U.S. patent application number 12/516896 was filed with the patent office on 2010-08-19 for metastasis-specific peptides and their diagnostic and therapeutic applications.
This patent application is currently assigned to UNIVERSITA DEGLI STUDI DI TORINO. Invention is credited to Federico BUSSOLINO, Serena MARCHIO.
Application Number | 20100210563 12/516896 |
Document ID | / |
Family ID | 39313019 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210563 |
Kind Code |
A2 |
BUSSOLINO; Federico ; et
al. |
August 19, 2010 |
METASTASIS-SPECIFIC PEPTIDES AND THEIR DIAGNOSTIC AND THERAPEUTIC
APPLICATIONS
Abstract
The present invention concerns peptide sequences that
specifically recognize cells of human hepatic metastases. The
invention comprises also the use of nucleic acids coding for such
peptides, as well as conjugates and formulations of such peptides
for diagnostic and therapeutic purposes.
Inventors: |
BUSSOLINO; Federico;
(Torino, IT) ; MARCHIO; Serena; (Torino,
IT) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
UNITED STATES
2027836040
2027836031
PTO-PAT-Email@rfem.com
|
Assignee: |
UNIVERSITA DEGLI STUDI DI
TORINO
Via Verdi 8
Torino
IT
I-10100
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100041614 A1 |
February 18, 2010 |
|
|
Family ID: |
39313019 |
Appl. No.: |
12/516896 |
Filed: |
November 30, 2007 |
PCT Filed: |
November 30, 2007 |
PCT NO: |
PCT/EP2007/010428 |
371 Date: |
May 29, 2009 |
Current U.S.
Class: |
514/1.1;
435/235.1; 435/7.21; 514/13.3; 530/317; 530/329; 536/23.1 |
Current CPC
Class: |
A61K 47/642 20170801;
A61P 35/04 20180101; A61K 47/64 20170801; A61K 49/14 20130101 |
Class at
Publication: |
514/016; 530/329;
530/317; 435/235.1; 536/023.1; 435/007.21 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 7/06 20060101 C07K007/06; C07K 7/64 20060101
C07K007/64; C12N 7/00 20060101 C12N007/00; C07H 21/04 20060101
C07H021/04; G01N 33/567 20060101 G01N033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2006 |
IT |
TO2006A000852 |
Claims
1.-36. (canceled)
37. A peptide capable of selectively binding to metastatic cells
having the sequence motif LRS, a length of 6 to 100 amino acids and
comprising an amino acid sequence selected from the group composed
by: ARPGLRS (SEQ ID NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID
NO. 3), LRSGSGS (SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS
(SEQ ID NO. 7), LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110),
SWYTLRS (SEQ ID NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID
NO. 115), VRPGLRS (SEQ ID NO. 117), LRSGRGS (SEQ ID NO. 119).
38. A peptide of claim 37, wherein said metastatic cells are human
hepatic metastasis cells.
39. A peptide of claim 37, which is a cyclic peptide.
40. A peptide of claim 37, which comprises at least a modified
amino acid, an unusual amino acid and/or an amino acid in
D-conformation.
41. A conjugate comprising at least a peptide capable of
selectively binding to metastatic cells of claim 37, and at least
one molecule.
42. A conjugate of claim 41, wherein said at least one molecule is
selected among a drug, a chemotherapic agent, a radioisotope, a
pro-apoptotic agent, an anti-angiogenic agent, an hormone, a
cytokine, a cytotoxic agent, a cytostatic agent, a peptide, a
protein, an antibody, an antibody fragment such as a Fab
fragment
43. A conjugate of claim 42, wherein said anti-angiogenic agent is
selected from the group consisting of thrombospondin, angiostatin,
pigment epithelium-derived factor, angiotensin, laminin peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12 (IL-12),
platelet factor 4, IP-10, 2-methoxyoestradiol, proliferin-related
protein, carboxiamidotriazole, CM101, Marimastat, pentosan
polysulphate, angiopoietin 2, interferon-alpha, herbimycin A,
PNU145156E, 16K prolactin fragment, Linomide, thalidomide,
pentoxifylline, genistein, TNP-470, endostatin, paclitaxel,
Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor,
a signaling peptide, accutin, cidofovir, vincristine, bleomycin,
AGM-1470, platelet factor 4 and minocycline.
44. A conjugate of claim 42, wherein said pro-apoptotic agent is
selected from the group consisting of etoposide, ceramide
sphingomyelin, Bax, Bid, Bik, Bad, caspase-3, caspase-8, caspase-9,
fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper, apoptin,
interleukin-2 converting enzyme and annexin V.
45. A conjugate of claim 42, wherein said cytokine is selected from
the group consisting of 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), and GM-CSF
(granulocyte macrophage colony stimulating factor).
46. A conjugate of claim 41, wherein said at least one molecule is
selected from a virus, a bacteriophage, a bacterium, a liposome, a
microparticle, a magnetic bead, a nanoparticle, a yeast cell, and a
mammalian cell.
47. A conjugate of claim 46, wherein said virus is selected from an
adenovirus, a retrovirus, an adeno-associated virus, and a
lentivirus.
48. A conjugate of claim 41, wherein said at least one molecule is
a diagnostic agent.
49. A conjugate of claim 48, wherein said diagnostic agent is a
diagnostic agent for in vivo use.
50. A conjugate of claim 49, wherein said diagnostic agent is
selected from paramagnetic ions or radioisotopes.
51. A conjugate of claim 48, wherein said diagnostic agent is a
diagnostic agent for in vitro assays.
52. A nucleic acid coding for a peptide capable of selectively
binding to metastatic cells having the sequence motif LRS, a length
of 6 to 100 sequence amino acids and comprising at least one amino
acid sequence selected from the group composed by: ARPGLRS (SEQ ID
NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS
(SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7),
LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID
NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115),
VRPGLRS (SEQ ID NO. 117), LRSGRGS (SEQ ID NO. 119).
53. A formulation comprising at least one peptide capable of
specifically binding to metastatic cells of claim 37.
54. A formulation of claim 53, wherein said at least one peptide is
conjugated with a drug.
55. A formulation of claim 54, wherein said drug is a therapeutic
agent capable of having a cytotoxic, cytostatic, pro-apoptotic, or
anti-angiogenic effect on hepatic metastasis cells.
56. A formulation of claim 54, wherein said drug is an alkylating
agent, an anti-metabolite, or an antibiotic.
57. A formulation of claim 54, wherein said at least one peptide is
conjugated with a diagnostic agent.
58. A formulation of claim 57, wherein said diagnostic agent is a
diagnostic agent for in vivo use.
59. A formulation of claim 57, wherein said diagnostic agent is a
diagnostic agent for in vitro assays.
60. A formulation of claims 53, which is a pharmaceutical
formulation.
61. A formulation of claim 53, which includes at least one
acceptable carrier and/or excipient.
62. Use of a peptide of claim 53 for the manufacture of a
diagnostic formulation for the localization of metastatic cells in
a subject with a tumor, particularly with a colon tumor.
63. Use of claim 62, wherein said metastatic cells are hepatic
metastasis cells.
64. Use of claim 62, wherein said formulation is for in vivo
use.
65. Use of claim 62, wherein said formulation is for in vitro
assays.
66. Use of a peptide of claim 37 for the manufacture of a
medicament for the anti-tumor therapy in a tumor-bearing
subject.
67. Use of a nucleic acid of claim 52 for the manufacture of a
medicament for the anti-tumor therapy in a tumor-bearing
subject.
68. Use of claim 67, wherein said anti-tumor therapy is a gene
therapy.
69. A process for obtaining a peptide capable of selectively
binding to a metastatic cell, which has a sequence motif LRS and
which has a length of 6 to 100 amino acids, whereby the process
comprises (1) contacting the metastatic cell or a tissue containing
metastatic cells with a plurality of phages, where each phage
presents heterologous peptide sequences incorporated into a capsid
protein, (2) removing phages that do not bind to the cells or
tissues, (3) isolating the phages that bind the cell or tissue, and
optionally (4) identifying the heterologous peptide sequences.
70. A process of claim 69, wherein said metastatic cells are
hepatic metastatic cells.
71. A process of claim 70, wherein said hepatic metastatic cells
are derived from a primary colorectal tumor.
72. A process of claim 69, wherein said peptide comprises at least
one sequence selected from the group composed by: ARPGLRS (SEQ ID
NO. 1), MRYALRS (SEQ ID NO. 2), LRPGLRS (SEQ ID NO. 3), LRSGSGS
(SEQ ID NO. 4), GIYRLRS (SEQ ID NO. 6), GVYSLRS (SEQ ID NO. 7),
LRSGRGS (SEQ ID NO. 96), RREGLRS (SEQ ID NO. 110), SWYTLRS (SEQ ID
NO. 111), LAYRLRS (SEQ ID NO. 113), LTYRLRS (SEQ ID NO. 115),
VRPGLRS (SEQ ID NO. 117), LRSGRGS (SEQ ID NO. 119).
Description
STATE-OF-THE ART OF THE INVENTION
[0001] The present invention comprises peptides that are highly
specific for tumor metastatic cells, in particular cells of hepatic
metastases, and their application in the diagnostic and therapeutic
fields.
TECHNICAL BACKGROUND OF THE INVENTION
Tumor and Metastatization
[0002] Tumorigenesis is a multi-stage process in which some cells
progressively evolve toward malignity. The actual knowledge in the
field of neoplasia underlines that cancer is a disease induced by
dynamic changes of the genome. Through these variations, tumor
cells acquire independence from various mechanisms that control the
physiological functions of the organism. As a consequence, they
become able of (1) growing continuously, (2) inducing the
recruitment of endothelial cells for the formation of new blood
vessels and (3) colonizing organs different from that of
origin.
[0003] The proliferative capability of tumor cells is fundamentally
due to two mechanisms. First, while normal cells need mitogenic
factors to switch from a quiescent condition to an active,
proliferating one, in tumor cells mutations and/or overexpression
of growth factor receptors can induce the proliferation cascade
independently from the presence of a ligand. Moreover, in many
cases, tumor cells acquire the ability to synthesize soluble
factors they are sensitive to. So, an autocrine stimulation is set
up, which further enhances the tumor growth. The other phenomenon
of deregulation of the cell growth in tumors is the resistance to
apoptosis (programmed cell death). This mechanism, fundamental in
the growth and remodeling of organs during the physiological
development, is induced also in the case of non-reversible genome
damages, to avoid the expansion of aberrant cell population. Some
cells, however, can escape this kind of protection and become
independent, thus favoring the propagation of mutations and the
consequent neoplastic progression.
[0004] A primordial tumor mass is constituted by a small cell
number, and could not develop over 2 mm in diameter if not
sustained by adequate feed and oxygen support. This is the phase in
which angiogenesis, the formation of new blood capillaries from
pre-existing blood vessels, is strongly stimulated by tumor cells
themselves. The unbalance between positive and negative signals of
the angiogenesis regulation leads to the neo-formation of a
vascular network that penetrates and feeds the actively
proliferating tumor mass. Tumor blood vessels, besides providing
the nutrition, have the function of carrying malignant cells toward
other body districts.
[0005] Tumor progression evolves toward an irreversibility stage,
whose characteristic feature is metastatization. In this process,
pioneer cells escape from the primary tumor mass. Once entered into
the capillaries that fill the tumor, they reach the bloodstream,
which carries them in regions distant from the site of derivation,
where they will give raise to a secondary tumor.
