK leads to the binding of
SH2-domain proteins including Src-family kinases and the Grb-2-Sos
complex. One plausible hypothesis is that integrin-mediated
tyrosine phosphorylation of FAK leads to activation of the Ras
cascade and ultimately to MAP kinase activation. However,
integrin-mediated MAP kinase activation has been shown to be
independent of FAK, indicating that at least two distinct integrin
signaling pathways might exist: (i) MAP kinase activation, which
may play a role in mitogenic and survival signals, and (ii) FAK
tyrosine phosphorylation, which is clearly involved in cytoskeletal
organization (Lin et al., 1997).
[0286] Previous studies of peptidic substrates and homology-based
molecular models suggested that about 9-13 residues of a peptidic
substrate contact the active-site cleft of the kinase domain
(Bossemeyer et al., 1993). The phage display technique offers an
alternative approach to generating and selecting diverse
combinatorial-peptide libraries (Smith, 1991; Wells and Lowman,
1992). Because the chemical diversity in a phage display library is
encoded by DNA that can be replicated and amplified, selection of a
phage library can be performed over multiple rounds, allowing even
rare motifs to be identified. In contrast to synthetic chemical
libraries, phage display permits the analysis of single species
instead of pooled species. The power of this technique has been
demonstrated mainly through the selection of rare antibodies or
peptides from large combinatorial libraries.
[0287] A combinatorial phage library has been used for determining
the preferred substrate sequences for different protein-tyrosine
kinases (PTKs), all of them closely related members of the Src
family and one more distantly related PTK, Syk (Schmitz et al.,
1996). Subsequent phosphorylation by recombinant PTKs and selection
of phosphorylated phage by an anti-phosphotyrosine antibody were
used to enrich for phage that displayed substrate peptides. After
several rounds of selection, distinct substrate sequences were
found for each of the PTKs tested. For the PTKs related to the Src
family, critical features of these canonical sequences were
recapitulated in known or presumed protein substrates. Most
notably, amino acids directly flanking the invariant tyrosine
residue were found to be highly conserved and specific for each of
the PTKs tested. The identified motifs could, therefore, provide a
rational basis for developing small and specific inhibitors of the
catalytic domain of PTKs (Schmitz et al., 1996).
[0288] Further studies extended the scope of phage display
technology by showing how peptide libraries can be used to
investigate the substrate specificity of Fyn, a protein kinase of
the Src family (Dente et al., 1997; Gram et al., 1997). Modified
peptides displayed by phage were used to determine the
phosphotyrosine specificity of the phosphotyrosine-binding domain
(PTB) of the protein Shc (Dente et al., 1997). Other related
experiments focused on identifying phosphopeptide ligands that
interact with the Src homology 2 (SH2) domain of the adapter
protein Grb2 by screening a random peptide library established on
phage. Phage were phosphorylated in vitro at an invariant tyrosine
residue by a mixture of the phosphotyrosine kinases c-Src, Blk, and
Syk. Binding motifs were selected by interaction of the library
with the recombinant SH2 domain of Grb2 expressed as a
glutathione-S-transferase (GST) fusion protein. Several subsequent
cycles of selection led to the enrichment of phage that bound to
the GST-Grb2 SH2 domain only when previously phosphorylated.
Sequence analysis revealed that all of the selected phage displayed
peptides with the consensus motif Y*MINW (Y* denotes
phosphotyrosine). One peptide bound the Grb2 SH2 domain with 3-fold
higher affinity than the peptide motif Y*VNV, which is derived from
the natural ligand Shc. These findings show that phage display can
be used to rapidly identify high-affinity ligands to SH2 domains
and other interacting proteins involved in signal transduction.
[0289] The cytoplasmic domain of .beta.5 is structurally and
functionally unique with regard to other integrin subunits (Table
8) and shares only 38% homology to the cytoplasmic domain of
.beta.3 (Hemler et al., 1994). It has been proposed that the
structural requirements for association with .alpha.v prevented
further primary sequence divergence between 83 and 135; yet the
existing differences are likely to account for the reduced
interaction of .alpha.v.beta.5 with talin (Lewis et al., 1996). The
cytoplasmic domain of .beta.5, when expressed in Chinese hamster
ovary (CHO) cells as a chimera with the extracellular domain of
.beta.1, led the chimeric receptor to behave like .beta.5,
promoting cell migration and loss of receptor localization to focal
adhesions (Pasqualini and Hemler, 1994). The cytoplasmic domains of
integrin .beta.1 and .beta.3 subunits, however, were shown to be
functionally interchangeable (Solowska et al., 1991). Other studies
have shown that .alpha.v.beta.3 and .alpha.v.beta.5 seem to differ
in terms of localization to focal adhesion and their contribution
to cell migration (Delannet et al., 1994; Filardo et al., 1995).
However, these reported functional divergences have not been mapped
to specific domains.
10TABLE 8 Alignment of similar integrin .beta. subunit cytoplasmic
domains. The main differences between the .beta.3 and .beta.5
cytoplasmic are highlighted. .beta.1 H D R R E F A K F E K E K M N
A K W D T G E N P I Y K S A V T T V V N P K Y E G K .beta.2 S D L R
E Y R R F E K E K L K S Q W N N-D N P L F K S A T T T V M N P K F A
E S .beta.3 H D R K E F A K F E E E R A R A K W D T A N N P L Y K E
A T S T F T N I T Y R G .beta.5 H D R R E F A K F Q S E R S R A R Y
E M A S N P L Y R K P I S T H T V D F T F N K S Y N G T V D
[0290] After the angiogenesis switch is triggered, distinct
molecules are likely to associate with either .beta.3 or .beta.5.
Moreover, selective associations with the .alpha.v cytoplasmic
domain may also be possible, in the context of each of the
heterodimers. For example, a .beta. turn in the cytoplasmic tail of
the integrin .alpha.v subunit has been shown to influence
conformation and ligand binding of .alpha.v.beta.3 (Filardo and
Cheresh, 1994). The basis of selective signaling properties may be
the assembly of specific molecules that associate with the
respective cytoplasmic domains. The present study defines the
molecules involved in .alpha.v.beta.3- and
.alpha.v.beta.5-selective angiogenic signaling by exploring a novel
strategy, panning of phage display peptide libraries on .beta.3 and
.beta.5 cytoplasmic domains and determining the biological
properties of the cytoplasmic domain-binding peptides.
[0291] The disclosed methods have several advantages over previous
approaches: (i) the ability to characterize the intracellular
molecules that directly or indirectly interact with integrin
cytoplasmic domains; (ii) the development of antibodies against
molecules that bind to integrin cytoplasmic domains in very low
amounts; and (iii) the phage display library screenings will lead
to the identification of peptides that mimic cytoplasmic-domain
binding proteins.
[0292] Methods
[0293] Two Dimensional Cell Culture
[0294] Three human endothelial cell lines that express .beta.3 and
.beta.5 integrins were used: KS 1767 cells (Herndier et al., 1996),
HUVECs (ATCC), and BCE cells (Solowska et al., 1991). Sterile glass
coverslips covered with different proteins (i.e. vitronectin,
fibronectin, collagen, or laminin) were used as substrates. After
cells attached and spread, the monolayers were rendered quiescent
by a 12-hour incubation in medium containing 0.05% fetal calf
serum. Peptides were introduced into the cells using the penetratin
membrane-permeable tag (see below). The cells were plated onto ECM
proteins for adhesion and spreading. The monolayer was stimulated
for 6 hours with each of the growth factors involved in
.alpha.v-mediated angiogenesis, including bFGF, TNF.alpha., VEGF,
and TGF.beta.. Untreated cells were the negative controls.
[0295] Three-Dimensional Cell Culture:
[0296] 150 .mu.l of Matrigel were added per well of 24-well tissue
culture plates and allowed to gel at 37.degree. C. for 10 min.
HUVECs starved for 24 h in M199 medium supplemented with 2% FCS
before being trypsinized were used. 10.sup.4 cells were gently
added to each of the triplicate wells and allowed to adhere to the
gel coating for 30 min at 37.degree. C. Then, medium was replaced
with peptides in complete medium. The plates were monitored and
photographed after 24 h with an inverted microscope (Canon).
[0297] Chemotaxis Assay:
[0298] Cell migration assays were performed as follows: 48-well
microchemotaxis chambers were used. Polyvinylpyrrolidone-free
polycarbonate filters (Nucleopore, Cambridge, Mass.) with 8-.mu.m
pores were coated with 1% gelatin for 10 min at room temperature
and equilibrated in M199 medium supplemented with 2% FCS. Peptides
were placed in the lower compartment of a Boyden chamber in M199
supplemented with 2% FCS, 20 ng/ml VEGF-A (R&D System), and 1
U/ml heparin. Overnight-starved subconfluent cultures were quickly
trypsinized, and resuspended in M199 containing 2% FCS at a final
concentration of 2.times.10.sup.6 cells/ml. After the filter was
placed between lower and upper chambers, 50 .mu.l of the cell
suspension was seeded in the upper compartment. Cells were allowed
to migrate for 5 h at 37.degree. C. in a humidified atmosphere with
5% CO.sub.2. The filter was then removed, and cells on the upper
side were scraped with a rubber policeman. Migrated cells were
fixed in methanol and stained with Giemsa solution (Diff-Quick,
Baxter Diagnostics, Rome, Italy). Five random high-power fields
(magnitude 40.times.) were counted in each well.
[0299] Proliferation Assay:
[0300] Cell proliferation was measured as described (Pasqualini and
Hemler, 1994). Briefly, 4.times.10.sup.4 HUVECs were incubated in
24-wells plates. The cells were starved for 24 h, and then the
medium was removed and replaced in the presence of VEGF and 15
.mu.M of each peptide and incubated for 18 h. Then, 50 .mu.l of
media containing [.sup.3H]thymidine (1 .mu.Ci/ml) was added to the
wells, and after 6 additional hours of incubation at 37.degree. C.,
the medium was removed and the cells were fixed in 10% TCA for 30
min at 4.degree. C., washed with ethanol, and solubilized in 0.5 N
NaOH. Radioactivity was counted by liquid scintillation with an LS
6000SC Beckman scintillation counter. Each experiment was performed
three times with triplicates, and the results are expressed as the
mean SD.
[0301] Apoptosis Assay (Propidium Iodide Staining Subdiploid
Population)
[0302] Approximately 1.times.10.sup.6 cells were harvested in
complete media and 15 .mu.M of peptide added for 4, 8, or 12 h. The
cells were then washed in PBS and resuspended in 0.5 ml propidium
iodide solution (50 .mu.g/ml PI, 0.1% Triton X-100, 0.1% sodium
citrate). After a 24-h incubation at 4.degree. C., cells were
counted with a XL Coulter (Coulter Corporation) with a 488-nm
laser; 12,000 cells were counted for each histogram, and cell cycle
distributions were analyzed with Multicycle program.
[0303] After microinjection or penetratin-mediated internalization
of the peptides and appropriate controls, cell apoptosis was
monitored using the ApopTag kit. Experiments were performed in the
presence of caspase inhibitors and antibodies against specific
caspases.
[0304] Cytokine- and Tumor-Induced Angiogenesis Assays
[0305] Angiogenic factors and tumor cells implanted into CAM
stimulate growth of new capillaries. Angiogenesis was induced in
CAMs from 10-day chicken embryos by VEGF or bFGF filters implanted
in regions that were previously avascular. Different treatments
(penetratin peptides and controls) were applied topically, and
after 3 days, the filters and surrounding CAMs were resected and
fixed in formalin. The number of blood vessels entering the disk
was quantified within the focal plane of the CAM with a
stereomicroscope. The mean number of vessels and standard errors
from 8 CAMs in each group were compared.
[0306] Phosphorylation and Panning of Phosphorylated Phage
Libraries:
[0307] Phosphorylation of peptide libraries with src family protein
kinases (Fyn, c-Src, Lyn, and Syc) and serine/threonine kinases
such as a MAP kinase were performed as described previously
(Schmitz et al., 1996; Dente et al., 1997; Gram et al., 1997).
Briefly, phage particles were collected from culture supernatants
by double precipitation with 20% polyethylene glycol 8000 in 2.5 M
NaCl. Particles were dissolved at 10.sup.12 particles/ml. Purified
phage (10 .mu.l) were incubated for 3 hours at room temperature
with different concentrations (35 to 3,500 units) of protein
kinases in a reaction buffer volume of 50 .mu.l. The reaction
mixtures were transferred to tubes containing 10 .mu.g of
agarose-conjugated anti-P-Tyr, anti-P-Ser, or anti-P-Thr monoclonal
antibodies to select phage displaying phosphorylated peptides.
Bound phage were eluted by washing the column with 0.3 ml of
elution buffer (0.1 M NaCl/glycine/1 mg/ml BSA, pH 2.35). The
eluates were neutralized with 2 M Tris-base and incubated with 2 ml
of a mid-log bacteria culture. Aliquots of 20 g were removed for
plating, and phage were harvested as described. The
phosphorylation-selection step was repeated. Phosphorylated
peptides binding to .beta.3 and .beta.5 cytoplasmic domains were
analyzed as described in the previous section.
[0308] Matrix-assisted laser desorption time-of-flight (MALDI-TOF)
mass spectrometry was used to map in vitro phosphorylation sites on
the .beta.3 and .beta.5 cytoplasmic domains and cytoplasmic
domain-binding peptides. The fusion proteins or peptides were
phosphorylated in vitro as described and purified by RP-BPLC or RP
microtip columns. Phosphorylated peptides were identified by three
methods: (1) 80-Da mass shifts after kinase reactions; (2) loss of
80 Da after phosphatase treatment; or (3) loss of 80 Da or 98 Da in
reflector vs. linear mode for tyrosine phosphorylated or serine,
threonine phosphorylated peptides, respectively. Where needed,
peptides were purified by RP-HPLC and subjected to carboxypeptidase
and aminopeptidase digestions to produce sequence ladders. This was
particularly useful where one peptide may harbor two or more
phosphorylation sites.
[0309] Panning on Phosphorylated GST-Fusion Proteins.
[0310] GST fusion proteins were phosphorylated in vitro as
described (Schmitz et al., 1996; Dente et al., 1997; Gram et al.,
1997). Briefly, 10 /g/ml was incubated for 3 h at room temperature
with 5.5 units of Fyn protein kinase in reaction buffer (50 mM
Tris, 5mM MgCl.sub.2, 500 .mu.M Na.sub.3VO.sub.4, 500 .mu.M ATP in
a total volume of 50 .mu.l). The reaction was stopped by adding 40%
of TCA. After the kinase substrate protein was precipitated, it was
resuspended in PBS and coated on microtiter wells at 10 .mu.g/well.
An aliquot of CX7C library (2.5.times.10.sup.11 transducing units
was incubated on the GST fusion proteins. Phage were sequenced from
randomly selected clones.
[0311] Mass Spectrometry Studies
[0312] Mass spectrometric peptide mass mapping was used to identify
novel ligands for .beta.3 and/or .beta.5 cytoplasmic domains.
Polyclonal and monoclonal antibodies raised against the cytoplasmic
domain-binding peptides were used to purify target proteins
(cytoskeletal or signaling molecules). These proteins were resolved
by SDS-PAGE, cut out from the SDS gels, and digested in-gel with
trypsin. After extraction of the peptides, MALDI-TOF mass
spectrometry analysis was performed to produce a list of peptide
masses. This list of peptide masses, in combination with protease
specificity, produces a relatively specific "signature" that can be
used to search sequence databases. If the protein sequence is
present in a database, the protein can be identified with high
confidence by this method. The lower detection limit for this
approach is currently 1 pmol, at least 10-20-fold better than
N-terminal Edman sequencing methods.
[0313] Results
[0314] Panning of Phage Peptide Libraries on .beta.3 or .beta.5
Cytoplasmic Domains.
[0315] .beta.3 and .beta.5 cytoplasmic domain-binding peptides were
isolated by screening multiple phage libraries with recombinant GST
fusion proteins that contained either GST-.beta.3cyto or
GST-.beta.5cyto coated onto microtiter wells. Immobilized GST was
used as a negative control for enrichment during the panning of
each cytoplasmic domain. Phage were sequenced from randomly
selected clones after three rounds of panning as disclosed
elsewhere (Koivunen et al., 1995; Pasqualini et al., 1995).
Distinct sequences were isolated that interacted specifically with
the .beta.3 or with the .beta.5 cytoplasmic domains (Table 9).
Randomly selected clones from panning rounds II and III were
sequenced. Amino acid sequences of the phagemid encoded peptides
were deduced from nucleotide sequences. The most frequent motifs
found after panning with the indicated libraries are shown in Table
9. The ratios were calculated by dividing the number of colonies
recovered from .beta.3-GST-coated wells and those recovered from
GST or BSA.
11TABLE 9 Sequences displayed by phage binding to .beta.3 or
.beta.5 integrin cytoplasmic domain .beta.3/GST .beta.3/BSA Peptide
motif SEQ ID NO Ratio Ratio CX.sub.9 Library CEQRQTQEGC SEQ ID
NO:93 4.3 14 CARLEVLLPC SEQ ID NO:94 2.8 18.7 X.sub.4YX.sub.4
Library YDWWYPWSW SEQ ID NO:95 5.6 163 GLDTYRGSP SEQ ID NO:96 4.1
48 SDNRYIGSW SEQ ID NO:97 3.3 32 YEWWYWSWA SEQ ID NO:98 2.2 28.1
KVSWYLDNG SEQ ID NO:99 2.1 20 SDWYYPWSW SEQ ID NO:100 2.1 157
AGWLYMSWK SEQ ID NO:101 1.8 2.4 Pool Cyclic Libraries CFQNRC SEQ ID
NO:102 3.1 16 CNLSSEQC SEQ ID NO:103 2.7 62 CLRQSYSYNC SEQ ID
NO:104 2.4 3.2 .beta.5/GST .beta.5/BSA Peptide motif SEQ ID NO
Ratio Ratio Pool Cyclic Libraries CYIWPDSGLC SEQ ID NO:105 5.2 193
CEPYWDGWFC SEQ ID NO:106 3.1 400 CKEDGWLMTC SEQ ID NO:107 2.3 836
CKLWQEDGY SEQ ID NO:108 1.8 665 CWDQNYLDDC SEQ ID NO:109 1.5 100
X.sub.4YX.sub.4 Library DEEGYYMMR SEQ ID NO:110 11.5 29 KQFSYRYLL
SEQ ID NO:111 4.5 8 VVISYSMPD SEQ ID NO:112 3.8 28 SDWYYPWSW SEQ ID
NO:113 2.4 304 DWFSYYEL SEQ ID NO:114 1.7 153
[0316] The specificity of the interaction with .beta.3 or .beta.5
cytoplasmic domains was determined by calculating the ratios
between the number of phage bound to the cytoplasmic. domain
containing-fusion proteins (.beta.3 or .beta.5) versus GST alone
(negative control). FIG. 9 shows the results from binding assays
performed with the GST-.beta.3cyto binding phage. Six phage were
tested that displayed the motifs most frequently found during the
second and third rounds of panning. Each panel shows the results
from binding assays for the phage displaying different peptides
that bind to the .beta.3 cytoplasmic domain, as indicated.
Insertless phage or unselected libraries were used as negative
controls and did not show binding above background. Two plating
dilutions were shown for each assay.
[0317] A similar strategy was used to determine the specificity of
the phage isolated in the screenings involving the .beta.5
cytoplasmic domain fusion protein. The binding assays were
performed with individually amplified phage, shown in FIG. 10. Five
phage were tested that displayed the motifs found most frequently
during the second and third rounds of panning. Each panel shows the
binding assays for the phage displaying peptides that bind to the
.beta.5 cytoplasmic domain. Insertless phage or unselected
libraries were used as negative controls and did not show binding
above background in these assays.
[0318] To determine whether the binding of the selected motifs was
specific for each cytoplasmic domain, binding assays were performed
comparing the interaction of individual phage motifs with .beta.1,
.beta.3, or .beta.5 cytoplasmic domain fusion proteins. ELISA with
anti-GST antibodies showed that the three proteins can be coated
onto plastic at equivalent efficiency, and thus the differences in
binding do not reflect differences in coating concentrations (not
shown). Both the .beta.3- and .beta.5-selected phage selectively
interacted with the proteins on which they were originally
selected, with average binding selectivities observed of
.beta.3/.beta.1=3.9, .beta.3/.beta.5=3.7, .beta.5/.beta.1=4.8, and
.beta.5/.beta.3=6.9 (not shown). The average selectivity for
integrin cytoplasmic domains versus BSA was about one to two orders
of magnitude (not shown). None of the phage tested seemed to bind
strongly to the .beta.1 cytoplasmic domain (not shown).
[0319] Characterization of Synthetic Peptides Corresponding to the
Sequences Displayed By the Integrin-Cytoplasmic Domain-Binding
Phage.
[0320] Specific-phage were selected for further studies on the
basis of their binding properties. Synthetic peptides corresponding
to the sequence displayed by each phage were used to perform
binding inhibition studies. This assay determined whether phage
binding was entirely mediated by the targeting peptide displayed by
the phage or whether it also included a non-specific component. As
expected, the synthetic peptides inhibited the binding of the
corresponding phage in a dose-dependent manner (FIG. 11 and FIG.
12). A control peptide containing unrelated amino acids had no
effect on phage binding when tested at identical
concentrations.
[0321] Phosphorylation Events Modulate the Interaction of the
Selected Peptides With Cytoplasmic Domains
[0322] Events involving phosphorylation are important in regulating
signal transduction. The phage display system was used to evaluate
the effect of tyrosine phosphorylation at two levels. First,
recombinant fusion proteins containing .beta.3 or .beta.5
cytoplasmic domains were used for panning of phage libraries
displaying tyrosine-containing peptides. Second, the cytoplasmic
domains themselves were phosphorylated before phage selection was
performed. Experiments were performed to investigate the capacity
of specific tyrosine kinases to modulate the interaction of the
selected peptides with the cytoplasmic domains. The results
obtained in the panning of phage libraries displaying
tyrosine-containing peptides are shown in Table 10.
[0323] Randomly selected clones from rounds III and IV were
sequenced from a X4YX4 phosphorylated library with Fyn. Amino acid
sequences of the phagemid encoded peptides were deduced from
nucleotide sequences. Table 10 shows the motifs found most
frequently after the indicated libraries were panned with .beta.3
or .beta.5. The ratio of binding to .beta.3 or .beta.5 was
calculated by dividing the number of .beta.3 or .beta.5 colonies by
GST or BSA colonies found after panning. The ratio of binding to
.beta.3 or .beta.5 with phosphorylated phage by Fyn versus
unphosphorylated phage was calculated by dividing the number of
colonies found after the panning.
12TABLE 10 Sequences displayed by phosphorylated phage binding to
integrin cytoplasmic domains. Peptide Motif Phos/Unphos .beta.3 or
.beta.5/GST .beta.3 or .beta.5/BSA .beta.3 cytoplasmic GGGSYRHVE
SEQ ID NO:115 13.2 1.5 5.3 RAILYRLAN SEQ ID NO:116 2.8 1.3 20
MLLGYRFEK SEQ ID NO:117 2.5 3.5 2.7 .beta.5 cytoplasmic TMLRYTVRL
SEQ ID NO:118 14.3 3.4 2.2 TMLRYFMFP SEQ ID NO:119 4.2 2.3 3.8
TLRKYFHSS SEQ ID NO:120 3.8 3.8 15.2 TLRKYFHSS SEQ ID NO:121 1.8
5.6 7.3
[0324] The effect of phosphorylation on the affinity and
specificity of the cytoplasmic domain-binding was examined. Phage
displaying the .beta.3 and .beta.5 cytoplasmic domain-binding
peptides were phosphorylated in vitro as previously described
(Schmitz et al., 1996; Dente et al., 1997; Gram et al., 1997),
using Fyn kinase. Specific phosphorylation of the
tyrosine-containing peptide on the surface of the phage was
confirmed by using .sup.32P-gamma dATP in the kinase reaction and
by separating the phage pIII protein by SDS-PAGE.
[0325] Phage phosphorylated in vitro showed increased binding
affinity and specificity to the .beta.3integrin cytoplasmic domain
(FIG. 13). The TLRKYFHSS (SEQ ID NO: 120) phage was also tested in
assays that included other GST-cytoplasmic domain fusion proteins
to determine specificity (FIG. 14).
[0326] Sequence Similarity of Integrin Binding Peptides With Known
Cytoskeletal and Signaling Proteins.
[0327] The peptides displayed by integrin cytoplasmic
domain-binding phage were similar to certain regions found within
cytoskeletal proteins and proteins involved in signal transduction
(Table 11). The similarity of some of the isolated peptides to a
region of mitogen-activated protein kinase 5 (MAPK5, amino acids
227-234) was particularly interesting. A connection involving the
MAPK cascade, cell adhesion, migration and proliferation has been
proposed (Lin et al., 1997)
13TABLE 11 Sequence similarity of integrin binding peptides with
known cytoskeletal and signaling proteins. Region (AA Homology
Isolated Motif Candidate Proteins #) % .beta.3 cytoplasmic
GLDTYRGSP (SEQ ID NO:96) Ras-related protein 124-133 75 SDNRYIGSW
(SEQ ID NO:97) Ser/Thr kinase (K-11) 18-25 75 CEQRQTQEGC (SEQ ID
NO:93) PDGF receptor 985-992 85 CLRQSYSYNC (SEQ ID NO:104)
Phosphatidylinositol 4 phosphatase 233-241 85 5 Receptor protein
kinase 185-191 85 Protein kinase clk2 71-79 63 MAPK5 227-234 75
Phophatidylinositol 3-kinase 494-503 78 Cyclin-dependent kinase 5
(cdk5) 230-239 75 .beta.5 cytoplasmic VVISYSMPD (SEQ ID NO:112)
Ser/Thr kinase 479-485 83 DEEGYYMMR (SEQ ID NO:110) IFN (.beta.
chain) 27-35 70 Actin 240-248 67 Focal adhesion kinase 43-51 75
Tubulin 60-66 100 Putative Ser/Thr kinase 292-299 86
[0328] Membrane-Permeable Peptides
[0329] Penetratin is a peptide that can translocate hydrophilic
compounds across the plasma membrane. Fusion to the penetrating
moiety allows oligopeptides to be targeted directly to the
cytoplasm, nucleus, or both without apparent degradation (Derossi
et al., 1994). This membrane-permeable peptide consists of 16
residues (RQIKIWFQNRRMKWKK, SEQ ID NO:122) corresponding to amino
acids 43-58 of the homeodomain of Antennapedia, a Drosophila
transcription factor (Joliet et al., 1991a, 1991b; Le Roux et al.,
1993). Internalization mediated by penetratin occurs at both
37.degree. C. and 4.degree. C., and the internalized peptide can be
retrieved intact from cells.
[0330] Peptides were designed containing penetratin sequences fused
to the sequences of motifs found to bind .beta.3 or .beta.5
cytoplasmic domains. The peptides were synthesized on a 431 Applied
Biosystems peptide synthesizer using p-hydroxymethylphenoxy methyl
polystyrene (HMP) resin and standard Fmoc chemistry. Peptide
internalization and visualization was performed as described
(Derossi et al., 1994; Hall et al., 1996; Theodore et al.,
1995).
[0331] Briefly, 10-50 .mu.g/ml of the biotinylated peptide was
added to cells in culture. Peptides were incubated with plated
cells. After 24 hours, the cultures were washed three times with
tissue culture media, fixed and permeabilized using ethanol:acetic
acid (9:1) for 5 min at -20.degree. C. Nonspecific protein binding
sites were blocked by incubating the cultures for 30 min with
Tris-buffered saline (TBS) containing 10% fetal calf serum (FCS)
and 0.02% Tween. The cultures were incubated in the same buffer
containing FITC-conjugated Streptavidin (1:200 dilution) and washed
with TBS before being mounted for viewing by confocal microscopy.