[0006] The development of metastasis represents a complex
biological event, related to the interactions between intrinsic
factors of the organism (general conditions, integrity of the
immune response) and specific features of tumor cells
(localization, size and histological patterns). From the
microscopic primary site, the diffusion of tumor cells is first
local, through a centrifugal spreading. By producing proteases that
degrade the intercellular connections and the extracellular matrix,
tumor cells invade anatomic structures and tissues that are
scarcely resistant (fat tissue, nerve sheaths, bone marrow).
[0007] A first obstacle to the metastatic diffusion is offered by
the presence of relatively impenetrable structures, such as the
organ capsules, cartilage or periostium, the meninx. Due to the
difficulty of going beyond these boundaries, metastatization in
distant sites must follow steps that can be summarized as follows:
(1) entrance into the tumor capillary network by a mechanism called
"intravasation", (2) transport through the bloodstream, (3)
specific recognition of the destination endothelium, (4) exit from
the capillary by a mechanism called "extravasation" and (5)
metastasis development, supported by active angiogenesis.
[0008] The success of dissemination depends on the anatomical
features and on the hemodynamic factors of the host organism, and
on the interactions that tumor cells undergo with the endothelium
lining the blood vessels. The most common pathways of diffusion are
the vessels (lymphatic and blood) and the celomatic cavities.
Lymphatic vessels are quite easily penetrated, because of the
absence of a basal lamina. So, tumor cells can easily transit into
the lymph nodes, before entering the venous system through the
lymphatic-venous connections. The transport into the vessels can
affect both arterious and venous system, even if the venous
invasion is more common, because the venous circulation collects
the flux exiting from organs. Typical examples are the systemic
vein for the lung or the port vein for the liver. The
trans-celomatic dissemination instead concerns the pleural cavity
of the chest and the peritoneal spaces of the abdomen and pelvis.
The most commonly involved site is peritoneum, where, after pouring
liquids due to the obstruction of the hepatic veins, tumor cells
are collected in the ascitic fluid. Stomach, colon, pancreas and
ovary cancers usually take this system.
[0009] Tumor metastatic cells express specific molecular
determinants that contribute in various ways to the metastasis
itself. The distribution of metastasis is not casual, but each
tumor has preferential addresses, this is known as organo-tropism.
Liver is a target organ for colorectal tumors; bones for prostate
and ovary tumors; lungs for testis, bone and breast tumors. Lungs
and liver, due to their filter function and to the presence of a
huge number of capillaries, can receive metastases virtually from
every organ and also send tumor colonies, mainly toward brain and
bones.
[0010] The liver is a common site for metastatic lesions. The
reason has to be searched in the functional and structural
organization of the hepatic district. The port vein, which drains
the blood to the abdominal viscera, represents the conduct through
which the cells coming from the primary tumors are veiculated to
the liver. The adhesion of circulating tumor cells to liver
endothelium is a critical step for the beginning of
metastatization. Hepatic metastases develop as a consequence of the
invasion of the hepatic parenchyma by these cell thrombi.
[0011] The high volume of hepatic blood flux (about 25% of the
cardiac flux), and the particular microscopic anatomy of the
sinusoids are the factors that favor the hepatic dissemination. The
primary tumor may be localized in the gastro-intestinal tract, i.e.
colon, rectum, stomach, pancreas, biliary tract and bowel. To
those, also tumors of the breast and lung may be added.
Colorectal Tumor
[0012] Different kinds of classification exist that, in general,
divide the progressive evolution of the disease in steps
characterized by the degree of body invasion of that tumor. The
Dukes and MAC (Modified Astler-Coller) classifications, proposed at
the beginning of the clinical studies, are now the less used.
Generally, the TNM (Tumor Node Metastasis) classification is
preferred, which includes four successive stages: [0013] stage I:
tumor limited to the mucosa and the sub-mucosa; [0014] stage II:
extension to deeper layers of the intestinal wall; [0015] stage
III: invasion of sub-sierosa and lymph nodes; [0016] stage IV:
metastasis.
[0017] The therapeutic approaches more common by now are surgery,
chemotherapy and radiotherapy. The kind of clinical strategy is
chosen based on the stage in which the pathology is. In general,
the following protocols are used: [0018] stage I: surgery
(colostomy); [0019] stage II: surgery can be associated to
chemotherapy; [0020] stage III: surgery is in any case associated
with chemotherapy; [0021] stage IV: palliative treatment with
surgery and/or chemotherapy.
[0022] Liver is the most frequent site of colonization by primary
colorectal cancer. Currently, the only treatment with a curative
potential is surgical removal of metastases. However, despite the
increasingly effective means of the hepatic surgery, most patients
with liver metastases are not amenable for surgery, because of the
extension of their tumor mass.
A Different Approach to Cancer Therapy: Attacking Tumor Blood
Vessels
[0023] The chemotherapic drugs currently used are between the drugs
with the most narrow therapeutic window in the whole medical field.
As a consequence, the dose of antitumor drugs that can be
administered is limited by the toxic effects on normal tissues.
This difficulty can be overcome by targeting cytotoxic drugs to the
tumor itself. Even if this has been a goal for long time in cancer
biology and in oncological medicine, right now only few examples
are known in which the target administration of a drug is possible.
For example, the use of monoclonal antibodies against tumor
antigens had a limited success, since only a few tumor antigens are
known and generally antibodies poorly penetrate into tissues.
Moreover, since tumor cells are genetically instable and
growth-advantageous mutations accumulate, tumor cell-targeted
treatments are generally followed by clonal selection of resistant
cells.
[0024] The targeting of therapy to the tumor vascular network
allows to overcome some of the problems related to traditional
therapy. Endothelial cells in the tumor vascular system express
molecules peculiar of anogiogenic vessels. Vascular targeting
offers several advantages. First, endothelial lining is easily
accessible. On the contrary, a tumor-targeted drug needs to diffuse
on long distances, penetrate into tightly bound tumor cells and in
a very dense stroma, and contrast a very high interstitial
pressure. Second, since tumor cells depend on blood supply for
their growth, a tumor therapy addressed to the vessels does not
need to lead to the destruction of all the endothelial cells.
Indeed, endothelium-target therapy has an intrinsic amplification
mechanism. Finally, since endothelial cells are diploid and not
transformed, it is improbable that they loose the expression of a
surface receptor or acquire drug resistance through mutations and
clonal evolution. Some endothelial markers have been recently
identified. Among these molecules there are some integrins,
particularly .alpha.v.beta.3 and .alpha.v.beta.5 and endothelial
tyrosine kinase receptors with their cognate ligands (VEGF
receptors and the various VEGFs, Tie1, Tie2 and angiopietins).
Peptides that Target a Mouse Model of Human Tumor: Discovery of
Tumor Endothelial Markers
[0025] By phage display studies performed in vivo in different
animal models peptide sequences have been identified which are able
to selectively target tumor vascularization. These sequences proved
to be a valid instrument to characterize tumor endothelium and its
specific molecular determinants, and to develop biotechnological
applications in tumor therapy.
[0026] In this way, recurrent peptide sequences have been
identified, such as RGD (Arginin-Glycin-Aspartic acid) and NGR
(Asparagin-Glycin-Arginin). The RGD motif is embedded in the
sequence of several proteins of the extracellular matrix and
represents their interaction site with integrins. A phage that
presents the CDRGDCFC (SEQ ID NO: 203) sequence, named RGD-4C, is
able to specifically target breast tumors, and to selectively bind
the .alpha.v.beta.3 and .alpha.v.beta.5 integrins. In vitro
experiments demonstrated that RGD-containing peptides inhibit
cell-cell adhesion thus inducing apoptosis. So, it has been thought
that the RGD peptide, without further modification, can act as an
antiangiogenic drug, leading to cell death after disruption of the
cell-matrix interactions. Also the NGR peptide binds integrins,
even if with minor affinity compared to RGD. The specific receptor
for the NGR sequence has been successively identified in another
membrane protein, aminopeptidase N (APN), overexpressed in vascular
structures in active angiogenesis and not detectable in quiescent
endothelium. It has been demonstrated that APN specific antibodies
can inhibit retinal neovascularization induced by hypoxia in the
mouse. In the same way, mice treated with anti-APN antibodies have
breast tumors strongly regressed compared to the control group.
[0027] In another set of studies peptides that specifically bind
the NG2 proteoglycan have been identified, a mouse homolog of HMP
(human melanoma proteoglycan), also known as Molecular Weight
Melanoma-Associated Antigen. This proteoglycan is mainly expressed
by glial progenitor cells, skeletal muscle and cartilage. After the
differentiation, the NG2 surface expression is lost. In adults, its
presence is limited to vessels in active angiogenesis in some tumor
kinds, among which glioblastoma, condrosarcoma, melanoma, and some
leukemias. In a nude mice bearing a malignant melanoma, an anti-NG2
antibody conjugated with doxorubicin suppresses tumor growth.
Peptides as Antitumor Drugs
[0028] Remodeling of the extracellular matrix is common both to
endothelial activation and neoplastic invasion, and need the action
of particular enzymes called Matrix Metallo-Proteases (MMP). These
proteases, overexpressed in the tumor, are almost absent in normal
tissues, except in cell migration and tissue remodeling events
during morphogenesis. Synthetic inhibitors of two such proteases,
MMP-2 (Gelatinase A; 72 Kd) and MMP-9 (Gelatinase B; 92 Kd), which
are the more strictly involved proteases in angiogenesis and
metastatic potential, have been isolated by phage display. From
this study, it has been shown that the most represented clones
express the LRSGRG (SEQ ID NO: 204) sequence derived from a CX6C
library. Another protein family, identified from a CX9 collection,
is the one with the HWGF (SEQ ID NO: 205) motif. Soluble peptides
containing the HWGF (SEQ ID NO: 205) motif show in vitro inhibitory
activity against MMP-9. These peptides inhibit the migration of
tumor cell lines and of endothelial cells derived from human
umbilical cord. In vivo, they are efficient in inhibiting tumor
growth and in preventing the appearance of metastases.
Use of Peptides in Biotechnologically Innovative Antitumor
Therapies
[0029] As described previously, peptides specifically associated to
tumor endothelial markers or tumor cells have been successfully
employed in therapeutic protocols in the mouse. A second approach
has been investigated, conjugating RGD-4C and CNGRC (SEQ ID NO:
207) peptides to the chemotherapic drug doxorubicin, and using this
compound for the treatment of breast tumors in mice. Animals
subjected to this therapy survived up to six months, demonstrating
that this compound is able to inhibit both primary tumor and
metastasis development with higher efficacy and lower toxicity
compared to systemic administration.
[0030] In a third set of applications, chimeric peptides have been
made, which possess two functional domains. The former can
selectively bind to the target cell and be internalized; the latter
is pro-apoptotic, non toxic in body fluids but only in the
intracellular environment. More than 100 peptides exist that act
causing the destruction of mitochondrial membranes and induction of
apoptosis. Among these, a 14 aa sequence has been selected,
KLAKLAKKLAKLAK (SEQ ID NO: 206), which demonstrated to have a
strong antibiotic potential in the form of D-enantiomer. The
peptides RGD-4C and CNGRC (SEQ ID NO: 207) have been coupled to
this peptide. It has been found that these compounds cause
mitochondrial alterations and lead to morphological variations
typical of an apoptotic status, such as condensation and
fragmentation of the nuclear structures. These results have been
confirmed in vivo: mice to which the antitumor agent has been
administered show tumors of reduced size and survive for several
months.