The penetratin-linked peptides were internalized quite efficiently
(data not shown).
[0332] Functional data showed that the cytoplasmic domain-binding
peptides selected on .beta.3 or .beta.5 can interfere with
integrin-mediated signaling and subsequent cellular responses
(i.e., endothelial cell adhesion, spreading, proliferation,
migration). A commercial panel of "internalizable" versions of the
synthetic motifs found by phage screenings (SDNRYIGSW, SEQ ID
NO:97; and CEQRQTQEGC, SEQ ID NO:93; .beta.3 binding peptides and
VVISYSMPD, SEQ ID NO:112; a .beta.5-binding peptide) were obtained.
These complex chimeric peptides consist of the most selective of
the .beta.3 or .beta.5-cytoplasmic domain-binding peptides coupled
to penetratin, plus a biotin moiety to allow the peptides to be
tracked once they were internalized into intact cells. These
membrane-permeable forms of the peptides are internalized, may
affect .beta.3 and .beta.5 post-ligand binding cellular events and
can induce massive apoptosis (data not shown).
[0333] Endothelial Cell Proliferation, Chemotaxis and Apoptosis
[0334] The effect of .beta.3 and .beta.5 integrin cytoplasmic
domain-binding motifs on endothelial cell proliferation was
evaluated after stimulation with factors that activate endothelial
cells (FIG. 15). Cell proliferation was measured according to
Pasqualini and Hemler (1994). Briefly, 4.times.10.sup.4 HUVECs were
incubated in 24-well plates and starved for 24 h, after which the
medium was removed and replaced in the presence of VEGF and 15
.mu.M of each peptide. After another 18 h of incubation, 50 .mu.l
of medium containing [.sup.3H]thymidine (1 .mu.Ci/ml) was added to
the wells. After 6 additional hours of incubation at 37.degree. C.,
the medium was removed and the cells were fixed in 10% TCA for 30
min at 4.degree. C., washed with ethanol and solubilized in 0.5 N
NaOH. Radioactivity was counted by liquid scintillation by using a
LS 6000SC Beckman scintillation counter. Each experiment was
performed three times with triplicates, and the results were
expressed as the mean .+-.SD.
[0335] The effect of .beta.3 and .beta.5 integrin cytoplasmic
domain-binding motifs in endothelial cell migration was evaluated
after stimulation with factors that activate endothelial cells. The
peptides tested affected cell function in a dose-dependent and
specific way. Their properties seem to be intrinsic to the .beta.3
or to the .beta.5 cytoplasmic domain (FIG. 16).
[0336] Chemotaxis Assay.
[0337] Cell migration was assayed in a 48-well microchemotaxis
chamber. Polyvinylpyrrolidone-free polycarbonate filters with
8-.mu.m pores were coated with 1% gelatin for 10 min at room
temperature and equilibrated in M199 medium supplemented with 2%
FCS. Peptides were placed in the lower compartment of a Boyden
chamber in M199 supplemented with 2% FCS, 20 ng/ml VEGF-A (R&D
System), and 1 U/ml heparin. Overnight-starved subconfluent
cultures were quickly trypsinized, and resuspended in M199
containing 2% FCS at a final concentration of 2.times.10.sup.6
cells/ml. After the filter was placed between lower and upper
chambers, 50 .mu.l of the cell suspension was seeded in the upper
compartment. Cells were allowed to migrate for 5 h at 37.degree. C.
in a humidified atmosphere with 5% CO.sub.2. The filter was then
removed, and cells on the upper side were scraped with a rubber
policeman. Migrated cells were fixed in methanol and stained with
Giemsa solution. Five random high-power fields (magnitude
40.times.) were counted in each well. The results show that both
.beta.3-integrin cytoplasmic domain binding peptides increased cell
migration but penetratin did not affect the cells.
[0338] Apoptosis Assay (Propidium Iodide (PI) Staining Subdiploid
Population).
[0339] Approximately 1.times.10.sup.6 cells were harvested in
complete medium, and 15 .mu.M of peptide was added for 4, 8, or 12
hours. The cells were then washed in PBS and resuspended in 0.5 ml
propidium iodide solution (50 .mu.g/ml PI, 0.1% Triton X-100, 0.1%
sodium citrate). After a 24-h incubation at 4.degree. C., the cells
were counted with an XL Coulter (Coulter Corporation) with a 488 nm
laser; 12,000 cells were counted for each histogram, and cell cycle
distributions were analyzed with the Multicycle program.
[0340] Treatment of cells with VISY-penetratin chimera resulted in
induction of apoptosis (FIG. 17, panel d) Pro-apoptotic effects
were not observed when the cells were exposed to other growth
factors (not shown). Penetratin alone and the other penetratin
chimeras also could not induce similar effects (FIG. 17, panel c).
This finding shows that novel approaches for inhibiting
angiogenesis can be developed based on the use of integrin
targeting peptides.
[0341] Immunization With Cytoplasmic Domain Binding Peptides and
Characterization of the Resulting Antibodies
[0342] Polyclonal antibodies that recognize .alpha.v.beta.3 and
.alpha.v.beta.5-binding peptides were generated using KLH
conjugates made with the synthetic peptides, according to standard
techniques. Antibodies against two different synthetic peptides
have been produced (FIG. 18). The sera not only recognize the
immobilized peptides, but also recognize specific proteins in total
cell extracts, as shown by western blot analysis (FIG. 19).
[0343] Rabbits were immunized with SDNRYIGSW (SEQ ID NO:97) or
GLDTYRGSP (SEQ ID NO:96)-KLH conjugates. Each rabbit was injected
with 200 .mu.g of peptide conjugated with KLH in Complete Freund's
Adjuvant. Between 20 and 60 days later, the rabbits were injected
with 100 .mu.g Incomplete Freund's Adjuvant. After the third
immunization, sera was collected. Preimmune serum obtained before
the first immunization was used as an additional control in the
experiments.
[0344] The polyclonal antibodies were tested by ELISA, Western blot
and immunoprecipitation. In the ELISA assays, microtiter well
plates were coated with 10 .mu.g/ml of peptides. The plates were
dried at 37.degree. C., blocked with PBS+3% BSA, and incubated with
different serum dilutions in PBS+1% BSA. After washing and
incubation with the secondary antibody, an alkaline phosphate
substrate was added and antibody binding detected calorimetrically
at 405 nm. The reactivity observed both in the mouse and rabbit
polyclonal sera was highly specific. In all cases, antibody binding
could be abrogated by preincubation with the corresponding peptide
that was used for immunization, but not by a control peptide (FIG.
18 and FIG. 19). Antibodies raised against two of the .beta.3
cytoplasmic domain binding peptides recognize specific bands on
total cell extracts and in immunoprecipitation experiments using
35S-abeled extracts. Similar results were obtained with polyclonal
sera and purified IgG's (not shown).
[0345] The present example shows that targeting peptides against
specific domains of cell receptors can be identified by phage
display. Such peptides may be used to identify the endogenous
ligands for cell receptors, such as endostatin. In addition, the
peptides themselves may have therapeutic effects, or may serve as
the basis for identification of more effective therapeutic agents.
The endostatin targeting peptides identified herein, when
introduced into cells, showed effects on cell proliferation,
chemotaxis and apoptosis. The skilled artisan will realize that the
present invention is not limited to the disclosed peptides or
therapeutic effects. Other cell receptors and ligands, as well as
inhibitors or activators thereof, may be identified by the
disclosed methods.
Example 7
Induction of Apoptosis with Integrin Binding Peptides
(Endothanos)
[0346] Example 9 showed that the VISY peptide (VVISYSMPD, SEQ ID
NO:112), imported into cells by attachment to penetratin, could
induce apoptosis in HUVEC cells. Antibodies raised against the VISY
peptide were used to identify the endogenous cell analog of the
peptide, identified herein as Annexin V. The results indicate that
Annexin V is an endogenous ligand for the integrins that is
involved in a novel pathway for apoptosis.
[0347] Methods
[0348] Protein Purification
[0349] Polyclonal antibodies against the VISY peptide (VVISYSMPD,
SEQ ID NO:112) were prepared using the methods described in Example
9 above. MDA-MB-435 breast carcinoma cells were used for
purification of the endogenous VISY peptide analog. Cells were
washed three times with ice cold PBS and lysed with chilled water
for 20 mn. Cell extracts were centrifuged for 30 min at
100,000.times.g to separate the cytoplasmic fraction from the
membrane fraction. The cytoplasmic fraction was subjected to column
chromatography on a gel filtration column (10-50 kDa) and an anion
exchange column (mono Q). The anion exchange column was eluted with
a salt gradient from 50 mM to 1 M NaCl. One ml fractions were
collected, run on SDS-PAGE and tested by Western blotting for the
presence of endogenous proteins reactivce with the anti-VISY
antibody. The fraction of interest, containing a 36 kDa antibody
reactive band, eluted at about 300 mM NaCl.
[0350] The 36 kDa always appeared in fractions that showed positive
reactivity with the anti-VISY antibody. The fractions were analyzed
by SDS-PAGE and 2-D gel electrophoresis, followed by Western
blotting. A substantial enrichment of the 36 kDa protein was seen
after column chromatography (not shown). The 36 kDa peptide was cut
from the SDS-PAGE gel and analyzed by mass spectroscopy to obtain
its sequence. All five peptide sequences that were obtained by mass
spectroscopy showed 100% homology to the reported sequence of
Annexin V (GenBank Accession No. GI.sub.--468888). In addition to
its presnece in 435 cells, the 36 kDa band was also seen in Kaposi
sarcoma, SKOV and HUVEC cells (not shown).
[0351] Commercial antibodies against Annexin V were obtained (Santa
Cruz Biologics, Santa Cruz, Calif.). Comparative Western blots were
performed using the anti-VISY antibody and the anti-Annexin V
antibody. Both antibodies showed reactivity with the 36 kDa protein
(not shown). These results indicate that the endogenous protein
analog of the VISY peptide is Annexin V.
[0352] Protein-Protein Interaction with Annexin V and .beta.5
Cytoplasmic Domain.
[0353] Competitive binding assays were performed to examine the
binding of Annexin V to .beta.5 integrin and the effect of the VISY
peptide. Plates were coated with GST fusion proteins of the
cytoplasmic domains of various integrins and Annexin V was added to
the plates. Binding of Annexin V was determined using anti-Annexin
V antibodies. As shown in FIG. 20A, Annexin V did not bind to
either the GST-.beta.1 or GST-.beta.3 integrins. Annexin V bound
strongly to the GST-.beta.5 integrin, but binding was dependent on
the buffer used (FIG. 20A). Low binding was observed in
Tris-buffered saline (TBS), while high binding was observed in
"cytoplasmic buffer" (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl.sub.2, 10
mM PIPES, 3 mM DTT) with or without added calcium (2 mM) (FIG.
20A). Calcium was used because Annexin V activity has been reported
to be modulated by calcium. Binding of Annexin V to GST-.beta.5 was
blocked by addition of the VISY peptide (FIG. 20A). FIG. 20B shows
the relative levels of binding of anti-Annexin V antibody to
purified Annexin V and to VISY peptide.
[0354] A reciprocal study was performed, using Annexin V to coat
plates and adding GST fusion proteins of integrin cytoplasmic
domains. Binding was assessed using anti-GST fusion protein
antibodies. As expected, only GST-.beta.5 showed substantial
binding to Annexin V, while GST-.beta.1 and GST-.beta.3 showed low
levels of Annexin V binding (not shown). In some studies, calcium
ion appeared to interfere with the binding interaction between
GST-.beta.5 and Annexin V, with decreased binding observed in the
presence of calcium (not shown). A greater degree of inhibition of
Annexin V binding to GST-.beta.5 by the VISY peptide was observed
in the presence of calcium (67% inhibition) than in the absence of
calcium (45%) (FIG. 20A).
[0355] Penetratin Peptide Chimera Binding to the .beta.5
Cytoplasmic Domain Induces Programmed Cell Death.
[0356] The induction of apoptosis by VISY peptide was shown in
Example 9 was confirmed. 10.sup.6 HUVEC were treated with 15 .mu.M
of VISY antennapedia (penetratin) chimera or 15 .mu.M of
antennapedia peptide (pentratin) alone for 2-4 hours and chromatin
fragmentation was analyzed by electrophoresis in an agarose gel.
FIG. 21 shows the induction of apoptosis by VISY-Ant (penetratin),
as indicated by chromatin fragmentation. Neither VISY or penetratin
alone induced apoptosis. Induction of apoptosis was inhibited up to
70% when a caspase inhibitor (zVAD, caspase inhibitor I, Calbiochem
#627610, San Diego, Calif.) was added to the media at the same time
as the VISY chimeric peptide.
[0357] A distinction between the mechanism of cell death induced by
VISY peptide and other pro-apoptosis agents is that other apoptotic
mechanisms evaluated in cell culture typically involve detachment
of the cells from the substrate, followed by cell death. In
contrast, in VISY induced cell death, the cells do not detach from
the substrate before dying. Thus, endothanos (death from inside)
appears to differ from anoikis (homelessness).
[0358] Example 9 and the present results show that VISY peptides
activate an integrin dependent apoptosis pathway. The present
example shows that the endogenous analog for VISY peptide in
Annexin V. These results demonstrate the existence of a novel
apoptotic pathway, mediated through an interaction between Annexin
V and .beta.5 integrin and dependent on caspase activity. This
novel apoptotic mechanism is termed endothanos. The skilled artisan
will realize that the existence of a novel mechanism for inducing
or inhibiting apoptosis is of use for a variety of applications,
such as cancer therapy.