GENERAL DESCRIPTION OF THE INVENTION
[0031] The appearance of metastases is a prognostic factor
unfavorable in tumor progression. So, it is fundamental to develop
methods that allow to detect and attack early (also sub-clinical
dimensioned) metastases. In most cases, the histopathological
methods currently employed for diagnosis allow to follow the
localization of metastases when they are no longer treatable.
Further, from a therapeutic point of view, present approaches are
limited mainly due to the unspecific toxicity of chemotherapic
drugs.
[0032] Metastatic cells have peculiar characteristic, compared both
to the primary tumors and to the tissues in which they localize. In
the same way, tumor blood vessel cells (endothelial cells) are
different from normal quiescent ones. In particular, significant
modifications involve the cell surfaces, on which molecules are
expressed or modified to favor the adaptation to the new
environment. Classical methods of study, however, have been proven
inefficient to front the problem of the multiplicity of these
modifications.
[0033] The present invention has the aim of providing a solution to
overcome the deficiencies of the state of the art.
[0034] According to the present invention, this aim is achieved by
peptides as defined in the appended claims, particularly with
peptides comprising a sequence as shown in SEQ ID NO: 1-201. The
invention also concerns the use of such peptides in the therapeutic
and diagnostic field. The appended claims are part of the technical
advance given here in relation to the invention.
[0035] Preferably, the present invention concerns peptides that
specifically recognize hepatic metastatic cells. The invention also
concerns the use of conjugates and formulations of such peptides,
when they are bound to a diagnostic agent (for example, a label) or
to a therapeutic agent (for example, a chemotherapic, a radioactive
isotope, a toxin), respectively, for the localization both in vitro
and in vivo of hepatic metastatic cells and for the therapy in a
tumor bearing subject.
[0036] The present invention can provide peptides with high binding
selectivity toward metastatic cells, particularly to hepatic
metastatic cells thus allowing an efficient localization of such
cells both in vitro and in vivo, so that they can be successfully
employed both for diagnosis and for therapy of tumors that
metastasize in the liver, more particularly, primary colorectal
cancers.
[0037] Some peptides of the invention share common sequence motifs,
such as GGG, RGL, GRL, GSG, LGR, GLS, SAD, YED, LRS and/or GSGS
(SEQ ID NO: 208). A preferred common sequence motif is LRS.
[0038] Beyond the therapeutic approach, peptides that selectively
recognize hepatic metastatic cells represent a useful mean to
identify metastases themselves. The small size of these peptides is
very advantageous for this kind of application. For example,
radionuclide- or fluorescent-conjugated peptides according with the
present invention can be used in patients with occult tumors or
with non-specific radiological results. Moreover, they can be used
for in vivo applications, such as for example magnetic resonance or
TAC, after conjugation with suitable molecules for their
visualization by any known visualization technique, particularly a
technique suitable for an individual body region.
[0039] Details on formulation techniques and conjugate
administration are known in the art and do not need a detailed
description here, being dispensable for the understanding of the
invention.
[0040] The inventors of the present invention used a proteomic
approach (phage display) to characterize the molecular determinants
expressed on the surface of cells derived from human hepatic
metastases secondary to colorectal carcinomas. Such technique
allowed the isolation of peptides that can interact with membrane
molecules exclusively present on these cells. The identification of
peptides that recognize molecular determinants not present in
normal tissues or in the primary tumor allows to use such peptides
both for diagnostic and therapeutic applications. From a diagnostic
point of view, these peptides, suitably labeled, can be used for
the detection of hepatic metastases also in pre-clinical stages.
From a therapeutic point of view, it is possible to conjugate them
with chemotherapeutic drugs in order to set up protocols of target
therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 illustrates the sequences of the peptides that bind
the hepatic metastatic cells according with the present invention,
particularly the SEQ ID NO:1-7 represent the peptides that have
been deeply studied, selected in the experiments on patients 16, 17
and 18; SEQ ID NO:8-19 represent the peptides selected in the III
round of selection on sample from patient 2; SEQ ID NO:20-39
represent the peptides selected in the II round of selection on
sample from patient 6; SEQ ID NO:40-64 represent the peptides
selected in the III round of selection on sample from patient 7;
SEQ ID NO:65-78 represent the peptides selected in the II round of
selection on sample from patient 8; SEQ ID NO:79-95 represent the
peptides selected in the II round of selection on sample from
patient 16; SEQ ID NO:96-107 represent the peptides selected in the
III round of selection on sample from patient 16; SEQ ID NO:108-109
represent the peptides selected in the III round of selection on
sample from patient 17; SEQ ID NO:110-118 represent the peptides
selected in the III round of selection on sample from patient 18;
SEQ ID NO:119-122 represent the peptides selected in the IV round
of selection on sample from patient 19; SEQ ID NO:123-140 represent
the peptides selected in the IV round of selection on sample from
patient 9; SEQ ID NO:141-152 represent the peptides selected in the
IV round of selection on sample from patient 21; SEQ ID NO:153-170
represent the peptides selected in the II round of selection on
sample from patient 23; SEQ ID NO:171-186 represent the peptides
selected in the IV round of selection on sample from patient 5; SEQ
ID NO:187-201 represent the peptides selected in the II round of
selection on sample from patient 8.
[0042] FIG. 2 illustrates the nucleotide sequence of the primer
used for sequencing the oligonucleotide insert in the phage
DNA;
[0043] FIG. 3 illustrates a picture of the polyacrylamide gel in
which proteins bound to the peptide GIYRLRS (SEQ ID NO: 6) fused to
a GST sequence have been separated.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The invention will be now described in details, as a
non-limiting example.
[0045] Peptides identified in this invention can be used as
molecular tools both in diagnostic and in therapeutic fields. It is
well known that actual therapeutic approaches in clinical oncology
are characterized by low selectivity. A chemotherapeutic agent
circulating into the bloodstream affects, other than the tumor
masses, all the body cell populations in active proliferation. On
the contrary, a peptide that is specifically recognized by surface
receptors specific for a particular cell type will be able to
address a chemotherapeutic drug preferentially to that kind of
cells. Peptides described in the present invention can therefore be
successfully employed as drug targeting agent to hepatic
metastases.
[0046] Moreover, peptides labeled with a detection molecule can be
used in the diagnostic field. Presently, detection techniques are
used that allow the resolution of a very precocious metastatic
lesion from the surrounding tissues. The technology that exploits
the use of labeled peptides for the detection of tumor cells is
instead based on the molecular differences that distinguish these
cells from the others. The peptides according with the present
invention can detect even single cells of human hepatic
metastases.
[0047] Data collected on peptides of the present invention can be
summarized as follows: [0048] 1) peptides selected in the present
application share a high sequence homology between each other,
indicating the specificity of the selection; [0049] 2) peptides of
the present invention have high homology with motifs present in
proteins specific for the hepatic tissue and/or related to
neoplastic pathologies; [0050] 3) from the binding assays it
appears that the peptides have high specificity for surface
molecules exposed on human hepatic metastatic cells, both primary
and in culture, while they preferably do not show affinity for
primary cells of normal liver or for cell lines of primary tumors
or other kinds of metastases; [0051] 4) peptides of the present
invention bind universally to cells of hepatic metastases
independently from the metastatic stage, clinical parameters to
other characteristics related to each patient, thus being good
diagnostic-prognostic and therapeutic candidate tools.
A. Definitions
[0052] 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.
1. Targeting Moiety
[0053] A "targeting moiety" is a term that encompasses various
types of affinity reagents that may be used to enhance the
localization or binding of a substance to a particular location in
an animal, including organs, tissues, particular cell types,
diseased tissues or tumors. Targeting moieties may include
peptides, peptide mimetics, polypeptides, antibodies, antibody-like
molecules, nucleic acids, aptamers, and fragments thereof. In
certain embodiments, a targeting moiety will enhance the
localization of a substance to cells of hepatic metastases
secondary to colon carcinoma, through the binding to surface
protein of these cells, i.e. through the binding to transmembrane
or surface-associated or secreted or extracellular
matrix-associated proteins. Selective binding of a targeting moiety
of the present invention, e.g., a targeting peptide or antibody, as
well as variants and fragments thereof is when the targeting moiety
binds a target (e.g. cells of the hepatic metastasis secondary to
colon cancer) and does not significantly bind to unrelated cells. A
targeting moiety is still considered to selectively bind even if it
also binds to other proteins that are not substantially homologous
with the target so long as such proteins share homology with a
fragment or domain of the peptide target of the antibody. In this
case, it would be understood that target moiety binding to the
target is still selective despite some degree of cross-reactivity.
Typically, the degree of cross-reactivity can be determined and
differentiated from binding to the target.
2. Targeting Peptide
[0054] A "targeting peptide" is a peptide comprising a contiguous
sequence of amino acids, which is characterized by selective
localization to an organ, tissue or cell type, which includes
specific binding with an extracellar protein or molecule that is
specifically expressed or produced in a specific tissue or cell
type(s).
3. Receptor
[0055] A "receptor" for a targeting peptide includes but is not
limited to any molecule or molecular complex that binds to a
targeting peptide. Non-limiting examples of receptors include
peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids,
carbohydrates, multi-molecular structures, and specific
conformation of one or more molecules. In preferred embodiments, a
"receptor" is a naturally occurring molecule or complex of
molecules that is present on the luminal surface of cells forming
blood vessels within a target organ, tissue or cell type. More
specifically, a "receptor" is a naturally occurring molecule that
is present on the luminal surface of cells that form blood vessels
into a target organ, tissue or cell type.
4. Amino Acid Residue
[0056] An "amino acidic residue" refers to any natural amino acid,
any amino acid derivative or any amino acid mimetic that is known
in the art. Protein residues are generally consecutive, without
non-amino acids that interrupt the sequence of amino acid residues.
In particular embodiments, the amino acidic sequence may include
one or more non-amino acids. In particular embodiments, the amino
acidic sequence may include one or more non-amino acids. In
particular embodiments, the sequence of a peptide of the present
invention may be interrupted by one or more non-amino acids.
Modified or unusual amino acids include but are not limited to:
Aad, 2-Aminoadipic acid; EtAsn, N-Ethylasparagine; Baad,
3-Aminoadipic acid, Hyl, Hydroxylysine; Bala, beta-alanine,
beta-Amino-propionic acid; AHyl, allo-Hydroxylysine; Abu,
2-Aminobutyric acid; 3Hyp, 3-Hydroxyproline; 4Abu, 4-Aminobutyric
acid, piperidinic acid; 4Hyp, 4-Hydroxyproline; Acp, 6-Aminocaproic
acid, Ide, Isodesmosine; Ahe, 2-Aminoheptanoic acid; Alle,
allo-Isoleucine; Aib, 2-Aminoisobutyric acid; MeGly,
N-Methylglycine, sarcosine; Baib, 3-Aminoisobutyric acid; Melle,
N-Methylisoleucine; Apm, 2-Aminopimelic acid; MeLys,
6-N-Methyllysine; Dbu, 2,4-Diaminobutyric acid; MeVal,
N-Methylvaline; Des, Desmosine; Nva, Norvaline; Dpm,
2,2'-Diaminopimelic acid; Nle, Norleucine; Dpr,
2,3-Diaminopropionic acid; Orn, Ornithine; and EtGly,
N-Ethylglycine. Also included are the D-amino acids.