Example 8
Identification of Receptor/Ligand Pairs: Aminopeptidase A Regulates
Endothellal Cell Function and Angiogenesis
[0359] Endothelial cells in tumor vessels express specific
angiogenic markers. Aminopeptidase A (APA, EC 3.4.11.7) is
upregulated in microvessels undergoing angiogenesis. APA is a
homodimeric, membrane-bound zinc metallopeptidase that hydrolyzes
N-terminal glutamyl or aspartyl residues from oligopeptides (Nanus
et al., 1993). In vivo, APA converts angiotensin II to angiotensin
III. The renin-angiotensin system plays an important role in
regulating several endocrine, cardiovascular, and behavioral
functions (Ardaillou, 1997; Stroth and Unger, 1999). Recent studies
also suggest a role for angiotensins in angiogenesis (Andrade et
al., 1996), but the function of APA in the angiogenic process has
not been investigated so far.
[0360] In the present example, targeting peptides capable of
binding APA were identified by screening phage libraries on
APA-expressing cells. APA-binding peptides containing the motif
CPRECESIC (SEQ ID NO: 123) specifically inhibited APA enzyme
activity. Soluble CPRECESIC (SEQ ID NO:123) peptide inhibited
migration, proliferation, and morphogenesis of endothelial cells in
vitro and interfered with in vivo angiogenesis in a chick embryo
chorioallantoic membrane (CAM) assay. Furthermore, APA null mice
had a decreased amount of retinal neovascularization compared to
wild type (wt) mice in hypoxia-induced retinopathy in premature
mice. These results may lead to a better understanding of the role
of APA in angiogenesis and to development of new anti-tumor
therapeutic strategies.
[0361] Materials and Methods
[0362] Cell Cultures
[0363] The renal carcinoma cell line SK-RC49 was transfected with
an expression vector encoding full-length APA cDNA (Geng et al.,
1998). Cells were maintained in MEM (Irvine Scientific, Santa Ana,
Calif.), supplemented with 2 mM glutamine, 1% nonessential amino
acids, 1% vitamins (Gibco BRL), 100 U/ml streptomycin, 100 U/ml
penicillin (Irvine Scientific), 10 mM sodium pyruvate
(Sigma-Aldrich), and 10% fetal calf serum (FCS) (Tissue Culture
Biological, Tulare, Calif.). Stably transfected cells were
maintained in G418-containing medium. HUVECs were isolated by
collagenase treatment and used between passages 1 to 4. Cells were
grown on gelatin-coated plastic in M199 medium (Sigma) supplemented
with 20% FCS, penicillin (100 U/ml), streptomycin (50 .mu.g/ml),
heparin (50 .mu.g/ml), and bovine brain extract (100 .mu.g/ml). All
media supplements were obtained commercially (Life Technologies,
Inc., Milan, Italy).
[0364] Antibodies and Peptides
[0365] The anti-APA mAb RC38 (Schlingemann et al., 1996) was used
to immunocapture APA from transfected cell lysates. CPRECESIC (SEQ
ID NO:123) and GACVRLSACGA (SEQ ID NO:124) cyclic peptides were
chemically synthesized, spontaneously cyclized in non-reducing
conditions, and purified by mass spectrometry (AnaSpec San Jose,
Calif.). The mass spectrometer analysis of the CPRECESIC (SEQ ID
NO:123) peptide revealed six different peaks, possibly reflecting
different positions of disulfide bounds and the formation of
dimers. Due to the similar biochemical behavior of the different
fractions on APA enzyme activity, a mix of the six peaks was used
in all procedures described below.
[0366] APA Immunocapture
[0367] Cells were scraped from semi-confluent plates in cold PBS
containing 100 mM N-octyl-.beta.-glucopyranoside (Calbiochem),
lysed on ice for 2 h, and centrifuged at 13,000.times.g for 15 min.
Microtiter round-bottom wells (Falcon) were coated with 2 .mu.g of
RC38 for 4 h at room temperature and blocked with PBS/3% BSA
(Intergen, Purchase, N.Y.) for 1 h at room temperature, after which
150 .mu.l of cell lysate (1 mg/ml) was incubated on the mAb-coated
wells overnight at 4.degree. C., washed five times with PBS/0.1%
Tween-20 (Sigma), and washed twice with PBS.
[0368] APA Enzyme Assay
[0369] Cells and immunocaptured proteins were tested for specific
enzyme activity according to Liln et al. (1998). Briefly, adherent
cells or RC38-inmmunocaptured cell extracts were incubated for 2 h
at 37.degree. C. with PBS containing 3 mM
.alpha.-L-glutamyl-p-nitroanilide (Fluka) and 1 mM CaCl.sub.2.
Enzyme activity was determined by reading the optical absorbance
(O.D.) at 405 nm in a microplate reader (Molecular Devices,
Sunnyvale, Calif.).
[0370] Cell Panning
[0371] A CX.sub.3CX.sub.3CX.sub.3C (C, cysteine; X, any amino acid)
library was prepared (Rajotte et al., 1998). Amplification and
purification of phage particles and DNA sequencing of
phage-displayed inserts were performed as described above. Cells
were detached by incubation with 2.5 mM EDTA in PBS, washed once in
binding medium (DMEM high glucose supplemented with 20 mM HEPES and
2% FCS), and resuspended in the same medium at a concentration of
2.times.10.sup.6 cells/ml. 10.sup.10 TU of phage were added to 500
.mu.l of the cell suspension, and the mixture was incubated
overnight (first round) or for 2 h (successive rounds) at 4.degree.
C. with gentle rotation. Cells were washed five times in binding
medium at room temperature and resuspended in 100 .mu.l of the same
medium. Phage were rescued by adding 1 ml of exponentially growing
K91Kan Escherichia coli bacteria and incubating the mixture for 1 h
at room temperature. Bacteria were diluted in 10 ml of LB medium
supplemented with 0.2 ag/ml tetracycline and incubated for another
20 min at room temperature. Serial dilutions were plated on LB
plates containing 40 .mu.g/ml tetracycline, and plates were
incubated at 37.degree. C. overnight before colonies were
counted.
[0372] Phage Binding Specificity Assay
[0373] The cell binding assay was performed with an input of
10.sup.9 TU as described for the cell panning. The specificity was
confirmed by adding CPRECESIC (SEQ ID NO:123) peptide to the
binding medium in increasing concentrations. For phage binding on
immunocaptured APA, wells were blocked for 1 h at room temperature
with PBS/3% BSA and incubated with 10.sup.9 TU for 1 h at room
temperature in 50 .mu.l PBS/3% BSA. After eight washes in PBS/1%
BSA/0.01% Tween-20 and two washes in PBS, phage were rescued by
adding 200 .mu.l of exponentially growing K91 Kan E. coli. Each
experiment was repeated at least three times.
[0374] In Vivo Tumor Homing of APA-Binding Phage
[0375] MDA-MB435-derived tumor xenografts were established in
female nude mice 2 months old (Jackson Labs, Bar Harbor, Me.). Mice
were anesthetized with Avertin and injected intravenously through
the tail vein with 10.sup.9 TU of the phage in a 200 .mu.l volume
of DMEM. The phage were allowed to circulate for 5 min, and the
animals were perfused through the heart with 5 ml of DMEM. The
tumor and brain were dissected from each mouse, weighed, and equal
amounts of tissue were homogenized. The tissue homogenates were
washed three times with ice-cold DMEM containing a proteinase
inhibitor cocktail and 0.1% BSA. Bound phage were rescued and
counted as described for cell panning. Fd-tet phage was injected at
the same input as a control. The experiment was repeated twice. In
parallel, part of the same tissue samples were fixed in Bouin
solution, and imbedded in paraffin for preparation of tissue
sections. An antibody to M-13 phage (Amersham-Pharmacia) was used
for the staining.
[0376] Cell Growth Assay
[0377] HUVECs were seeded in 48-well plates (10.sup.4 cells/well)
and allowed to attach for 24 h in complete M199 medium. The cells
were then starved in M199 medium containing 2% FCS for 24 h.
CPRECESIC (SEQ ID NO:123) or control GACVRLSACGA (SEQ ID NO:124)
peptide (1 mM) was added to the wells in medium containing 2% FCS
and 10 ng/ml VEGF-A (R&D System, Abingdom, UK). After
incubation for the indicated times, cells were fixed in 2.5%
glutaraldehyde, stained with 0.1% crystal violet in 20% methanol,
and solubilized in 10% acetic acid. All treatments were done in
triplicate. Cell growth was evaluated by measuring the O.D. at 590
nm in a microplate reader (Biorad, Hercules, Calif.). A calibration
curve was established and a linear correlation between O.D. and
cell counts was observed between 10.sup.3 and 10.sup.5 cells.
[0378] Chemotaxis Assay
[0379] A cell migration assay was performed in a 48-well
microchemotaxis chamber NeuroProbe, Gaithersburg, Md.) according to
Bussolini et al. (1995). Polyvinylpyrrolidone-free polycarbonate
filters (Nucleopore, Cambridge, Mass.) with 8-.mu.m pores were
coated with 1% gelatin for 10 min at room temperature and
equilibrated in M199 medium supplemented with 2% FCS. CPRECESIC
(SEQ ID NO: 123) or control GACVRLSACGA (SEQ ID NO: 124) peptide (1
mM) was placed in the lower compartment of a Boyden chamber in M199
medium supplemented with 2% FCS and 10 ng/ml VEGF-A (R&D
System). Subconfluent cultures that had been starved overnight were
harvested in PBS containing 2.5 mM EDTA, washed once in PBS, and
resuspended in M199 medium containing 2% FCS at a final
concentration of 2.times.10.sup.6 cells/ml. After the filter was
placed between the lower and upper chambers, 50 .mu.l of the cell
suspension was seeded in the upper compartment, and cells were
allowed to migrate for 5 h at 37.degree. C. in a humidified
atmosphere with 5% CO.sub.2. The filter was then removed, and cells
on the upper side were scraped with a rubber policeman. Migrated
cells were fixed in methanol and stained with Giemsa solution
(Diff-Quick, Baxter Diagnostics, Rome, Italy). Five random
high-power fields (magnitude 100.times.) were counted in each well.
Each assay was run in triplicate.
[0380] Three-Dimensional Cell Culture
[0381] Matrigel (Collaborative Research, Bedford, Mass.) was added
at 100 .mu.l per well to 48-well tissue culture plates and allowed
to solidify for 10 min at 37.degree. C. HUVECs were starved for 24
h in M199 medium supplemented with 2% FCS before being harvested in
PBS containing 2.5 mM EDTA. 10 cells were gently added to each of
the triplicate wells and allowed to adhere to the gel coating for
30 min at 37.degree. C. Then, medium was replaced with indicated
concentrations of CPRECESIC (SEQ ID NO:123) or GACVRLSACGA (SEQ ID
NO: 124) peptides in complete medium. The plates were photographed
after 24 h with an inverted microscope (Canon). The assay was
repeated three times.
[0382] CAM Assay
[0383] In vivo angiogenesis was evaluated by a CAM assay (Ribatti
et al., 1994). Fertilized eggs from White Leghorn chickens were
maintained in constant humidity at 37.degree. C. On the third day
of incubation, a square window was opened in the eggshell and 2-3
ml of albumen was removed to detach the developing CAM from the
shell. The window was sealed with a glass plate of the same size
and the eggs were returned to the incubator. At day 8, 1 mm.sup.3
sterilized gelatin sponges (Gelfoam, Upjohn Co, Kalamazoo, Milan)
were adsorbed with VEGF-A (20 ng, R&D System) and either
CPRECESIC (SEQ ID NO:123) or control GACVRLSACGA (SEQ ID NO:124)
peptide (1 mM) in 3 .mu.l PBS and implanted on the top of the
growing CAMs under sterile conditions. CAMs were examined daily
until day 12 and photographed in ovo with a Leica stereomicroscope.
Capillaries emerging from the sponge were counted. The assay was
repeated twice.
[0384] Induction of Retinal Neovascularization
[0385] APA null mice have been described (Lin et al., 1998). Mice
pups on P7 (7.sup.th day post-partum) with their nursing mothers
were exposed to 75% oxygen for 5 days. Mice were brought back to
normal oxygen (room air) on P12. For histological analysis mice
were killed between P17 and P21 and eyes were enucleated and fixed
in 4% paraformaldehyde in PBS overnight at +4.degree. C. Fixed eyes
were imbedded in paraffin and 5 .mu.m serial sections were cut.
Sections were stained with hematoxylin/eosin (h/e) solution.
Neovascular nuclei on the vitreous side of, the internal limiting
membrane were counted from 20 h/e-stained sections per each eye.
The average number of neovascular nuclei per section was calculated
and compared between animal groups using Student's t-test.
[0386] Results
[0387] Cell Panning With Phage Display Select an APA-Binding
Motif
[0388] To identify a peptide capable of binding to APA, cells were
screened with a random peptide phage library. First, SK-RC49 renal
carcinoma cells, which do not express APA, were transfected with
full-length APA cDNA to obtain a model of APA expression in the
native conformation. APA expressed as a result of transfection was
functionally active, as evidenced by an APA enzyme assay (not
shown), but parental SK-RC49 cells showed neither APA expression
nor activity (not shown).
[0389] The CX.sub.3CX.sub.3CX.sub.3C phage library (10.sup.10
transducing units [TU]) was preadsorbed on parental SK-RC49 cells
to decrease nonspecific binding. Resuspended SK-RC-49/APA cells
were screened with phage that did not bind to the parent cells.
SK-RC-49/APA-bound phage were amplified and used for two
consecutive rounds of selection. An increase in phage binding to
SK-RC49/APA cells relative to phage binding to SK-RC49 parental
cells was observed in the second and third rounds (not shown).
[0390] Subsequent sequencing of the phage revealed a specific
enrichment of a peptide insert, CYNLCIRECESICGADGACWTWCADGCSRSC
(SEQ ID NO:125), with a tandem repetition of the general library
sequence CX.sub.3CX.sub.3CX.sub.3C. This sequence represented 50%
of 18 randornly selected phage inserts from round 2 and 100% of
phage inserts from round 3. Four peptide inserts derived from round
2 shared sequence similarity with the tandem phage (Table 12, in
bold font). Several other apparently conserved motifs were observed
among round 2 peptides (Table 12, underlined or italicized). One of
these overlapped in part with the tandem repeated sequence. A
search for sequence homology of the selected peptides against human
databases did not yield a significant match.
14TABLE 12 APA-binding peptide sequences. Peptide sequences
.sup.(a) Round 2 (%) Round 3 (%) CYNLCIRECESICGADGACWTWCADGCSRSC
(SEQ ID NO:125) 50 100 CLGQCASICVNDC (SEQ ID NO:126) 5 --
CPKVCPRECESNC (SEQ ID NO:127) 5 -- CGTGCAVECEVVC (SEQ ID NO:128) 5
-- CAVACWADCQLGC (SEQ ID NO:129) 5 -- CSGLCTVQCLEGC (SEQ ID NO:130)
5 -- CSMMCLEGCDDWC (SEQ ID NO:131) 5 -- OTHER 20 --
[0391] Selected Phage Inserts are Specific APA Ligands.
[0392] Phage displaying the peptide inserts
CYNLCIRECESICGADGACWTWCADGCSRS- C (SEQ ID NO:125), CPKVCPRECESNC
(SEQ ID NO: 127) or CLGQCASICVNDC (SEQ ID NO: 126) were
individually tested for APA binding. AU three phage specifically
bound to the surface of SK-RC-49/APA cells (not shown), with a
similar pattern of 6-fold enrichment relative to SK-RC-49 parental
cells. Control, insertless phage showed no binding preference (not
shown). CGTGCAVECEVVC (SEQ ID NO:128) and the other phage selected
in round 2 showed no selective binding to SK-RC-49/APA cells (data
not shown). A soluble peptide, CPRECESIC (SEQ ID NO: 123)
containing a consensus sequence reproducing the APA-binding phage
inserts was synthesized.
[0393] Binding assays were performed with CPKVCPRECESNC (SEQ ID
NO:127) phage in the presence of the CPRECESIC (SEQ ID NO: 123)
peptide. Soluble CPRECESIC (SEQ ID NO:123) peptide competed with
CPKVCPRECESNC (SEQ ID NO:127) phage for binding to SK-RC-49/APA
cells, but had no effect on nonspecific binding to SK-RC49 parental
cells (not shown. The unrelated cyclic peptide GACVRLSACGA (SEQ ID
NO:124) had no competitive activity (not shown). Binding of
CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:125) phage was also
displaced by CPRECESIC (SEQ ID NO:123) peptide, but the binding of
CLGQCASICVNDC (SEQ ID NO: 126) phage was not affected (data not
shown).
[0394] To further confirm the substrate specificity of the selected
peptide inserts, APA was partially purified from APA-transfected
cell extracts by immunocapture with mAb RC38. The APA protein
immobilized on RC38-coated microwells was functional, as confirmed
by enzyme assay (not shown). The CYNLCIRECESICGADGACWTWCADGCSRSC
(SEQ ID NO:125), CPKVCPRECESNC (SEQ ID NO:127), and CLGQCASICVNDC
(SEQ ID NO:126) phage selectively bound immunocaptured APA, with a
10- to 12-fold enrichment compared to phage binding to
RC38-immunocaptured cell lysates from SK-RC49 parental cells (not
shown).
[0395] APA-Binding Phage Target Tumors In Vivo.
[0396] The ability of the identified peptide to home to tumors was
evaluated, using nude mice implanted with human breast tumor
xenografts as a model system. Phage were injected into the tail
vein of tumor-bearing mice, and targeting was evaluated by phage
recovery from tissue homogenates. CPKVCPRECESNC (SEQ ID NO:127)
phage was enriched 4-fold in tumor xenografts compared to brain
tissue, which was used as a control (FIG. 22). Insertless phage did
not target the tumors (FIG. 22). Neither
CYNLCIRECESICGADGACWTWCADGCSRSC (SEQ ID NO:125) nor CLGQCASICVNDC
(SEQ ID NO:126) phage showed any tumor-homing preference (data not
shown).
[0397] The homing of CPKVCPRECESNC (SEQ ID NO: 127) was confirmed
by anti-M13 immunostaining on tissue sections (not shown). Strong
phage staining was apparent in tumor vasculature but not in normal
vasculature (not shown). Insertless phage did not bind to tumor
vessels.
[0398] CPRECESIC (SEQ ID NO:123) is a Specific Inhibitor of APA
Activity.
[0399] To investigate the effect of CPRECESIC (SEQ ID NO:123) on
APA enzyme activity, SK-RC49/APA cells were incubated with the APA
specific substrate .alpha.-glutamyl-p-nitroanilide in the presence
of increasing concentrations of either CPRECESIC (SEQ ID NO: 123)
or control GACVRLSACGA (SEQ ID NO: 124) peptides. Enzyme activity
was evaluated by a colorimetric assay after 2 h incubation at
37.degree. C. CPRECESIC (SEQ ID NO:123) inhibited APA enzyme
activity, reducing the activity by 60% at the highest concentration
tested (FIG. 23). The IC.sub.50 of CPRECESIC (SEQ ID NO: 123) for
enzyme inhibition was calculated to be 800 .mu.M. CPRECESIC (SEQ ID
NO:123) did not affect the activity of a closely related protease,
aminopeptidase N (data not shown).
[0400] CPRECESIC (SEQ ID NO:123) Inhibits Migration and
Proliferation of Endothelial Cells.
[0401] The potential use of CPRECESIC (SEQ ID NO:123) peptide as an
anti-angiogenic drug was determined. First, the effect of APA
inhibition by CPRECESIC (SEQ ID NO:123) peptide in vitro on the
migration and proliferation of human umbilical vein endothelial
cells (HUVECs) stimulated with VEGF-A (10 ng/ml) was examined. The
presence of functional APA on HUVECs was evaluated by enzyme assay
(not shown). At the highest concentration tested (1 mM), CPRECESIC
(SEQ ID NO:123) peptide inhibited chemotaxis of HUVECs by 70% in a
Boyden chamber assay (FIG. 24). At the same peptide concentration,
cell proliferation was inhibited by 50% (FIG. 25). Lower
concentrations of CPRECESIC (SEQ ID NO:123) peptide or the
GACVRLSACGA (SEQ ID NO:124) control peptide had no significant
effect on cell migration or proliferation (not shown).
[0402] CPRECESIC (SEQ ID NO:123) Inhibits Angiogenesis In Vitro and
In Vivo
[0403] The inhibitory effect of CPRECESIC (SEQ ID NO:123) peptide
in different in vitro and in vivo models of angiogenesis was
examined. HUVECs plated on a three-dimensional matrix gel
differentiate into a capillary-like structure, providing an in
vitro model for angiogenesis. Increasing concentrations of
CPRECESIC (SEQ ID NO:123) peptide resulted in a progressive
impairment of the formation of this network (not shown). At a
peptide concentration of 1 mM, vessel-like branching structures
were significantly fewer and shorter, and as a result, the cells
could not form a complete network organization (not shown). The
control peptide GACVRLSACGA (SEQ ID NO: 124) did not affect HUVEC
morphogenesis (not shown).
[0404] A commonly used model of simplified in vivo angiogenesis is
the chicken chorioallantoic membrane (CAM), in which
neovascularization can be stimulated during embryonic development.
An appropriate stimulus, adsorbed on a gelatin sponge, induces
microvessel recruitment to the sponge itself, accompanied by
remodeling and ramification of the new capillaries. Eight-day-old
chicken egg CAMs were stimulated with VEGF-A alone (20 ng) or with
VEGF-A plus CPRECESIC (SEQ ID NO:123) or GACVRLSACGA (SEQ ID
NO:124) (1 mM) peptides. The CAMs were photographed at day 12.
Neovascularization induced by VEGF-A was inhibited by CPRECESIC
(SEQ ID NO:123) by 40% based on the number of capillaries emerging
from the sponge (Table 13). The neovessels did not show the highly
branching capillary structures typically seen after VEGF-A
stimulation (not shown). Treatment with control peptide GACVRLSACGA
(SEQ ID NO: 124) or with lower peptide concentrations of CPRECESIC
(SEQ ID NO: 123) had no effect on the number of growing vessels
(not shown).
15TABLE 13 CAM assay for angiogenesis TREATMENT BLOOD VESSEL
NUMBERS No VEGF-A 12.0 .+-. 2.82* VEGF-A 57.0 .+-. 1.41* VEGF-A +
control 56.5 .+-. 2.12 VEGF-A + CPRECESIC 5.5 .+-. 1.41* (SEQ ID
NO:123) *p < 0.01 with the Student-Newman-Keuls test. The
results are expressed as the mean and standard error from two
independent experiments.
[0405] APA-Deficient Mice Show Impaired Neovascularization
[0406] The ability of APA.sup..+-. and APK.sup.-/- null mice to
undergo neovascularization was examined in a model of hypoxic
retinopathy in premature mice. Induction of retinal
neovascularization by relative hypoxia was already present in
APA.sup..+-. mice compared to wild type mice (not shown).
Neovascularization was almost undetectable in APA null mice (not
shown). Neovascularization was quantified by counting vitreous
protruding neovascular nuclei from 20 sections of hypoxic eyes.
Significant induction of retinal neovascularization (16.17.+-.1.19
neovascular nuclei/eye section) was seen in the wild type mice on
postnatal day 17 (P17) after 75% oxygen treatment from P7 to P12.
Decreased amounts of neovascular nuclei were seen in the retinas of
APA.sup..+-. (10.76.+-.1.03 neovascular nuclei/eye section) and APA
null (4.25.+-.0.45 neovascular nuclei/eye section) mice on P17
after exposure to 75% oxygen from P7 to P12.
[0407] Discussion
[0408] In vivo, APA is overexpressed by activated microvessels,
including those in tumors, but it is barely detectable in quiescent
vasculature, making it a suitable target for vessel-directed tumor
therapy. The present example identified a novel targeting peptide
ligand for APA, CPRECESIC (SEQ ID NO:123). Soluble CPRECESIC (SEQ
ID NO: 123) peptide inhibited APA enzyme activity with an IC.sub.50
of 800 .mu.M.
[0409] Using cultured HTVECs as an in vitro model of angiogenesis,
soluble CPRECESIC (SEQ ID NO:123) peptide inhibited VEGF-A-induced
migration and proliferation of HUVECs. These data are consistent
with a requirement for migration and proliferation of endothelial
cells during angiogenesis. CPRECESIC (SEQ ID NO: 123) also blocked
the formation of capillary-like structures in a Matrigel model and
inhibited angiogenesis in VEGF-A-stimulated CAMs.
[0410] APA was shown to be important player in neovascularization
induced by relative hypoxia, since APA null mice had
significatively less retinal neovascularization compared to wt
mice. These results strengthen the potential of using APA as a
specific target for the inhibition of tumor angiogenesis.
[0411] In summary, the soluble peptide CPRECESIC (SEQ ID NO: 123)
is a selective APA ligand and inhibitor. The inhibition of APA by
CPRECESIC (SEQ ID NO:123) led to the inhibition of angiogenesis in
different in vitro and in vivo assays, demonstrating for the first
time a prominent role for APA in the angiogenic process.
Furthermore, APA-binding phage can home to tumor blood vessels,
suggesting possible therapeutic uses of CPRECESIC (SEQ ID NO:123)
as an inhibitor of tumor neovascularization. The endogenous analog
of CPRECESIC (SEQ ID NO:123) may be identified by antibody based
purification or identification methods, similar to those disclosed
above.
Example 9
Screening Phage Libraries by PALM
[0412] In certain embodiments, it is desirable to be able to select
specific cell types from a heterogeneous sample of an organ or
tissue. One method to accomplish such selective sampling is by PALM
(Positioning and Ablation with Laser Microbeams).
[0413] The PALM Robot-MicroBeam uses a precise, computer-guided
laser for microablation. A pulsed ultra-violet (UV) laser is
interfaced into a microscope and focused through an objective to a
beam spot size of less than 1 micrometer in diameter. The principle
of laser cutting is a locally restricted ablative
photodecomposition process without heating (Hendrix, 1999). The
effective laser energy is concentrated on the minute focal spot
only and most biological objects are transparent for the applied
laser wavelength. This system appears to be the tool of choice for
recovery of homogeneous cell populations or even single cells or
subcellular structures for subsequent phage recovery. Tissue
samples may be retrieved by circumcising a selected zone or a
single cell after phage administration to the subject. A clear-cut
gap between selected and non-selected area is typically obtained.
The isolated tissue specimen can be ejected from the object plane
and catapulted directly into the cap of a common microfuge tube in
an entirely non-contact manner. The basics of this so called Laser
Pressure Catapulting (LPC) method is believed to be the laser
pressure force that develops under the specimen, caused by the
extremely high photon density of the precisely focused laser
microbeam. This tissue harvesting technique allows the phage to
survive the microdissection procedure and be rescued.
[0414] PALM was used in the present example to select targeting
phage for mouse pancreatic tissue, as described below.
[0415] Materials and Methods
[0416] In Vivo and In Situ Panning
[0417] A CX.sub.7C peptide phage library (10.sup.9 TU) was injected
into the tail vein of a C57BL/6 male mouse, and the pancreas was
harvested to recover the phage by bacterial infection. Phage from
246 colonies were grown separately in 5 mls LB/kanamycin (100
.mu.g/ml)/tetracycline (40 .mu.g/ml) at 37.degree. C. in the dark
with agitation. Overnight cultures were pooled and the phage
purified by NaCl/PEG precipitation for another round of in vivo
bio-panning. Three hundred colonies were picked from the second
round of panning, and the phage were recovered by precipitation.
Phage from the second bio-panning round was then used for another
round of in vivo panning and also was incubated with thawed frozen
murine pancreatic sections for one in situ panning round.