5. Protein or Peptide
[0057] The term "protein or peptide" includes amino acid sequences
constituted by at least one of the 20 common amino acids that can
be found in natural proteins, or at least a modified or unusual
amino acid.
6. Cross-Linking Reagents
[0058] 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.
7. Antibodies
[0059] As used herein, the term "antibody" is intended to refer
broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD
and IgE or antibody like molecule. Generally, IgG and/or IgM are
preferred because they are the most common antibodies in the
physiological situation and because they are most easily made in a
laboratory setting. Means for preparing and characterizing
antibodies are also well known in the art. It is here defined
"antibody" any molecule similar to an antibody that has an antigen
binding region, including antibody fragments such as Fab', Fab,
F(ab')2, single-domain antibodies (DABs), Fv, single chain
antibodies (scFv).
8. Nucleic Acids
[0060] "Nucleic acids" according to the present invention may
encode a targeting peptide, a targeting antibody, a therapeutic
polypeptide a fusion protein or other protein or peptide. The
nucleic acid may be derived from genomic DNA, complementary DNA
(cDNA) or synthetic DNA. The term "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 almost any size, determined in part
by the length of the encoded protein or peptide. It is contemplated
that targeting peptides, targeting antibodies, and fusion proteins
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.
9. Delivery Tools
[0061] Several delivery tools can be used for the administration of
target peptides according with the present invention; among the
others, liposomes and oil-in-water or water-in-oil micro-emulsion
systems. The liposomes and the micro-emulsions, and other
micro-delivery systems, can be prepared by procedures well known in
the art. Ligands may be bound covalently to sites on the liposome
surfaces. The number and surface density of these sites may be
adjusted by employing specific liposome formulations and/or
liposome types. 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).
B. Proteins and Peptides
1. Peptides
[0062] In one embodiment, the present invention involves the use of
a peptide capable of selectively binding to metastatic cells,
preferably hepatic metastatic cells. The peptide may comprise a
single copy of a sequence as defined in SEQ ID NO. 1-201 or
multiple identical or different copies of such sequences optionally
connected by amino acid linker sequences.
[0063] Due to their relatively small size, target peptides of the
present invention can be synthesized in solution or on solid
supports, according to well known techniques. Short peptides,
generally from about 6 to 35-40 amino acids, can be easily produced
with these techniques. Alternatively, recombinant cDNA technology
can be used, in which a nucleotidic sequence coding for a peptide
of the invention is inserted in an expression vector, transformed
or transfected in proper host cells, and cultured in conditions
suitable for protein expression.
[0064] The peptides of the present invention can consist of natural
amino acid residues or may comprise at least one modified or
unusual amino acid residue. The peptides of the present invention
may be linear or cyclic peptides. The peptides of the present
invention preferably have a length of about 6 to about 100,
preferably to about 35-40 amino acids or amino acid mimetics.
2. Peptidomimetics
[0065] Another embodiment of the present invention involves the use
of "peptidomimetics". Mimetics are peptides containing molecules
that mimic elements of the secondary structure of the proteins. The
ratio at the basis of peptidomimetics is in the fact that protein
peptide backbone has mainly the function of orienting the side
chains of the amino acids, in order to favor the molecular
interactions, such as those of the antibodies and antigens. A
peptidomimetic allows the molecular interactions in a same way as
in the natural molecule. These principles can be exploited to
engineer second generation molecules, having most of the natural
properties of the target peptides described in the present
invention, but with modified and possibly improved characteristics.
An example of peptidomimetics is a retroinverted peptide, formed by
D-amino acids in inverted sequence compared to the peptide sequence
that it mimics. The peptidomimetics of the present invention
preferably have a length of about 6 to about 100, preferably to
about 35-40 amino acids or amino acid mimetics.
3. Fusion Proteins
[0066] Peptides of the present invention can also be used as one of
the components of a fusion protein.
[0067] Fusion proteins comprise the whole sequence or a portion of
the target peptide fused at its N- and/or C-terminus optionally via
a peptidic linker to a second polypeptide or protein, which is
heterologous to the target peptide. For example, fusion proteins
may comprise signal sequences of other proteins, to allow the
expression of recombinant proteins in an heterologous host. Other
useful fusion proteins comprise an immunologically active domain,
such as an antibody epitope, to facilitate the purification of the
fusion protein. The incorporation of a cleavage site, e.g. a
proteolytic cleavage site, at the fusion site or in the immediate
vicinity will favor the removal of the exogeneous domain after
purification. Other useful fusion proteins comprise functional
domains, such as active sites of enzymes, glycosylation domains,
cell addressing signals, or transmembrane regions.
[0068] In one embodiment of the present invention, fusion proteins
are made by target peptides fused to a protein or a peptide with
therapeutic activity. Examples of proteins or peptides that can be
incorporated in a fusion protein include: cytostatic proteins,
cytotoxic proteins, pro-apoptotic agents, antiangiogenic agents,
hormones, cytokines, growth factors, peptide drugs, antibodies, Fab
fragments of antibodies, antigens, receptor proteins, enzymes,
lectins, proteins of the major histocompatibility complex, cell
adhesion proteins and binding proteins. Such examples are not
intended to be limiting, but it is understood that, accordingly
with the present invention, virtually any protein or peptide can be
incorporated into a fusion protein that includes a target peptide.
Methods to produce fusion proteins are well known. Such proteins
can be produced, for example, by chemical bound using bifunctional
cross-linking reagents, by de novo synthesis of the whole fusion
protein, or by attachment of a sequence of DNA coding for the
target peptide to a sequence of DNA coding for the second protein
or peptide, followed by the expression of the whole fusion
protein.
4. Antibodies
[0069] In a different embodiment of the present invention, it may
be desirable to produce antibodies against target peptides object
of the present invention.
[0070] For this purpose, the target peptides, or the molecules they
bind, can be coupled, bound, conjugated or chemically linked to one
or more agents by spacers, poly-spacers, or derivatized amino acids
to produce a complex comprising at least one target peptide or
molecule which binds to a target peptide. This can be done in such
a way that a bi- or multivalent complex is produced, or a vaccine.
Methods for producing such complexes are familiar to those skilled
in the art, and can be adapted to human administration, i.e.
pharmacologically acceptable. Preferred agents are carriers, like
hemocyanin (KLH) and bovine serum albumin (BSA). The resulting
antibodies can be used both for diagnosis and therapy, for example
by binding and/or inhibiting functional proteins on the surface of
metastatic cells.
[0071] To improve the efficiency of antibody molecules, they may be
bound or complexed to at least one system or molecule, for example
a molecule that allows its detection. Non-limiting examples of such
molecules include enzymes, radionuclides, aptamers, fluorescent
labels, phosphorescent molecules, chemiluminescent molecules,
chromophores, luminescent molecules, colored particles or ligands
such as biotin.
C. Diagnostic and Therapeutic Conjugates
[0072] In an embodiment of the present invention, it may be
desirable to couple specific bioactive agents to one or more target
peptides accordingly to the present invention for the specific
release into an organ, tissue or cell type. Below are indicated
some examples of agents that can be coupled to target peptides
accordingly to the present invention.
[0073] Conjugates according to the present invention can be
produced by direct conjugation of the target peptide to the
therapeutic or diagnostic agent of interest, or using cross-linking
reagents to establish a binding between a peptide and the molecule
of interest.
1. Cytokines
[0074] The term "cytokine" is a generic term for proteins released
by one cell population that 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-1alpha, 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.
2. Chemokines
[0075] "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.
3. Imaging Agents
[0076] In certain embodiments, the targeting moieties of the
present invention may be attached to imaging agents of use for
imaging and diagnosis of hepatic metastases.
[0077] Several imaging agents are well known, as are the methods to
bind them to proteins or peptides. Non-limiting examples of imaging
agents include paramagnetic ions such as 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).
[0078] Radioisotopes of use as imaging or therapeutic agents
include .sup.211astatine, .sup.14-carbon, .sup.51chromium,
.sup.36-chlorine, .sup.57cobalt, .sup.58cobalt, .sup.67copper,
.sup.152Eu, .sup.67gallium, .sup.3hydrogen, .sup.123iodine,
.sup.125iodine, .sup.131iodine, .sup.111indium, .sup.59iron,
phosphorus, .sup.186rhenium, .sup.188rhenium, .sup.75selenium,
.sup.35sulphur, .sup.99mtechneticum and .sup.90yttrium.
[0079] 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.
[0080] In still further embodiments, a targeting moiety may be
operatively coupled to a nanoparticle. Nanoparticles include, but
are not limited to colloidal gold and silver nanoparticles. Metal
nanoparticles exhibit colors in the visible spectral region.
Further examples of nanoparticles are magnetic nanoparticles.
4. Therapeutic Agents
[0081] In certain embodiments, therapeutic agents may be
operatively coupled to a targeting peptide or fusion protein for
selective delivery to, for example, tumor vasculature of the
hepatic metastases. Agents or factors suitable for use may include
any chemical compound that induces apoptosis, cell death, cell
stasis and/or anti-angiogenesis, such as: [0082] Regulators of
Programmed Cell Death or Apoptosis. The Bcl-2 protein and other
members of the family are involved in apoptosis, and can be
classified as agonists or antagonists of apoptosis. For example,
Bcl-2 and other members of the family (e.g., Bcl-XL, Bcl.-W, Bcl-S,
Mcl-1, A1, Bfl-1) are pro-apoptotic, while others (e.g., Bax, Bak,
Bik, Bim, Bid, Bad, Harakiri) are anti-apoptotic. [0083] Inhibitors
of angiogenesis. In certain embodiments the present invention may
concern administration of targeting moieties operatively coupled 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, thrombospondin, 2-methoxyestradiol,
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.
[0084] Cytotoxic agents. Chemotherapeutic (cytotoxic) agents may be
used to treat various disease states, including cancer. Most
chemotherapeutic agents fall into the categories of alkylating
agents, antimetabolites, antitumor antibiotics, corticosteroid
hormones, mitotic inhibitors, and nitrosoureas, hormone agents,
miscellaneous agents, and any analog or derivative variant thereof.
[0085] Alkylating agents. Alkylating agents are drugs that directly
interact with genomic DNA to prevent cells 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 ethyleneimine, a methylmelamine, an alkyl sulfonate, a
nitrosourea or a triazine. They include but are not limited to:
busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),
dacarbazine, ifosfamide, mechlorethamine (mustargen), and
melphalan. [0086] Antimetabolites. 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. Antimetabolites include but are not limited to,
5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine,
gemcitabine, and methotrexate. [0087] Natural products. Natural
products generally refer to compounds originally isolated from a
natural source, and identified as 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. [0088] Mitotic inhibitors. 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. 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.
Vinca alkaloids are a type of plant alkaloid identified to have
pharmaceutical activity. They include such compounds as vinblastine
(VLB) and vincristine. [0089] Antibiotics. It is well known that
certain 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 cytotoxic antibiotics include, but are not
limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin
(Adriamycin), plicamycin (mithramycin) and idarubicin. [0090]
Miscellaneous Cytotoxic Agents. Miscellaneous cytotoxic agents that
do not fall into the previous categories include, but are not
limited to, platinum coordination complexes, anthracenediones,
substituted ureas, methyl hydrazine derivatives, amsacrine,
L-asparaginase, and tretinoin. Platinum coordination complexes
include such compounds as carboplatin and cisplatin (cis-DDP). An
exemplary anthracenedione is mitoxantrone. An exemplary substituted
urea is hydroxyurea. An exemplary methyl hydrazine derivative is
procarbazine (N-methylhydrazine, MIH). These examples are not
limiting and it is contemplated that any known cytotoxic,
cytostatic or cytocidal agent may be attached to targeting peptides
and administered to a targeted organ, tissue or cell type within
the scope of the invention.
D. Nucleic Acids
[0091] Nucleic acids accordingly to the present invention can code
for a target peptide, a target antibody, a therapeutic polypeptide,
a fusion protein or other proteins or peptides. The nucleic acid
can be selected from genomic DNA, complementary DNA (cDNA),
synthetic DNA or RNA.
[0092] In one embodiment, the present invention involves the use of
vectors expressing a peptide according to the present invention for
gene therapy. Gene therapy vectors can include several transgenes
including a DNA or RNA sequence coding for at least a peptide or
polypeptide of the present invention operatively linked to
expression control sequences.
[0093] Gene therapy can be used to express a therapeutic gene, for
example to enhance or decrease neo-vascularization. DNA may be in
form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a
plasmid DNA, genetic material derived from a virus, linear DNA,
vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression
cassettes, chimeric sequences, recombinant DNA, chromosomal DNA, an
oligonucleotide, anti-sense DNA, or derivatives of these groups.
RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA),
snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger
RNA), in vitro polymerized RNA, recombinant RNA, chimeric
sequences, anti-sense RNA, siRNA (small interfering RNA),
ribozymes, or derivatives of these groups. An anti-sense
polynucleotide is a polynucleotide that interferes with the
function of DNA and/or RNA. Antisense polynucleotides include, but
are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA,
RNA and the like. SiRNA comprises a double stranded structure
typically containing 15-50 base pairs and preferably 21-25 base
pairs and having a nucleotide sequence identical or nearly
identical to an expressed target gene or RNA within the cell.
Interference may result in suppression of expression. The
polynucleotide can also be a sequence whose presence or expression
in a cell alters the expression or function of cellular genes or
RNA. In addition, DNA and RNA may be single, double, triple, or
quadruple stranded.
Materials and Methods
The Phage Display Methodology
[0094] Phage display is a technique developed in the middle 80' by
George Smith of the University of Missouri. The principle consists
in selecting peptides from a collection, or library, in which
virtually all the possible amino acid permutations are represented.
Such peptides are selected based on their ability to specifically
bind a target of whatever nature and complexity. The phage display
methodology involves rounds of screening and amplification of bound
particles, with the aim of obtaining a reduction in diversity and
an increase in binding specificity.
[0095] The construction of a phage library involves the use of M13
filamentous bacteriophages that can infect Escherichia coli
bacteria. Peculiar characteristic of these phages is to have a
circular single stranded DNA genome, which can be manipulated with
the molecular biology techniques. In such a library, the peptides
derive from the transcription and translation of random exogenous
oligonucleotides, which are cloned into the viral DNA upstream from
the gene for the capsid protein pill. Bacteria are transformed with
these constructs by electoporation; so, they will produce a
population of recombinant phages, each of which will include a
different peptide as a fusion with the pill protein. With this
system it is possible to produce a library with a diversity of
about 10.sup.8-10.sup.9 peptides, in which each sequence is
represented up to 100-1,000 times. If the degeneration of the
sequence is complete (Xn, where X=any amino acid, n=number of the
amino acids), each of the 20 amino acids has the same theoretic
probability of being included into the sequence. Another
possibility is to establish fixed positions for an amino acid.
Libraries are frequently characterized by cysteins placed in
preferential positions, at both ends of the peptide or intercalated
into the random residues, among which intermolecular disulfide
bridges are formed that render the peptide circular.
Circularization of the insert allows a better exposition of the
sequence.
ABBREVIATIONS AND SOLUTIONS
[0096] AEC 3-Amino-9-Ethyl Carbazole
[0097] Amp Ampicillin
[0098] BSA Bovine Serum Albumin
[0099] DMEM Dulbecco's Minimal Essential Medium
[0100] DMEM/FCS/HEPES High Glucose DMEM/2% FCS/20 mM HEPES
[0101] DMSO Dimethylsulfoxide
[0102] DTT Dithiothreitol
[0103] EDTA Ethylene Diamino Tetracetic Acid
[0104] FCS Fetal Calf Serum
[0105] GST Glutathione Sulfo-Transferase
[0106] HEPES N2-Hydroxy Ethyl piperazino-N'-2-Ethyl Sulfonic
Acid
[0107] HRP Horseradish Peroxidase
[0108] Kan Kanamycin
[0109] IPTG Isopropyl-.beta.-Thio Galactoside
[0110] LB Luria Bertani Broth
[0111] PAF Paraformaldehyde
[0112] PBS Phosphate Buffer Saline, 150 mM NaCl, 10 mM
KH.sub.2PO.sub.4, pH 7.40
[0113] PEG/NaCl 20% Poly Ethylene Glycole-8,000, 4 M NaCl
[0114] PMSF Phenyl Methyl Sulfonyl Fluoride
[0115] SDS Sodium Dodecyl Sulfate
[0116] TAE 40 mM Tris-HCl, 0.12% Acetic Acid, 1 mM EDTA
[0117] Buffer A 50 mM Tris-HCl, pH 7.40, 150 mM NaCl, 5% Glycerol,
2 mM DTT
[0118] Buffer H 10 M Tris-HCl, Ph 7.40, 10 mM NaCl, 10 mM PMSF
[0119] TBS Tris Buffer Saline, 150 mM NaCl, 2.8 mM KCl, 25 mM Tris
base, pH 7.40
[0120] TBS-T TBS-0.1%, Tween-20
[0121] TB Terrific Broth
[0122] Tet Tetracylin
[0123] TE 10 mM Tris-HCl, 1 mM EDTA
Reagents
Disposable Plastic Material: Falcon, Eppendorf.
[0124] Media and other cell culture reagents: High Glucose DMEM and
RPMI-1640: Sigma; DMEM and Ham's F12: Biowhittaker Europe; FCS:
Gibco; Collagenase: Roche; L-Glutamine and Penicillin/Streptomycin
solution: Biowhittaker Europe; Broths and antibiotics for bacterial
cultures: LB: Sigma; TB: Gibco; Kan and Tet: Sigma; Reagents for
immunohistochemistry: DAKO Cytomation.
Surgical Samples
[0125] Surgical samples are derived from surgical patients of the
Institute for Cancer Research and Treatment (IRCC), Candiolo (TO),
Italy, Division of Oncological Surgery. Written consensus for the
participation in this study was obtained from all the donors.
[0126] For each patient one sample of normal liver and one of
hepatic metastases was obtained. Samples were morphologically
different for size, aspect, color, vascularization, presence of
necrotic regions, and accumulation of lipid aggregates (an index of
the degeneration degree induced by steatosis). The differences in
the tissues are related to the different stage of progression of
the disease, to the site of metastatization of the primary tumor,
to other eventual causes or diseases occurred in the pathogenic
process, or to other reasons related to an individual
variability.
[0127] Samples were processed immediately after surgical removal,
in order to disaggregate the tissues and extract single cells on
which to perform the experiments. All the manipulations were
performed under laminar flux in sterility. Samples were chopped
with a scalpel in a small volume of PBS. The suspension, collected
in PBS, was centrifuged for 3 minutes at 100 rpm at room
temperature and the pellet was resuspended in 5 ml of collagenase
(0.25% weight/volume in DMEM). The digestion of the tissue
fragments was done incubating this suspension for 2 hours at
37.degree. C. while shaking. The sample was again centrifuged, to
eliminate all the particulate under the cell size (lipid
aggregates, cell portions) or also smaller cells, of hematopoietic
origin. Pellet was washed twice in PBS. Cells were filtered on
filters with a diameter of 45 .mu.m, counted in Burker chamber and
resuspended at a concentration of 10.sup.6/ml in
DMEM/FCS/HEPES.
[0128] At the microscopic examination, after tissue disaggregation
and cell purification, the primary cell population appeared
heterogeneous and other cell types other that hepatocytes and tumor
cells could be distinguished. Among these, red blood cells, and
other cells of the hematopoietic origin; fibroblasts derived from
the connectival structures of the parenchyma; endothelial cells
that line the blood vessels of the analyzed tissue.
Cell Lines
[0129] For the experiments of the present invention the human cell
lines indicated in Table 1 were used. TABLE-US-00001 TABLE 1 Cell
line Description SW480 primary colorectal cancer (ATCC CCL228)
SW620 lymph node metastasis from colorectal cancer (ATCC CCL227)
NCI-H630 hepatic metastasis from colorectal cancer (ATCC CRL5833)
HepG2 primary hepatic cancer (ATCC HB8065) AGS primary stomach
cancer (ATCC CRL1739) NCI-N87 hepatic metastasis from stomach
cancer (ATCC CRL5822) Capan-2 primary pancreas cancer (ATCC HTB80)
Capan-1 hepatic metastasis from pancreas cancer (ATCC HTB79) BT-474
primary breast cancer (ATCC HTB20) MCF-7 pleural effusion from
breast cancer (ATCC HTB22) A549 primary lung cancer (ATCC CCL185)
NCI-H1688 hepatic metastasis from lung cancer (ATCC CCl257)
Media for Cell Culture
[0130] For the maintenance and the growth of cell lines different
culture media were used, depending on cell type: [0131] SW480,
SW620, HepG2, BT-474, and MCF-7 cells were cultured in DMEM, with
10% FCS, 20 mM HEPES, L-glutamine (40 mM), Penicillin (200 U/ml),
and Streptomycin (200 .mu.g/ml). [0132] NIC-H630, NCI-H87,
NCI-H1688, Capan-1, and Capan-2 cells were cultured in RPMI-1640,
with 10% FCS, L-glutamine (40 mM), Penicillin (200 U/ml), and
Streptomycin (200 .mu.g/ml). [0133] A549 and AGS cells were
cultured in Ham's F12, with 10% FCS, L-glutamine (40 mM),
Penicillin (200 U/ml), and Streptomycin (200 .mu.g/ml). Cell
Cultures
[0134] Cultures were started from cells stored in liquid nitrogen
in a solution of FCS with 10% DMSO. Cells, after quick thawing at
37.degree. C., were cultured in 100.times.20 mm dishes, in
humidified incubator at 37.degree. C. with 5% CO.sub.2. The
complete replacement of the culture medium was done every 3-4 days.
When the 80-90% of confluence was reached, cells were washed in PBS
and detached by incubating with a solution of 0.05% Trypsin, 2 mM
EDTA at 37.degree. C. for 3 minutes. An excess volume of medium
with 10% FCS was then added and cells were collected by
precipitation at 1,000 rpm for 3 minutes. Supernatant was removed,
the pellet was resuspended in complete medium and aliquoted in 4
new dishes.
[0135] For the phage display experiments, cells were washed in PBS
with 10 mM EDTA, and incubated in the same solution for 3 minutes
at 37.degree. C. The cell suspension was then harvested in PBS in a
total volume of 10 ml. After counting in the Burker chamber, cells
were resuspended in DMEM/FCS/HEPES at a final concentration of
1.times.10.sup.6 ml.
Phage Libraries
[0136] For the phage display experiments, two cyclic libraries of
the CX.sub.7C and CX.sub.3CX.sub.3CX.sub.3C types, and one linear
library of the CX.sub.9 type were used. In these libraries, the
insert is expressed in 5 identical copies as a fusion peptide at
the N-terminal of the pill protein. The cysteine of the insert,
close to the capsid surface, is covalently bound to the phage
protein and, in the case of the CX.sub.7C library, can form a
di-sulfide bond with the cysteine at the opposite side. As a
consequence, the peptide is cyclized. In the
CX.sub.3CX.sub.3CX.sub.3C library, instead, different combinations
of di-sulfide bridges can form, which lead to multiple cyclizations
and to the exposure of tri-peptide motifs.