[0418] For the third in vivo panning round, 10.sup.9 TU phage from
the second round were injected into a third mouse and allowed to
circulate for six minutes, followed by an intravenous injection of
50 .mu.l of FITC-lectin (Vector Laboratories, Inc.). After a
two-minute circulation, the mouse was perfused through the left
ventricle with 3 mls MEM Earle salts. The pancreas was harvested,
frozen at -80.degree. C. in Tissue Tek (Sakura), and sectioned onto
prepared slides.
[0419] For the third in situ round, purified phage, isolated from
the second round, were incubated with 4-14 .mu.m thawed murine
pancreatic sections on ice for 30 minutes. Sections were rinsed
with 100 .mu.l ice-cold PBS 8.times. at room temperature (RT).
Bound phage were recovered from each section by adding 100 .mu.l
K91 Kan.sup.R (OD.sub.600=2.03) to infect at RT for 30-60 minutes.
Infected K91 Kan.sup.R were withdrawn from each section and allowed
to recover in 10 mls LB/Kan/Tet (0.2 .mu.g/ml) for 20 minutes in
the dark. Aliquots from the each culture were plated out onto
LB/Kan/Tet (40 .mu.g/ml) plates and incubated overnight in the dark
at 37.degree. C. The tetracycline concentration of the remainder of
each culture was increased to 40 .mu.g/ml and the cultures were
incubated overnight at 37.degree. C. in the dark with agitation for
phage amplification and purification.
[0420] DNA Amplification
[0421] Phage were recovered from cryo-preserved FIFC-lectin stained
mouse pancreatic islets and surrounding acinar cells that were
microdissected from 14 .mu.m sections using the PALM (ositioning
and Ablation with Laser Microbeams) cold laser pressure catapulting
system. Pancreatic islet and control sections were catapulted into
1 mM EDTA, pH 8, and frozen at -20.degree. C. until enough material
was collected for PCR amplification. Phage DNA was amplified with
fUSE5 primers: forward primer 5' TAA TAC GAC TCA CTA TAG GGC AAG
CTG ATA AAC CGA TAC AATT 3'(SEQ ID NO:132), reverse primer 5' CCC
TCA TAG TTA GCG TAA CGA TCT 3' (SEQ ID NO:133). The PCR products
were subjected to another round of PCR using a nested set of
primers. The 3' end of the second primer set was tailed with the
M13 reverse primer for sequencing purposes. The nested primer set
used was: forward nested primer 5' CCTTTCTATTCTCACTCGGCCG 3' (SEQ
ED NO:134), reverse nested primer 5'
CAGGAAACAGCTATGACCGCTAAACAACTTTCAACAGTTTCGGC 3' (SEQ ID NO:135). To
generate peptide insert sequence containing flanking SfiI
restriction sites, two more primers were used: forward library
primer 5' CACTCGGCCGACGGGGC 3' (SEQ ID NO:136), reverse primer 5'
CAGTTTCGGCCCCAGCGGCCC 3' (SEQ ID NO:137). PCR products generated
from the nested primers were gel purified (Qiag,en), and confirmed
for the presence of a CX.sub.7C peptide insert sequence using the
M13 reverse primer by automated sequencing. PCR products generated
from the library primers were gel purified (Qiagen), ligated into
CsCl.sub.2 purified fUSE5/SfiI, electroporated into
electrocompetent MC1061 cells, and plated onto LB/streptomycin (100
.mu.g/ml)/tetracycline (40 .mu.g/ml) agar plates. Single colonies
were subjected to colony PCR using the fUSE5 primers to verify the
presence of a CX.sub.7C insert sequence by gel electrophoresis.
Positive clones were sequenced using BigDye terminators (Perkin
Elmer)
[0422] Phage Infection
[0423] Pancreatic islet and control sections were catapulted into 1
mM AEBSF, 20 .mu.g/ml aprotinin, 10 .mu.g/ml leupeptin, 1 mM
elastase inhibitor 1, 0.1 mM TPCK, 1 nM pepstatin A in PBS, pH 7.4,
and frozen for 48 hours or less until enough material was
collected. The sections were thawed on ice and the volume adjusted
to 200 .mu.l with PBS, pH 7.4. Samples were incubated with 1 ml K91
Kan.sup.R (OD=0.22) for two hours at RT on a nutator. Each culture
was transferred to 1.2 mls LB/Kan/Tet (0.2 .mu.g/ml) and incubated
in the dark at RT for 40 minutes. The tetracycline concentration
was increased to 40 .mu.g/ml for each culture, and the cultures
were incubated overnight at 37.degree. C. with agitation. Each
culture was plated out the following day onto LB/Kan/Tet agar
plates and incubated for 14 hours at 37.degree. C. in the dark.
Positive clones were picked for colony PCR and automated
sequencing.
[0424] Results
[0425] The general schem for in vivo panning using PALM is
illustrated in FIG. 26. After an initial round of in vivo
selection, phage were either bulk amplified or else single colonies
of phage from pancreas, kidney, lung and adrenal glands were
amplified and subjected to additional rounds of in vivo screening.
Both bulk amplified and colony amplified phage from mouse pancreas
showed successive enrichment with increasing rounds of selection
(not shown). After three rounds of selection, the colony amplified
phage showed almost an order of magnitude higher enrichment than
bulk amplified phage (not shown).
[0426] Table 14 lists selected targeting sequences and consensus
motifs identified by pancreatic screening.
16TABLE 14 Pancreatic targeting peptides and motifs Motif Peptide
Sequence GGL (SEQ ID NO:138) CVPGLGGLC (SEQ ID NO:139) CGGLDVRMC
(SEQ ID NO:140) CDGGLDWVC (SEQ ID NO:141) LGG (SEQ ID NO:142)
CVPGLGGLC (SEQ ID NO:139)) CTWLGGREC (SEQ ID NO:143) CSRWGLGGC (SEQ
ID NO:144) CPPLGGSRC (SEQ ID NO:251) VRG (SEQ ID NO:145) CVGGVRGGC
(SEQ ID NO:146) CVGNDVRGC (SEQ ID NO:147) CESRLVRGC (SEQ ID NO:148)
CGGRPVRGC (SEQ ID NO:149) AGG (SEQ ID NO:150) CTPFIAGGC (SEQ ID
NO:151) CREWMAGGC (SEQ ID NO:152) CAGGSLRVC (SEQ ID NO:153) VVG
(SEQ ID NO:154) CEGVVGIVC (SEQ ID NO:155) CDSVVGAWC (SEQ ID NO:156)
CRTAVVGSC (SEQ ID NO:157) VGG (SEQ ID NO:158) CVGGARALC (SEQ ID
NO:159) CVGGVRGGC (SEQ ID NO:147) CLAHRVGGC (SEQ ID NO:160) GGL
(SEQ ID NO:161) CWALSGGLC (SEQ ID NO:162) CGGLVAYGC (SEQ ID NO:163)
CGGLATTTC (SEQ ID NO:164) GRV (SEQ ID NO:165) CGRVNSVAC (SEQ ID
NO:166) CAGRVALRC (SEQ ID NO:167) GGA (SEQ ID NO:168) CWNGGARAC
(SEQ ID NO:169) CLDRGGAHC (SEQ ID NO:170) GVV (SEQ ID NO:171)
CELRGVVVC (SEQ ID NO:172) GGV (SEQ ID NO:173) CIGGVHYAC (SEQ ID
NO:174) CGGVHALRC (SEQ ID NO:175) GMWG (SEQ ID NO:176) CIREGMWGC
(SEQ ID NO:177) CIRKGMWGC (SEQ ID NO:178) ALR (SEQ ID NO:179)
CGGVHALRC (SEQ ID NO:175) CAGRVALRC (SEQ ID NO:167) CEALRLRAC (SEQ
ID NO:180) ALV (SEQ ID NO:181) CALVNVHLC (SEQ ID NO:182) CALVMVGAC
(SEQ ID NO:183) GGVH (SEQ ID NO:184) CGGVHALRC (SEQ ID NO:175)
CIGGVHYAC (SEQ ID NO:174) VSG (SEQ ID NO:185) CMVSGVLLC (SEQ ID
NO:186) CGLVSGPWC (SEQ ID NO:187) CLYDVSGGC (SEQ ID NO:188) GPW
(SEQ ID NO:189) CSKVGPWWC (SEQ ID NO:190) CGLVSGPWC (SEQ ID NO:191)
none CAHHALMEC (SEQ ID NO:192) CERPPFLDC (SEQ ID NO:193)
[0427] FIG. 27 shows a general protocol for recovery of phage
insert sequences from PALM selected thin section materials. As
indicated, phage may be recovered by direct infection of E. coli
host bacteria, after protease digestion of the thin section sample.
Alternatively, phage inserts may be recovered by PCR amplification
and cloned into new vector DNA, then electroporated or otherwise
transformed into host bacteria for cloning.
[0428] Both methods of PALM recovery of phage were successful in
retrieving pancreatic targeting sequences. Pancreatic sequences
recovered by direct bacterial infection included CVPRRWDVC (SEQ ID
NO:194), CQHITSGRGC (SEQ ID NO: 195), CRARGWLLC (SEQ ID NO: 196),
CVSNPRWKC (SEQ ID NO: 197), CGGVHALRC (SEQ ID NO: 175), CFNRTWIGC
(SEQ ID NO: 198) and CSRGPAWGC (SEQ ID NO:199). Pancreatic
targeting sequences recovered by amplification of phage inserts and
cloning into phage include CWSRGQGGC (SEQ ID NO:200), CHVLWSTRC
(SEQ ID NO:201), CLGLLMAGC (SEQ ID NO:202), CMSSPGVAC (SEQ ID
NO:203), CLASGMDAC (SEQ ID NO:204), CHDERTGRC (SEQ ID NO:205),
CAHHALMEC (SEQ ID NO:206), CMQGAATSC (SEQ ID NO:207), CMQGARTSC
(SEQ ID NO:208) and CVRDLLTGC (SEQ ID NO:209).
[0429] FIG. 28 through FIG. 31 show sequence homologies identified
for selected pancreatic targeting sequences. Several proteins known
to be present in pancreatic tissues are identified. The results of
this example show that the PALM method may be used for selecting
cell types from tissue thin sections and recovering targeting phage
sequences. The skilled artisan will realize that this method could
be used with virtually any tissue to obtain targeting sequences
directed to specific types of cells in heterologous organs or
tissues.
[0430] All of the COMPOSITIONS, METHODS and APPARATUS disclosed and
claimed herein can be made and executed without undue
experimentation in light of the presentdisclosure. While the
compositions and methods of this invention have been described in
terms of preferred embodiments, it are apparent to those of skill
in the art that variations maybe applied to the COMPOSITIONS,
METHODS and APPARATUS and in the steps or in the sequence of steps
of the methods described herein without departing from the concept,
spirit and scope of the invention. More specifically, it are
apparent that certain agents that are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
251 1 14 PRT Artificial Sequence Peptide (1)..(14) synthetic
construct 1 Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys
1 5 10 2 14 PRT Artificial Sequence Peptide (1)..(14) synthetic
construct 2 Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala
1 5 10 3 14 PRT Artificial Sequence Peptide (1)..(14) synthetic
construct 3 Lys Ala Ala Lys Lys Ala Ala Lys Ala Ala Lys Lys Ala Ala
1 5 10 4 21 PRT Artificial Sequence Peptide (1)..(21) synthetic
construct 4 Lys Leu Gly Lys Lys Leu Gly Lys Leu Gly Lys Lys Leu Gly
Lys Leu 1 5 10 15 Gly Lys Lys Leu Gly 20 5 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 5 Cys Val Met Thr
Cys Ala Pro Arg Cys Phe Glu His Cys 1 5 10 6 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 6 Cys Asp Gly Val
Cys Ala Pro Arg Cys Gly Glu Arg Cys 1 5 10 7 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 7 Cys Thr Gly Gly
Cys Val Val Asp Cys Leu Ser Ile Cys 1 5 10 8 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 8 Cys Gly Val Pro
Cys Arg Pro Ala Cys Arg Gly Leu Cys 1 5 10 9 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 9 Cys Ala Gly Phe
Cys Val Pro Gly Cys His Ser Lys Cys 1 5 10 10 13 PRT Artificial
Sequence Peptide (1)..(13) synthetic construct 10 Cys Ala Gly Ala
Cys Pro Val Gly Cys Gly Thr Gly Cys 1 5 10 11 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 11 Ala Glu Arg Leu
Trp Arg Ser 1 5 12 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 12 Ser Gln His Val Val Ser Gly 1 5 13 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 13 Ile Ala
Trp Arg Leu Glu His 1 5 14 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 14 Trp Tyr Thr Val Met Ser Trp 1 5 15
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 15
Arg Leu Thr Tyr Lys Leu Gln 1 5 16 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 16 Trp Gln Arg Leu Tyr Ala Trp
1 5 17 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 17 Glu Phe Arg Leu Gly Ser Lys 1 5 18 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 18 Leu Gly Ser Asn
Ser Lys Ala 1 5 19 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 19 Cys Gly Val Val Lys Phe Ala 1 5 20 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 20 Arg Val
Gly Thr Trp Gly Arg 1 5 21 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 21 Gly Arg Gly Arg Trp Gly Ser 1 5 22
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 22
Val Gln Gly Ile Gly Arg Leu 1 5 23 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 23 Val Gly Ser Gly Arg Leu Ser
1 5 24 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 24 Gly Trp Thr Val Arg Asp Gly 1 5 25 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 25 Gly Ser Arg Ile
Arg Thr Pro 1 5 26 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 26 Gly Gly Gly Ser Arg Ile Ser 1 5 27 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 27 Val Met
Gly Gly Val Val Ser 1 5 28 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 28 Tyr Gly Asn Asp Arg Arg Asn 1 5 29
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 29
Ser Gly Lys Asp Arg Arg Ser 1 5 30 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 30 Tyr Ile Cys Pro Gly Pro Cys
1 5 31 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 31 Ser Tyr Gln Ser Pro Gly Pro 1 5 32 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 32 Ala Ala Ala Gly
Ser Lys His 1 5 33 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 33 Gly Ser Arg Ile Arg Thr Pro 1 5 34 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 34 Ser Trp
Gly Ser Arg Ile Arg 1 5 35 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 35 Gly Gly Gly Ser Arg Ile Ser 1 5 36
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 36
Arg Val Val Gly Ser Arg Ser 1 5 37 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 37 Asp Gly Ser Thr Asn Leu Ser
1 5 38 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 38 Val Gly Ser Gly Arg Leu Ser 1 5 39 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 39 Thr Pro Lys Thr
Ser Val Thr 1 5 40 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 40 Arg Met Asp Gly Pro Val Arg 1 5 41 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 41 Arg Ala
Pro Gly Gly Val Arg 1 5 42 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 42 Val Gly Leu His Ala Arg Ala 1 5 43
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 43
Tyr Ile Arg Pro Phe Thr Leu 1 5 44 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 44 Leu Gly Leu Arg Ser Val Gly
1 5 45 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 45 Pro Ser Glu Arg Ser Pro Ser 1 5 46 5 PRT Artificial
Sequence Peptide (1)..(5) synthetic construct 46 Cys Ala Arg Ala
Cys 1 5 47 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 47 Thr Arg Glu Val His Arg Ser 1 5 48 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 48 Thr Arg Asn Thr
Gly Asn Ile 1 5 49 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 49 Phe Asp Gly Gln Asp Arg Ser 1 5 50 6 PRT
Artificial Sequence Peptide (1)..(6) synthetic construct 50 Trp Gly
Pro Lys Arg Leu 1 5 51 6 PRT Artificial Sequence Peptide (1)..(6)
synthetic construct 51 Trp Gly Glu Ser Arg Leu 1 5 52 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 52 Val Met
Gly Ser Val Thr Gly 1 5 53 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 53 Lys Gly Gly Arg Ala Lys Asp 1 5 54
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 54
Arg Gly Glu Val Leu Trp Ser 1 5 55 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 55 His Gly Gln Gly Val Arg Pro
1 5 56 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 56 Gly Leu Ala Lys Leu Ile Pro 1 5 57 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 57 His Leu Ile Ser
Asp Met Ser 1 5 58 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 58 Leu Gln His Trp Leu Leu Ser 1 5 59 6 PRT
Artificial Sequence Peptide (1)..(6) synthetic construct 59 Ala Leu
Val Leu Gln Gly 1 5 60 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 60 Thr Gly Val Ala Leu Gln Ser 1 5 61 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 61 Tyr Val
Gln Ser Arg Glu Gly 1 5 62 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 62 Pro Leu Phe Trp Pro Tyr Ser 1 5 63
4 PRT Artificial Sequence Peptide (1)..(4) synthetic construct 63
Asp Gly Ser Gly 1 64 4 PRT Artificial Sequence Peptide (1)..(4)
synthetic construct 64 Glu Gly Ser Gly 1 65 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 65 Ser Ser Pro Arg
Pro Gly Val 1 5 66 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 66 Asp Gly Tyr Pro Ala Ile Ala 1 5 67 5 PRT
Artificial Sequence Peptide (1)..(5) synthetic construct 67 Gly His
Ala Ile Glu 1 5 68 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 68 Ile Trp Ser Thr Ser Glu Arg 1 5 69 5 PRT
Artificial Sequence Peptide (1)..(5) synthetic construct 69 Tyr Arg
Leu Arg Gly 1 5 70 5 PRT Artificial Sequence Peptide (1)..(5)
synthetic construct 70 Tyr Arg Ala Arg Gly 1 5 71 5 PRT Artificial
Sequence Peptide (1)..(5) synthetic construct 71 Ser Gln Pro Leu
Gly 1 5 72 5 PRT Artificial Sequence Peptide (1)..(5) synthetic
construct 72 Ser Gln Pro Trp Gly 1 5 73 6 PRT Artificial Sequence
Peptide (1)..(6) synthetic construct 73 Gln Arg Leu Val Thr Pro 1 5
74 6 PRT Artificial Sequence Peptide (1)..(6) synthetic construct
74 Gln Val Leu Val Thr Pro 1 5 75 6 PRT Artificial Sequence Peptide
(1)..(6) synthetic construct 75 Gln Arg Leu Val His Pro 1 5 76 6
PRT Artificial Sequence Peptide (1)..(6) synthetic construct 76 Gln
Val Leu Val His Pro 1 5 77 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 77 Ile Thr Arg Trp Arg Tyr Leu 1 5 78
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 78
Ser Leu Gly Gly Met Ser Gly 1 5 79 6 PRT Artificial Sequence
Peptide (1)..(6) synthetic construct 79 Ser Gln Leu Ala Ala Gly 1 5
80 6 PRT Artificial Sequence Peptide (1)..(6) synthetic construct
80 Ser Leu Leu Ala Ala Gly 1 5 81 6 PRT Artificial Sequence Peptide
(1)..(6) synthetic construct 81 Ser Gln Leu Val Ala Gly 1 5 82 6
PRT Artificial Sequence Peptide (1)..(6) synthetic construct 82 Ser
Leu Leu Ala Ala Gly 1 5 83 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 83 Gly Leu Pro Ser Gly Leu Leu 1 5 84
7 PRT Artificial Sequence Peptide (1)..(7) synthetic construct 84
His Gly Gly Ser Ala Asn Pro 1 5 85 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 85 Ser Leu Glu Ala Phe Phe Leu
1 5 86 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 86 Cys Val Pro Glu Leu Gly His Glu Cys 1 5 87 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 87 Cys Glu
Leu Gly Phe Glu Leu Gly Cys 1 5 88 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 88 Cys Phe Phe Leu Arg Asp Trp
Phe Cys 1 5 89 95 PRT Gallus gallus 89 Cys Gln Pro Ala Met Ala Ala
Val Thr Leu Asp Glu Ser Gly Gly Gly 1 5 10 15 Leu Gln Thr Pro Gly
Gly Ala Leu Ser Leu Val Cys Lys Ala Ser Gly 20 25 30 Phe Thr Phe
Asn Ser Tyr Pro Met Gly Trp Val Arg Gln Ala Pro Gly 35 40 45 Lys
Gly Leu Glu Trp Val Ala Val Ile Ser Ser Ser Gly Thr Thr Trp 50 55
60 Tyr Ala Pro Ala Val Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly
65 70 75 80 Gln Ser Thr Val Arg Leu Gln Leu Ser Asn Leu Arg Ala Glu
Asp 85 90 95 90 92 PRT Gallus gallus 90 Cys Gln Pro Ala Met Ala Ala
Val Thr Leu Asp Glu Ser Gly Gly Gly 1 5 10 15 Leu Gln Thr Pro Gly
Gly Thr Leu Ser Leu Val Cys Lys Ala Ser Gly 20 25 30 Ile Ser Ile
Gly Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys 35 40 45 Gly
Leu Glu Tyr Val Ala Ser Ile Ser Gly Asp Gly Asn Phe Ala His 50 55
60 Tyr Gly Ala Pro Val Lys Gly Arg Ala Thr Ile Ser Arg Asp Asp Gly
65 70 75 80 Gln Asn Thr Val Thr Leu Gln Leu Asn Asn Leu Arg 85 90
91 95 PRT Gallus gallus 91 Cys Gln Pro Ala Met Ala Ala Val Thr Leu
Asp Glu Ser Gly Gly Gly 1 5 10 15 Leu Gln Thr Pro Gly Gly Thr Leu
Ser Leu Val Cys Lys Gly Ser Gly 20 25 30 Phe Ile Phe Ser Arg Tyr
Asp Met Ala Trp Val Arg Gln Ala Pro Gly 35 40 45 Lys Gly Leu Glu
Trp Val Ala Gly Ile Asp Asp Gly Gly Gly Tyr Thr 50 55 60 Thr Leu
Tyr Ala Pro Ala Val Lys Gly Arg Ala Thr Ile Thr Ser Arg 65 70 75 80
Asp Asn Gly Gln Ser Thr Val Arg Leu Gln Leu Asn Asn Leu Arg 85 90
95 92 96 PRT Gallus gallus 92 Ala Asn Gln Pro Trp Pro Pro Leu Thr
Leu Asp Glu Ser Gly Gly Gly 1 5 10 15 Leu Gln Thr Pro Gly Gly Ala
Leu Ser Leu Val Cys Lys Ala Ser Gly 20 25 30 Phe Thr Met Ser Ser
Tyr Asp Met Phe Trp Val Arg Gln Ala Pro Gly 35 40 45 Lys Gly Leu
Glu Phe Val Ala Gly Ile Ser Ser Ser Gly Ser Ser Thr 50 55 60 Glu
Tyr Gly Ala Ala Val Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn 65 70
75 80 Gly Gln Ser Thr Val Arg Leu Gln Leu Asn Asn Leu Arg Ala Glu
Asp 85 90 95 93 10 PRT Artificial Sequence Peptide (1)..(10)
synthetic construct 93 Cys Glu Gln Arg Gln Thr Gln Glu Gly Cys 1 5
10 94 10 PRT Artificial Sequence Peptide (1)..(10) synthetic
construct 94 Cys Ala Arg Leu Glu Val Leu Leu Pro Cys 1 5 10 95 9
PRT Artificial Sequence Peptide (1)..(9) synthetic construct 95 Tyr
Asp Trp Trp Tyr Pro Trp Ser Trp 1 5 96 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 96 Gly Leu Asp Thr Tyr Arg Gly
Ser Pro 1 5 97 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 97 Ser Asp Asn Arg Tyr Ile Gly Ser Trp 1 5 98 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 98 Tyr Glu
Trp Trp Tyr Trp Ser Trp Ala 1 5 99 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 99 Lys Val Ser Trp Tyr Leu Asp
Asn Gly 1 5 100 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 100 Ser Asp Trp Tyr Tyr Pro Trp Ser Trp 1 5 101
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 101
Ala Gly Trp Leu Tyr Met Ser Trp Lys 1 5 102 6 PRT Artificial
Sequence Peptide (1)..(6) synthetic construct 102 Cys Phe Gln Asn
Arg Cys 1 5 103 8 PRT Artificial Sequence Peptide (1)..(8)
synthetic construct 103 Cys Asn Leu Ser Ser Glu Gln Cys 1 5 104 10
PRT Artificial Sequence Peptide (1)..(10) synthetic construct 104
Cys Leu Arg Gln Ser Tyr Ser Tyr Asn Cys 1 5 10 105 10 PRT
Artificial Sequence Peptide (1)..(10) synthetic construct 105 Cys
Tyr Ile Trp Pro Asp Ser Gly Leu Cys 1 5 10 106 10 PRT Artificial
Sequence Peptide (1)..(10) synthetic construct 106 Cys Glu Pro Tyr
Trp Asp Gly Trp Phe Cys 1 5 10 107 10 PRT Artificial Sequence
Peptide (1)..(10) synthetic construct 107 Cys Lys Glu Asp Gly Trp
Leu Met Thr Cys 1 5 10 108 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 108 Cys Lys Leu Trp Gln Glu Asp Gly
Tyr 1 5 109 10 PRT Artificial Sequence Peptide (1)..(10) synthetic
construct 109 Cys Trp Asp Gln Asn Tyr Leu Asp Asp Cys 1 5 10 110 9
PRT Artificial Sequence Peptide (1)..(9) synthetic construct 110
Asp Glu Glu Gly Tyr Tyr Met Met Arg 1 5 111 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 111 Lys Gln Phe Ser
Tyr Arg Tyr Leu Leu 1 5 112 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 112 Val Val Ile Ser Tyr Ser Met Pro
Asp 1 5 113 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 113 Ser Asp Trp Tyr Tyr Pro Trp Ser Trp 1 5 114 8 PRT
Artificial Sequence Peptide (1)..(8) synthetic construct 114 Asp
Trp Phe Ser Tyr Tyr Glu Leu 1 5 115 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 115 Gly Gly Gly Ser Tyr Arg
His Val Glu 1 5 116 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 116 Arg Ala Ile Leu Tyr Arg Leu Ala Asn 1 5 117
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 117
Met Leu Leu Gly Tyr Arg Phe Glu Lys 1 5 118 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 118 Thr Met Leu Arg
Tyr Thr Val Arg Leu 1 5 119 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 119 Thr Met Leu Arg Tyr Phe Met Phe