[0137] The CX.sub.9 library is linear and there is no cyclization
but in a case in which the last amino acid is a cysteine as well.
Phage libraries are conserved at 4.degree. C. in TBS, at a
concentration of 10.sup.10-10.sup.12 TU/ml.
Broths and Plates for Bacterial Cultures
[0138] LB: this medium was supplemented either with Kan, to a final
concentration of 20 .mu.g/ml, for the amplification of Escherichia
coli bacteria strain K91kan, or with both Kan and Tet, both 20
.mu.g/ml, for bacteria amplification after the infection.
[0139] TB: this broth was used to render the K91 kan bacteria
competent to infection, and was supplemented with Kan, to a final
concentration of 20 .mu.g/ml.
[0140] Plates: bacteria were amplified in Petri plates on a
semi-solid substrate composed as follows: LB with 15% weight/volume
bacteriological agar, and Kan (20 .mu.g/ml) for the growth of K91
kan, or Kan (20 .mu.g/ml) and Tet (40 .mu.g/ml) for the growth of
the infected bacteria.
Library Selection on the Cells
[0141] For all the procedures regarding phage display, protocols
known in the literature were used. In particular, the protocol for
the whole cell panning is derived from described methods, but it
was adapted to the system in analysis, after several tests, with
the aim of optimizing the application.
[0142] First round. A microliter of the library was incubated with
5.times.10.sup.5 fresh metastatic cells, in a total volume of 500
.mu.l in DMEM/FCS/HEPES, for 16 hours at 4.degree. C., under mild
shaking. Four washes in the same medium were then performed, to
eliminate the weakly bound phages or the phages left in solution.
The washes were performed in 1 ml of the same medium.
[0143] Successive rounds. In the successive selection rounds, 50
.mu.l of the phages obtained from round I were incubated with
5.times.10.sup.5 cells of normal liver from the same patient in 500
.mu.l of DMEM/FCS/HEPES. This negative pre-selection step lasted 1
hour at room temperature under mild shaking, and was repeated
twice. Then the supernatant was divided in two parts that were
added to 5.times.10.sup.5 cells of either normal liver or hepatic
metastatic cells, respectively. The two cell suspensions were
incubated for 2 hour at 4.degree. C. under mild shaking. Washes as
described followed. Bound phages were collected by infecting
competent bacteria.
Infection of the Bacteria and Phase Amplification
[0144] Bacteria were grown in 10 ml of TB with Kan, at 37.degree.
C. while shaking for 2-3 hours, until they reached the optical
density of 1.5-2.0 at the 600 nm wavelength. One milliliter of
competent bacteria was then added to the 100 .mu.l of cell
suspension after washing. Infection lasted 1 hour at room
temperature. At the end of the incubation, part of the bacteria
were plated, in duplicate, on Petri plates with LB-agar and Tet,
and incubated for 16 hours at 37.degree. C. This system allows to
grow only the phage-infected bacteria, since only the phages carry
the resistance to this antibiotic. The TU related to the substrate
bound phage were evaluated colony counting of each plate. Here we
refer to this value with the term "Output".
[0145] The remaining part of the bacteria was added to 10 ml of LB
with Tet and Kan and grown for 16 hours at 37.degree. C. while
shaking.
Phage Purification
[0146] This procedure was used to purify both phage populations
deriving from selection rounds and single phage clones. The
bacterial culture was centrifuged at 5,000 rpm for 10 minutes at
4.degree. C. to eliminate the bacteria. The phages, now in the
supernatant, were precipitated with 0.15 volumes of PEG/NaCl for 1
hour at 4.degree. C. and collected by centrifugation at 6,000 rpm
for 15 minutes at 4.degree. C. After having decanted the
supernatant, the pellet was compacted by further centrifuging at
6,000 rpm for 5 minutes at 4.degree. C. and then resuspended,
through shaking for 10 minutes, in 500 .mu.l of TBS. To eliminate
the debris, the suspension was then centrifuged at 12,000 rpm for
10 minutes at room temperature. The phage population was collected
and stored at 4.degree. C.
Phage Titration
[0147] The titration allows evaluating the amount of starting TU
for each round or the titer of the single clones (amount to which
we here refer as "Input"). To perform the titration, from the
original phage suspension the dilutions described in Table 2 have
been made. TABLE-US-00002 TABLE 2 sample (1) 1 .mu.l of the phage
suspension + 99 .mu.l of PBS (1 .times. 10.sup.-2 dilution) sample
(2) 10 .mu.l of sample (1) + 90 .mu.l of PBS (1 .times. 10.sup.-3
dilution) sample (3) 10 .mu.l of sample (2) + 90 .mu.l of PBS (1
.times. 10.sup.-4 dilution) sample (4) 10 .mu.l of sample (3) + 90
.mu.l of PBS (1 .times. 10.sup.-5 dilution) sample (5) 10 .mu.l of
sample (4) + 90 .mu.l of PBS (1 .times. 10.sup.-6 dilution) sample
(6) 20 .mu.l of sample (5) in a new tube sample (7) 2 .mu.l of
sample (6) + 18 .mu.l of PBS (1 .times. 10.sup.-7 dilution) sample
(8) 2 .mu.l of sample (7) + 18 .mu.l of PBS (1 .times. 10.sup.-8
dilution)
100 .mu.l of the samples (6), (7), and (8), that is of the
1.times.10.sup.-6, 1.times.10.sup.-7 and 1 10.sup.-8 dilutions,
were plated on Petri plates with agar and Tet. Plates were
incubated for 16 hours at 37.degree. C. The number of total TU was
then evaluated by colony counting and referred to the total volume.
Single Clone Binding Assays
[0148] These assays were performed with an Input of 10.sup.9 TU of
each clone, on cells from the hepatic metastasis cell line (target)
and on cells from normal liver (negative control). The Output of
these experiments was normalized on binding to an insertless phage,
fd-tet, giving a measure of the unspecific interaction due to the
phage itself. The binding increase was evaluated as ratio between
normalized Output of the target and normalized Output of the
negative control. All the experiments were repeated at least 3
times, when possible for material availability.
Isolation and Amplification of the Clones
[0149] When a significant increase was observed between the number
of phages bound to metastatic cells compared to those bound to the
normal liver cells, single clones were isolated to identify the
sequence of their insert and to evaluate their binding specificity.
For clone amplification, bacteria from single colonies were grown
in 5 ml of LB with Kan and Tet for 16 hours at 37.degree. C. while
shaking. Phages were then purified as described.
[0150] For mechanically disaggregating the phage capsid, resin
beads were used, named Strataclean Beads by the manufacturer.
Before their use, the beads were resuspended in TBS in a 1:1
volume/volume ratio. For each clone, 200 .mu.l of phage suspension
was added to 10 .mu.l of beads and vortexed for 30 seconds. After
centrifugation at 400 rpm for 3 minutes, 195 .mu.l of supernatant
were collected and subjected to the same disaggregation cycle.
Finally, 150 .mu..l of supernatant were filled up to 410 .mu.l with
TE, and DNA was precipitated by incubation with 0.1 volumes of
Sodium Acetate pH 5.5, and 2.2 volumes of 100% ethanol. DNA was
collected by centrifugation for 10 minutes at 12,000 rpm, washed in
70% ethanol and resuspended in ultrapure H.sub.2O. DNA amount was
evaluated both by reading its absorbance at a 260 nm wavelength and
by electrophoresis on 1% agarose gels in TAE.
Preparation of Samples for Sequencing
[0151] Ten microliters of the solution, corresponding to about 800
ng of phage DNA, were incubated with 3 pmol of the following
primer: 5'-CCCTCATAGTTAGCGTAACG-3' (SEQ ID NO. 202), which
corresponds to a zone immediately downstream from the
oligonucleotide insert.
Sequence Analysis
[0152] To translate the nucleotide sequences into peptide sequences
we used the software DNAs is V2.5.
Protocol of Immunohystochemistry with the Phages
[0153] Tissue samples were embedded in OCT and stored at
-80.degree. C. For the experiment, they were cut using a cryostate
at -20.degree. C.
[0154] Tissues were cut in 10 .mu.m slides. These slides were then
treated with PBS for 5 minutes, until OCT was completely removed.
Tissues were fixed in 4% PAF in PBS for 10 minutes at room
temperature, then washed for 5 minutes in PBS and incubated with 50
mM NH.sub.4Cl in PBS for 20 minutes.
[0155] Tissue peroxidases were then inactivated by treating with 3%
H.sub.2O.sub.2 in H.sub.2O for 10 minutes in the dark at room
temperature. One wash for 5 minutes in PBS followed.
[0156] The unspecific interaction sites were blocked by incubating
the samples in the "DAKO block" reagent for 30 minutes at room
temperature. Phages were then added (from 1.times.10.sup.6 to
5.times.10.sup.6 total TU) diluted into the "DAKO diluent" reagent;
incubation lasted overnight at 4.degree. C. After 4 washes of 5
minutes each in TBS, samples were stained with a rabbit polyclonal
anti M13 phage antibody (Sigma B7786), diluted 1:500 in the "DAKO
diluent" reagent, for 1 hour at room temperature.
[0157] The labeling was done using a secondary "DAKO envision"
anti-rabbit antibody, developed with the AEC substrate for 5
minutes and followed by a control-staining with Mayer's
hematoxylin.
GST-Fused Peptide Purification
[0158] Some of the selected peptides were produced in Escherichia
coli as a fusion protein with GST using standard purification
protocols.
Preparation and Lysis of the Bacteria:
[0159] 1. inoculate the bacteria and grow overnight in 20 TB/Amp
broth at 30.degree. C.; [0160] 2. transfer bacteria in 300 ml of
TB/Amp broth at 30.degree. C.; shake for 1 hour; [0161] 3. add IPTG
(final concentration 1 mM) and incubate for 2 hours; [0162] 4.
centrifuge at 5,000 rpm for 15 minutes at 4.degree. C.; [0163] 5.
resuspend bacteria in 10 ml of buffer A; [0164] 6. centrifuge at
3,000 rpm for 20 minutes at 4.degree. C.; [0165] 7. resuspend
pellet in 5 ml, sonicate bacteria with four pulses of 20 seconds
each at 35% power; [0166] 8. centrifuge at 11,000 rpm for 20
minutes at 4.degree. C. and collect supernatant.
Glutathione-Agarose Resin Preparation: [0167] 9. hydrate 250 .mu.l
of resin in distilled H.sub.2O, in rotation for 1 hour; [0168] 10.
wash the resin 3 times in buffer A and finally resuspend it in an
equal volume of buffer A. Purification of the Recombinant Proteins:
[0169] 11. add 250 .mu.l of resin to the sample of step 8; [0170]
12. rotate at 4.degree. C. for 1 hour; [0171] 13. wash 3 times in
buffer A; [0172] 14. evaluate the concentration by electrophoresis
followed by Coomassie blue staining.