Pro 1 5 120 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 120 Thr Leu Arg Lys Tyr Phe His Ser Ser 1 5 121 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 121 Thr
Leu Arg Lys Tyr Phe His Ser Ser 1 5 122 16 PRT Drosophila
melanogaster 122 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys 1 5 10 15 123 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 123 Cys Pro Arg Glu Cys Glu Ser Ile
Cys 1 5 124 11 PRT Artificial Sequence Peptide (1)..(11) synthetic
construct 124 Gly Ala Cys Val Arg Leu Ser Ala Cys Gly Ala 1 5 10
125 31 PRT Artificial Sequence Peptide (1)..(31) synthetic
construct 125 Cys Tyr Asn Leu Cys Ile Arg Glu Cys Glu Ser Ile Cys
Gly Ala Asp 1 5
10 15 Gly Ala Cys Trp Thr Trp Cys Ala Asp Gly Cys Ser Arg Ser Cys
20 25 30 126 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 126 Cys Leu Gly Gln Cys Ala Ser Ile Cys Val Asn Asp Cys 1
5 10 127 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 127 Cys Pro Lys Val Cys Pro Arg Glu Cys Glu Ser Asn Cys 1
5 10 128 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 128 Cys Gly Thr Gly Cys Ala Val Glu Cys Glu Val Val Cys 1
5 10 129 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 129 Cys Ala Val Ala Cys Trp Ala Asp Cys Gln Leu Gly Cys 1
5 10 130 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 130 Cys Ser Gly Leu Cys Thr Val Gln Cys Leu Glu Gly Cys 1
5 10 131 13 PRT Artificial Sequence Peptide (1)..(13) synthetic
construct 131 Cys Ser Met Met Cys Leu Glu Gly Cys Asp Asp Trp Cys 1
5 10 132 43 DNA Artificial Sequence misc_feature (1)..(43)
Oligonucleotide 132 taatacgact cactataggg caagctgata aaccgataca att
43 133 24 DNA Artificial Sequence misc_feature (1)..(24)
Oligonucleotide 133 ccctcatagt tagcgtaacg atct 24 134 22 DNA
Artificial Sequence misc_feature (1)..(22) Oligonucleotide 134
cctttctatt ctcactcggc cg 22 135 44 DNA Artificial Sequence
misc_feature (1)..(44) Oligonucleotide 135 caggaaacag ctatgaccgc
taaacaactt tcaacagttt cggc 44 136 17 DNA Artificial Sequence
misc_feature (1)..(17) Oligonucleotide 136 cactcggccg acggggc 17
137 21 DNA Artificial Sequence misc_feature (1)..(21)
Oligonucleotide 137 cagtttcggc cccagcggcc c 21 138 3 PRT Artificial
Sequence Peptide (1)..(3) synthetic construct 138 Gly Gly Leu 1 139
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 139
Cys Val Pro Gly Leu Gly Gly Leu Cys 1 5 140 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 140 Cys Gly Gly Leu
Asp Val Arg Met Cys 1 5 141 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 141 Cys Asp Gly Gly Leu Asp Trp Val
Cys 1 5 142 3 PRT Artificial Sequence Peptide (1)..(3) synthetic
construct 142 Leu Gly Gly 1 143 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 143 Cys Thr Trp Lys Gly Gly Arg Glu
Cys 1 5 144 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 144 Cys Ser Arg Trp Gly Leu Gly Gly Cys 1 5 145 3 PRT
Artificial Sequence Peptide (1)..(3) synthetic construct 145 Val
Arg Gly 1 146 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 146 Cys Val Gly Gly Val Arg Gly Gly Cys 1 5 147 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 147 Cys
Val Gly Asn Asp Val Arg Gly Cys 1 5 148 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 148 Cys Glu Ser Arg Leu Val
Arg Gly Cys 1 5 149 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 149 Cys Gly Gly Arg Pro Val Arg Gly Cys 1 5 150
3 PRT Artificial Sequence Peptide (1)..(3) synthetic construct 150
Ala Gly Gly 1 151 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 151 Cys Thr Pro Phe Ile Ala Gly Gly Cys 1 5 152
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 152
Cys Arg Glu Trp Met Ala Gly Gly Cys 1 5 153 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 153 Cys Ala Gly Gly
Ser Leu Arg Val Cys 1 5 154 3 PRT Artificial Sequence Peptide
(1)..(3) synthetic construct 154 Val Val Gly 1 155 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 155 Cys Glu Gly Val
Val Gly Ile Val Cys 1 5 156 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 156 Cys Asp Ser Val Val Gly Ala Trp
Cys 1 5 157 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 157 Cys Arg Thr Ala Val Val Gly Ser Cys 1 5 158 3 PRT
Artificial Sequence Peptide (1)..(3) synthetic construct 158 Val
Gly Gly 1 159 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 159 Cys Val Gly Gly Ala Arg Ala Leu Cys 1 5 160 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 160 Cys
Leu Ala His Arg Val Gly Gly Cys 1 5 161 3 PRT Artificial Sequence
Peptide (1)..(3) synthetic construct 161 Gly Gly Leu 1 162 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 162 Cys
Trp Ala Leu Ser Gly Gly Leu Cys 1 5 163 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 163 Cys Gly Gly Leu Val Ala
Tyr Gly Cys 1 5 164 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 164 Cys Gly Gly Leu Ala Thr Thr Thr Cys 1 5 165
3 PRT Artificial Sequence Peptide (1)..(3) synthetic construct 165
Gly Arg Val 1 166 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 166 Cys Gly Arg Val Asn Ser Val Ala Cys 1 5 167
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 167
Cys Ala Gly Arg Val Ala Leu Arg Cys 1 5 168 3 PRT Artificial
Sequence Peptide (1)..(3) synthetic construct 168 Gly Gly Ala 1 169
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 169
Cys Trp Asn Gly Gly Ala Arg Ala Cys 1 5 170 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 170 Cys Leu Asp Arg
Gly Gly Ala His Cys 1 5 171 3 PRT Artificial Sequence Peptide
(1)..(3) synthetic construct 171 Gly Val Val 1 172 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 172 Cys Glu Leu Arg
Gly Val Val Val Cys 1 5 173 3 PRT Artificial Sequence Peptide
(1)..(3) synthetic construct 173 Gly Gly Val 1 174 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 174 Cys Ile Gly Gly
Val His Tyr Ala Cys 1 5 175 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 175 Cys Gly Gly Val His Ala Leu Arg
Cys 1 5 176 4 PRT Artificial Sequence Peptide (1)..(4) synthetic
construct 176 Gly Met Trp Gly 1 177 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 177 Cys Ile Arg Glu Gly Met
Trp Gly Cys 1 5 178 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 178 Cys Ile Arg Lys Gly Met Trp Gly Cys 1 5 179
3 PRT Artificial Sequence Peptide (1)..(3) synthetic construct 179
Ala Leu Arg 1 180 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 180 Cys Glu Ala Leu Arg Leu Arg Ala Cys 1 5 181
3 PRT Artificial Sequence Peptide (1)..(3) synthetic construct 181
Ala Leu Val 1 182 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 182 Cys Ala Leu Val Asn Val His Leu Cys 1 5 183
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 183
Cys Ala Leu Val Met Val Gly Ala Cys 1 5 184 4 PRT Artificial
Sequence Peptide (1)..(4) synthetic construct 184 Gly Gly Val His 1
185 3 PRT Artificial Sequence Peptide (1)..(3) synthetic construct
185 Val Ser Gly 1 186 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 186 Cys Met Val Ser Gly Val Leu Leu Cys 1 5 187
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 187
Cys Gly Leu Val Ser Gly Pro Trp Cys 1 5 188 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 188 Cys Leu Tyr Asp
Val Ser Gly Gly Cys 1 5 189 3 PRT Artificial Sequence Peptide
(1)..(3) synthetic construct 189 Gly Pro Trp 1 190 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 190 Cys Ser Lys Val
Gly Pro Trp Trp Cys 1 5 191 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 191 Cys Gly Leu Val Ser Gly Pro Trp
Cys 1 5 192 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 192 Cys Ala His His Ala Leu Met Glu Cys 1 5 193 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 193 Cys
Glu Arg Pro Pro Phe Leu Asp Cys 1 5 194 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 194 Cys Val Pro Arg Arg Trp
Asp Val Cys 1 5 195 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 195 Cys Gln His Thr Ser Gly Arg Gly Cys 1 5 196
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 196
Cys Arg Ala Arg Gly Trp Leu Leu Cys 1 5 197 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 197 Cys Val Ser Asn
Pro Arg Trp Lys Cys 1 5 198 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 198 Cys Phe Asn Arg Thr Trp Ile Gly
Cys 1 5 199 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 199 Cys Ser Arg Gly Pro Ala Trp Gly Cys 1 5 200 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 200 Cys
Trp Ser Arg Gly Gln Gly Gly Cys 1 5 201 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 201 Cys His Val Leu Trp Ser
Thr Arg Cys 1 5 202 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 202 Cys Leu Gly Leu Leu Met Ala Gly Cys 1 5 203
9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct 203
Cys Met Ser Ser Pro Gly Val Ala Cys 1 5 204 9 PRT Artificial
Sequence Peptide (1)..(9) synthetic construct 204 Cys Leu Ala Ser
Gly Met Asp Ala Cys 1 5 205 9 PRT Artificial Sequence Peptide
(1)..(9) synthetic construct 205 Cys His Asp Glu Arg Thr Gly Arg
Cys 1 5 206 9 PRT Artificial Sequence Peptide (1)..(9) synthetic
construct 206 Cys Ala His His Ala Leu Met Glu Cys 1 5 207 9 PRT
Artificial Sequence Peptide (1)..(9) synthetic construct 207 Cys
Met Gln Gly Ala Ala Thr Ser Cys 1 5 208 9 PRT Artificial Sequence
Peptide (1)..(9) synthetic construct 208 Cys Met Gln Gly Ala Arg
Thr Ser Cys 1 5 209 9 PRT Artificial Sequence Peptide (1)..(9)
synthetic construct 209 Cys Val Arg Asp Leu Leu Thr Gly Cys 1 5 210
12 PRT Artificial Sequence Peptide (1)..(12) synthetic construct
210 Cys Leu Ser Arg Leu Val Thr Gly Asp Val Ile Cys 1 5 10 211 12
PRT Artificial Sequence Peptide (1)..(12) synthetic construct 211
Cys Gly Asn Met Gly Gly Ser Leu Tyr Tyr Val Cys 1 5 10 212 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 212 Cys
Leu His Trp Glu Ala Thr Phe Asn Pro Gln Cys 1 5 10 213 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 213 Cys
Arg Thr Glu Val Trp Arg Ser Asn Gln Arg Cys 1 5 10 214 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 214 Cys
His Val Arg Asp Glu His His Glu Gln Gly Cys 1 5 10 215 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 215 Cys
Pro Met Gln Ala Thr Arg Asn Leu Trp His Cys 1 5 10 216 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 216 Cys
Arg Asp Asp Ala Lys Val Met Arg Tyr Asn Cys 1 5 10 217 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 217 Cys
Asn Asn Trp Gly Glu Leu Leu Gly Phe Asn Cys 1 5 10 218 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 218 Cys
Glu Gly Gly Tyr Glu Asn Leu Val Leu Lys Cys 1 5 10 219 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 219 Cys
Arg Asn Ala Trp Asn Lys His Gly Ser Arg Cys 1 5 10 220 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 220 Cys
Lys Glu Arg Met Tyr Arg Glu Gln Arg Arg Cys 1 5 10 221 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 221 Cys
Arg Thr Ile Asp Ile Glu Asn Asn Glu Leu Cys 1 5 10 222 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 222 Cys
His Arg Gly Ile Asn Arg Ser Thr Thr Asp Cys 1 5 10 223 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 223 Cys
Glu Thr Gly Arg Glu Ile Asp Arg Ser Asp Cys 1 5 10 224 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 224 Cys
Cys Gly Arg Lys Thr Arg Gly Val Ala Ile Cys 1 5 10 225 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 225 Cys
Leu Ala Ser Met Leu Asn Met Ser Thr Leu Cys 1 5 10 226 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 226 Cys
Gly Gln Gly Phe Ala Pro Arg Asn Leu Val Cys 1 5 10 227 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 227 Cys
Leu Gly Lys Trp Lys Ser Ser Arg Gly Thr Cys 1 5 10 228 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 228 Cys
Gly Glu Gly Phe Gly Ser Glu Trp Pro Pro Cys 1 5 10 229 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 229 Cys
Lys Pro Asp Tyr Met Asp Ser Asn Lys Met Cys 1 5 10 230 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 230 Cys
Thr Arg Asn Ile Thr Lys Ser Arg Met Met Cys 1 5 10 231 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 231 Cys
Val Arg Asn Val Asp Gln Asn Thr Asn Thr Cys 1 5 10 232 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 232 Cys
Phe Trp Thr Arg Glu Asn Arg Gly Trp Thr Cys 1 5 10 233 12 PRT
Artificial Sequence Peptide (1)..(12) synthetic construct 233 Cys
Arg Ile Arg Gly Ile Gln Leu Arg Pro Ala Cys 1 5 10 234 13 PRT
Artificial Sequence Peptide (1)..(13) synthetic construct 234 Cys
Glu Val Gly Leu Ser Ala Ala Met Ala Tyr Cys Cys 1 5 10 235 5 PRT
Artificial Sequence misc_feature (3)..(3) Unidentified amino acid
235 Leu Arg Xaa Gly Asn 1 5 236 4 PRT Artificial Sequence Peptide
(1)..(4) synthetic construct 236 Arg Gly Ala Gly 1 237 4 PRT
Artificial Sequence Peptide (1)..(4) synthetic construct 237 Asp
Leu Leu Arg 1 238 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 238 Gly Val Met Leu Arg Arg Gly 1 5 239 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 239 Tyr
Ser Leu Arg Ile Gly Leu 1 5 240 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 240 Leu Arg Asp Gly Asn Gly Glu 1 5
241 8 PRT Artificial Sequence Peptide (1)..(8) synthetic construct
241 Cys Leu Arg Gly Gly Asn Leu Arg 1 5 242 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 242 Val Arg Gly Leu
Ala Ala Ala 1 5 243 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 243 Ala Arg Gly Ala Gly Leu Ala 1 5 244 8 PRT
Artificial Sequence Peptide (1)..(8) synthetic construct 244 Arg
Gly Ala Gly Thr Gly Trp Thr 1 5 245 7 PRT Artificial Sequence
Peptide (1)..(7) synthetic construct 245 Ala Arg Gly Val Asn Gly
Ala 1 5 246 7 PRT Artificial Sequence Peptide (1)..(7) synthetic
construct 246 Asp Leu Leu Arg Ala Arg Trp 1 5 247 7 PRT Artificial
Sequence Peptide (1)..(7) synthetic construct 247 Asp Leu Leu Arg
Thr Glu Trp 1 5 248 7 PRT Artificial Sequence Peptide (1)..(7)
synthetic construct 248 Glu Phe Asp Leu Val Arg Gln 1 5 249 7 PRT
Artificial Sequence Peptide (1)..(7) synthetic construct 249 Gly
Cys Asp Glu Gly Gly Gly 1 5 250 7 PRT Artificial Sequence Peptide
(1)..(7) synthetic construct 250 Gly Asp Ser Pro Val Glu Ser 1 5
251 9 PRT Artificial Sequence Peptide (1)..(9) synthetic construct
251 Cys Pro Pro Leu Gly Gly Ser Arg Cys 1 5
* * * * *
References