[0173] Polymerization mix for SDS-polyacrylamide gel
electrophoresis (Table 3). TABLE-US-00003 TABLE 3 12% running gel
Acrylamide/Bis-Acrylamide (4 ml) 1.5 M Tris pH 8.8 (3.75 ml) 10%
SDS (0.1 ml) Bidistilled water (2.15 ml) Ammonium Persulfate (100
mg/ml) (33 .mu.l) TEMED (8 .mu.l) 5% stacking gel
Acrylamide/Bis-Acrylamide (0.8 ml) 0.5 M Tris pH 6.8 (650 .mu.l)
10% SDS (0.05 ml) Bidistilled water (3.55 ml) Ammonium Persulfate
(100 mg/ml) (30 .mu.l) TEMED (5 .mu.l)
Coomassie Blue Staining [0174] 1. incubate the gel with Coomassie
Blue for 30-45 minutes in mild agitation; [0175] 2. destain in 45%
methanol-10% acetic acid; [0176] 3. rehydrate in water, eventually
dry on paper. Cell Lysis
[0177] Twenty 100.times.20 mm dishes of HepG2 (hepatoma) or
NCI-H630 (liver metastasis secondary to colorectal carcinoma) cells
were mechanically detached in PBS and resuspended in 2 volumes of
buffer H, with 10% glycerol and 0.1% Nonidet-P40. These suspensions
were incubated for 30 minutes at 4.degree. C. under agitation and
then centrifuged at 2,500 rpm for 30 minutes at 4.degree. C.
Protein concentration was evaluated by the BCA kit (Pierce),
following the instructions of the manufacturer.
Pull-Down Assay
[0178] The GST-peptides bound to the resin were incubated overnight
at 4.degree. C. with milk and washed 7 times in buffer A. 10 mg of
total protein lysate were incubated with 12 .mu.g of GST-resin at
4.degree. C. for 1 hour twice to eliminate the proteins that bind
non-specifically to GST or to the resin. The pull-down assay was
performed on the unbound proteins, with 12 .mu.g of
GST-peptide-resin, overnight at 4.degree. C.
[0179] After 4 washes in buffer A, proteins bound to the
GST-peptide-resin complex were eluted in 20 mM Glutathione for 30
minutes at 4.degree. C. and collected by centrifugation at 3,000
rpm for 2 minutes a 4.degree. C.
[0180] The supernatant was loaded on a 10% polyacrylamide
denaturing gel. This gel was then stained with a Coomassie Blue
solution and the specific bands were collected and analyzed by mass
spectrometry and micro-sequencing (with standard protocols).
Results
Search for Peptide Motifs Specific for Human Hepatic Metastases
[0181] To find peptides which specifically bind to human hepatic
metastases, phage library screenings were performed on suspended
cells derived from normal liver and metastasis samples, surgically
removed from the liver of patients. In this phase, 11 couples of
samples from different patients were used (patients 2, 5, 6, 7, 8,
16, 17, 18, 19, 21, 23). In almost all of the samples, the hepatic
metastases were secondary to primary tumors of the colon or rectum,
with the exclusion of patient 8, who had a brain hemangioma as a
primary tumor (Table 4). TABLE-US-00004 TABLE 4 Patient Sex Age
Locus.sup.1 TN.sup.2 Marker.sup.3 Virus.sup.4 Necrosis.sup.5 2 F 60
Colon T3N0 CEA No 40% 5 M 46 Colon T4N2 CEA No 60% 6 M 72 Colon
T3N2 CEA No 50% 8 F 31 Brain No 7 M 64 Colon T3N1 GICA No 15% 16 M
45 Colon T4N2 No 20% 17 M 70 Colon T3N0 No 80% 18 F 62 Colon T4N0
CEA, No 60% GICA 19 M 76 Colon T3N0 CEA No 50% 21 M 59 Retto T3N0
No <5% 23 F 49 Colon No 50% .sup.1site of primary tumor;
.sup.2TN classification of the primary tumor; .sup.3tumor markers;
.sup.4evidence of hepatitis B or C viruses in the liver;
.sup.5percent of necrosis into the metastasis.
[0182] In Table 4, the clinical parameters of the patients used for
the selection are shown. For the patients 2, 6, 7 (2 experiments),
16, 17, 18, 19 and 21 the CX.sub.7C library was used; for patient
8, both the CX.sub.7C and the CX.sub.3CX.sub.3CX.sub.3C libraries
on samples of two different metastases; for patient 23 the CX.sub.9
library. For each experiment, 4 rounds of selection and
amplification were performed.
[0183] Analysis of the Peptide Sequences Obtained in the Screening
Experiments
[0184] In each experiment in which we observed a significant
increase in the ratio of binding to the hepatic metastasis cells
and the negative control (macroscopically healthy liver tissue), 20
phage clones were amplified and purified. The DNA of each clone was
purified and sequenced to derive the peptide motif. Selected
peptides are shown in FIG. 1.
[0185] Some sequences are particularly represented, both in a same
experiment and in experiments performed on samples of different
patients. In the experiments performed on patients 2, 5, 6, 7, 8,
21, and 23 peptides were selected that share common sequences,
particularly tri-/tetra-peptide motifs (among which GGG, RGL, GRL,
GSG, LGR, GLS, SAD, YEG, GSGS (SEQ ID NO: 208)). In the experiments
performed on patients 16, 17 and 18 we found more repeated
sequences. In these experiments motifs with high homology with
those previously described came out as well. The most repeated
peptide is LRS.
Analysis of the Selected Sequences
[0186] The attention was focused to the study of the sequences
obtained in the experiments 16, 17 and 18, in particular: ARPGLRS
(SEQ ID NO. 1); MRYALRS (SEQ ID NO. 2); LRPGLRS (SEQ ID NO. 3);
LRSGSGS (SEQ ID NO. 4); VRSGRGS (SEQ ID NO. 5); GIYRLRS (SEQ ID NO.
6); and GVYSLRS (SEQ ID NO. 7). To identify sequence homologies
among these peptides and known proteins, a search in the BLAST data
bank was done. From these analyses it emerged that a significant
number of peptides share sequence homologies with proteins of the
extracellular matrix and with molecules of cell
adhesion/motility.
Binding Experiments on Cell Lines
[0187] To evaluate if the selected inserts were specific ligands
for surface determinant peculiarly expressed in the hepatic
metastases, the 7 clones were tested on the cell lines described in
Table 1. A summary of the results is shown in Tables 5 and 6.
[0188] In this study model, selected peptide sequences do not bind
to cells derived from primary tumors (with the exception of
BT-474). On the contrary, these sequences preferentially bind to
cells derived from hepatic metastases (6 out of 7 clones bind to
cells of hepatic metastasis secondary to primary colon tumor, 3 out
of 7 clones bind to cells of hepatic metastasis secondary to
primary stomach or lung tumor). TABLE-US-00005 TABLE 5 SW620
NCI-H630 NCI-N87 Lymph Hepatic Hepatic SW480 Node meta meta of AGS
meta of SEQ Hep-G2 Colon of colon colon Stomach stomach ID NO.
Liver tumor tumor tumor tumor tumor tumor 1 - - - - - - 2 - - + + -
+ 3 - - - + - - 4 - - - + - + 5 - - - + - + 6 - - - + - - 7 - + - -
- -
[0189] TABLE-US-00006 TABLE 6 Capan-1 MCF-7 Hepatic Pleural Capan-2
meta from BT-474 effusion A549 NCI-H1688 SEQ Pancreas pancreas
Breast of breast Lung Hepatic meta ID NO. tumor tumor tumor tumor
tumor of lung tumor 1 - - - - - + 2 - - - - - - 3 - - - - - - 4 - -
+ + + + 5 - - + - - - 6 - - - + + + 7 - - - - - -
Binding Experiments on Primary Cells
[0190] To evaluate if the selected inserts were specific for
ubiquitous surface determinants in human hepatic metastases, the 7
selected clones were tested on primary cells of hepatic metastasis,
comparing to normal liver of the same patients. For these binding
assays, samples from 9 patients were used (20, 21, 22, 25, 26, 27,
28, 31, 32), with the same conditions described for the cell lines.
A summary of the results is shown in Table 7. TABLE-US-00007 TABLE
7 SEQ ID NO. P#20 P#21 Pz.#22 P#25 P#26 P#27 P#28 P#31 P#32 1 + + +
+ + + 2 + + - - + + 3 + + + + + + + 4 - + + + + + + 5 - + + + + + 6
+ + + + + + 7 + + + + + + +
[0191] In the first experiments, due to the low cell numbers,
related to the set up phase of the purification procedure, the
binding of only some clones was evaluated. In general, 3 assays
have been performed for each sample. From all these experiments it
emerges that the less functional clone as universal diagnostic
marker is the one that displays the MRYALRS sequence (SEQ ID NO.
2), which gave negative results in two assays (27, 28), while the
clones that worked on all the samples are those exposing the
sequences ARPGLRS (SEQ ID NO. 1), LRPGLRS (SEQ ID NO. 3), GIYRLRS
(SEQ ID NO. 6), and GVYSLRS (SEQ ID NO. 7). It is interesting to
note that, in the experiments on fresh cells, binding increases are
much higher than in those performed with the cultured cell
lines.
Binding Overlay Experiments on Tissue Samples
[0192] Binding overlay assays with phages having the sequences
GIYRLRS (SEQ ID NO. 6), and GVYSLRS (SEQ ID NO. 7) were performed
on 64 tissue samples (tumor and metastasis tissues from 37
different patients): 18 samples of hepatic metastasis and cognate
healthy tissue; 4 samples of primary colon tumor; 2 samples of
primary rectum tumor; 2 samples of healthy colon; 3 samples of
primary breast tumor; 6 samples of primary ovary tumor with cognate
omental metastases (one with sigma metastasis); 2 lung metastases
secondary to colorectal tumors and 2 lung metastases secondary to
renal tumor. The results of all the assays is shown in Table 8.
TABLE-US-00008 TABLE 8 Tissue type Result % positive samples
hepatic meta secondary to colorectal tumor +++ 75 healthy colon - 0
healthy liver - 0 primary colorectal tumor - 0 primary ovary tumor
-+ 10 omental meta of ovary tumor -+ 10 sigma meta of ovary tumor -
0 lung meta of colorectal tumor - 0 lung meta of renal tumor -
0
Receptor Purification
[0193] The search for molecules specifically present on the
surfaces of the metastatic cells was performed by pull-down
experiments, using NCI-H630 (as a substrate, being positive to the
binding of 6 out of 7 phages) and HepG2 (as a control, being
negative to the binding of all the phages). The pull-down was
performed using the peptide GIYRLRS (SEQ ID NO. 6), present as a
fusion with the GST protein. This experiment was repeated three
times. In FIG. 3 a denaturing polyacrylamide gel is shown, in which
the GIYRLRS-GST-bound proteins (SEQ ID NO: 6) have been separated
and stained with Coomassie Blue. In the figure: MM, molecular
weight markers, HepG2, lysate of the HepG2 cells; NCI-H630, lysate
of the NCI-H630 cells; numbers 250, 150, 100, 75, 50, 37, 25
indicate the standard molecular weights; numbers from 1 to 9
indicate the bands analyzed. Proteins were identified by mass
spectrometry.
[0194] Obviously, the details of the realization and the
embodiments can be largely varied compared to what is here
described and illustrated, without exiting from the field of the
present invention, as defined by the claims included.
BIBLIOGRAPHY
[0195] 1. Arap, W., Pasqualini, R. & Ruoslahti, E. Chemotherapy
targeted to tumor vasculature. Curr. Opin. Oncol. 10, 560-565
(1998). [0196] 2. Pasqualini, R., Arap, W., Rajotte, D. &
Ruoslahti, E. in Phage display: a laboratory manual (eds. Barbas,
C. F., Burton, D. R., Scott, J. K. & Silverman, G. J.) 1-24
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2000). [0197] 3. Del Gatto, A. et al. Novel and selective alpha(v)
beta3 receptor peptide antagonist: design, synthesis, and
biological behavior. J Med Chem 49, 3416-20 (2006). [0198] 4.
Colombo, G. et al. Structure-activity relationships of linear and
cyclic peptides containing the NGR tumor-homing motif. J Biol Chem
277, 47891-7 (2002). [0199] 5. Corti, A. & Ponzoni, M. Tumor
vascular targeting with tumor necrosis factor alpha and
chemotherapeutic drugs. Ann N Y Acad Sci 1028, 104-12 (2004).
[0200] 6. Curnis, F. et al. Differential binding of drugs
containing the NGR motif to CD13 isoforms in tumor vessels,
epithelia, and myeloid cells. Cancer Res 62, 867-74 (2002). [0201]
7. Di Matteo, P. et al. Immunogenic and structural properties of
the Asn-Gly-Arg (NGR) tumor neovasculature-homing motif. Mol
Immunol 43, 1509-18 (2006). [0202] 8. Koivunen, E., Wang, B. &
Ruoslahti, E. Isolation of a highly specific ligand for the alpha 5
beta 1 integrin from a phage display library. J Cell Biol 124,
373-80 (1994). [0203] 9. Pasqualini, R. et al. Aminopeptidase N is
a receptor for tumor-homing peptides and a target for inhibiting
angiogenesis. Cancer Res 60, 722-7 (2000). [0204] 10. Pastorino, F.
et al. Vascular damage and anti-angiogenic effects of tumor
vessel-targeted liposomal chemotherapy. Cancer Res 63, 7400-9
(2003). [0205] 11. Burg, M. A., Pasqualini, R., Arap, W.,
Ruoslahti, E. & Stallcup, W. B. NG2 proteoglycan-binding
peptides target tumor neovasculature. Cancer Res. 59, 2869-2874
(1999). [0206] 12. Koivunen, E. et al. Tumor targeting with a
selective gelatinase inhibitor. Nat. Biotechnol. 17, 768-774
(1999). [0207] 13. Ellerby, H. M. et al. Anti-cancer activity of
targeted pro-apoptotic peptides. Nat. Med. 5, 1032-1038 (1999).
[0208] 14. Scott, J. K. & Smith, G. P. Searching for peptide
ligands with an epitope library. Science 249, 386-390 (1990).
[0209] 15. Smith, G. P. & Scott, J. K. Libraries of peptides
and proteins displayed on filamentous phage. Methods Enzymol. 217,
228-257 (1993).
Sequence CWU 1
1
208 1 7 PRT Homo sapiens PEPTIDE (1)..(7) 1 Ala Arg Pro Gly Leu Arg
Ser 1 5 2 7 PRT Homo sapiens PEPTIDE (1)..(7) 2 Met Arg Tyr Ala Leu
Arg Ser 1 5 3 7 PRT Homo sapiens PEPTIDE (1)..(7) 3 Leu Arg Pro Gly
Leu Arg Ser 1 5 4 7 PRT Homo sapiens PEPTIDE (1)..(7) 4 Leu Arg Ser
Gly Ser Gly Ser 1 5 5 7 PRT Homo sapiens PEPTIDE (1)..(7) 5 Val Arg
Ser Gly Arg Gly Ser 1 5 6 7 PRT Homo sapiens PEPTIDE (1)..(7)
MISC_FEATURE (6)..(6) May be fused with GST 6 Gly Ile Tyr Arg Leu
Arg Ser 1 5 7 7 PRT Homo sapiens PEPTIDE (1)..(7) 7 Gly Val Tyr Ser
Leu Arg Ser 1 5 8 7 PRT Homo sapiens PEPTIDE (1)..(7) 8 Lys Tyr Pro
Phe Asp Lys Leu 1 5 9 7 PRT Homo sapiens PEPTIDE (1)..(7) 9 Lys Val
Tyr Glu Ser Trp Ser 1 5 10 7 PRT Homo sapiens PEPTIDE (1)..(7) 10
Gly Leu Asp Thr Leu Leu Val 1 5 11 7 PRT Homo sapiens PEPTIDE
(1)..(7) 11 Gln Ser Arg Met Leu Arg Ile 1 5 12 7 PRT Homo sapiens
PEPTIDE (1)..(7) 12 Ala Ala Phe Leu Gln Gly Gly 1 5 13 7 PRT Homo
sapiens PEPTIDE (1)..(7) 13 Arg Ser Tyr Phe Glu Met Leu 1 5 14 7
PRT Homo sapiens PEPTIDE (1)..(7) 14 Tyr Leu His Leu Leu Pro Pro 1
5 15 7 PRT Homo sapiens PEPTIDE (1)..(7) 15 Arg Pro Thr Leu Ile Thr
Pro 1 5 16 7 PRT Homo sapiens PEPTIDE (1)..(7) 16 Ala Ser Arg Val
Arg Leu Pro 1 5 17 7 PRT Homo sapiens PEPTIDE (1)..(7) 17 Met Tyr
Val Val His Ala Asp 1 5 18 7 PRT Homo sapiens PEPTIDE (1)..(7) 18
Gly Pro Thr Leu Ile Lys Leu 1 5 19 7 PRT Homo sapiens PEPTIDE
(1)..(7) 19 Ala Pro Ala Leu Tyr His Val 1 5 20 7 PRT Homo sapiens
PEPTIDE (1)..(7) 20 Ser Val Asp Ser Gln Met Gly 1 5 21 7 PRT Homo
sapiens PEPTIDE (1)..(7) 21 Val Val Ser Met Val Gly Val 1 5 22 7
PRT Homo sapiens PEPTIDE (1)..(7) 22 His Leu Leu Ala Val Ser Tyr 1
5 23 7 PRT Homo sapiens PEPTIDE (1)..(7) 23 Pro Gly Cys Ala Leu Gly
Ser 1 5 24 7 PRT Homo sapiens PEPTIDE (1)..(7) 24 Ala Ala Gly Glu
Trp Ser Gly 1 5 25 7 PRT Homo sapiens PEPTIDE (1)..(7) 25 Pro Arg
Leu Gly His Gly Ser 1 5 26 7 PRT Homo sapiens PEPTIDE (1)..(7) 26
Arg Ala Gly Gly Gly Arg Leu 1 5 27 7 PRT Homo sapiens PEPTIDE
(1)..(7) 27 Leu Thr Val Arg Ala Val Asp 1 5 28 7 PRT Homo sapiens
PEPTIDE (1)..(7) 28 Pro Leu Gly Trp Leu Ser Tyr 1 5 29 7 PRT Homo
sapiens PEPTIDE (1)..(7) 29 Cys His Arg Thr Met Arg Asn 1 5 30 7
PRT Homo sapiens PEPTIDE (1)..(7) 30 Leu Arg Gly Gly Ile Gly Val 1
5 31 7 PRT Homo sapiens PEPTIDE (1)..(7) 31 Phe Phe Asp Gly Ala Gly
Ser 1 5 32 7 PRT Homo sapiens PEPTIDE (1)..(7) 32 Arg Arg Ile Asp
Asp Phe Arg 1 5 33 7 PRT Homo sapiens PEPTIDE (1)..(7) 33 His Leu
Ser Leu Ala Gly Leu 1 5 34 7 PRT Homo sapiens PEPTIDE (1)..(7) 34
Arg Pro Arg Thr Asp Thr Tyr 1 5 35 7 PRT Homo sapiens PEPTIDE
(1)..(7) 35 Phe Ser Gln Gly Lys Leu Ala 1 5 36 7 PRT Homo sapiens
PEPTIDE (1)..(7) 36 Thr Met Glu Thr Gly Gly Ser 1 5 37 7 PRT Homo
sapiens PEPTIDE (1)..(7) 37 Gly Val Arg Ser Val Arg Asn 1 5 38 7
PRT Homo sapiens PEPTIDE (1)..(7) 38 His Ser Gln Arg Phe Gly Lys 1
5 39 7 PRT Homo sapiens PEPTIDE (1)..(7) 39 Val Ser Ala Leu Glu Leu
Ser 1 5 40 7 PRT Homo sapiens PEPTIDE (1)..(7) 40 Ala Gly Met Val
Leu Trp Thr 1 5 41 7 PRT Homo sapiens PEPTIDE (1)..(7) 41 Pro Asp
Gly Arg Phe Gly Gly 1 5 42 7 PRT Homo sapiens PEPTIDE (1)..(7) 42
Glu Ser Pro Ser Arg His Thr 1 5 43 7 PRT Homo sapiens PEPTIDE
(1)..(7) 43 Ala Arg Gly Phe Pro Gly Val 1 5 44 7 PRT Homo sapiens
PEPTIDE (1)..(7) 44 Gln Ser Ser Ser Val Ile Leu 1 5 45 7 PRT Homo
sapiens PEPTIDE (1)..(7) 45 Arg Trp Thr Ser Ser Arg Ser 1 5 46 7
PRT Homo sapiens PEPTIDE (1)..(7) 46 Ala Tyr Thr Asn Phe Val Tyr 1
5 47 7 PRT Homo sapiens PEPTIDE (1)..(7) 47 Ser Val Leu Glu Asn Ala
Ile 1 5 48 7 PRT Homo sapiens PEPTIDE (1)..(7) 48 Leu Val Gly Asn
Phe Gly Leu 1 5 49 7 PRT Homo sapiens PEPTIDE (1)..(7) 49 Gly Leu
Val Gly Ser Arg Val 1 5 50 7 PRT Homo sapiens PEPTIDE (1)..(7) 50
Arg Thr Phe Ser Lys Leu Gly 1 5 51 7 PRT Homo sapiens PEPTIDE
(1)..(7) 51 Gly Ser Ile Val Met Leu Ser 1 5 52 7 PRT Homo sapiens
PEPTIDE (1)..(7) 52 Ala Gly Gly Gly Leu Leu Arg 1 5 53 7 PRT Homo
sapiens PEPTIDE (1)..(7) 53 Gly Val Arg Leu Leu Thr Ala 1 5 54 7
PRT Homo sapiens PEPTIDE (1)..(7) 54 Trp Gly Ala Glu Trp Ser Ser 1
5 55 7 PRT Homo sapiens PEPTIDE (1)..(7) 55 Val Arg Glu Asp Lys Gly
Ile 1 5 56 7 PRT Homo sapiens PEPTIDE (1)..(7) 56 Leu Phe Ile Leu
Val Ser Gly 1 5 57 7 PRT Homo sapiens PEPTIDE (1)..(7) 57 Ala Ser
Trp Thr Ala Arg Val 1 5 58 7 PRT Homo sapiens PEPTIDE (1)..(7) 58
Gly Arg Phe Met Gly Ala Phe 1 5 59 7 PRT Homo sapiens PEPTIDE
(1)..(7) 59 Asn Arg Thr Arg Phe Ser Ser 1 5 60 7 PRT Homo sapiens
PEPTIDE (1)..(7) 60 Val Leu Gly Ile Ala Val Ser 1 5 61 7 PRT Homo
sapiens PEPTIDE (1)..(7) 61 Glu Leu Ala Gln Ala Ile Ser 1 5 62 7
PRT Homo sapiens PEPTIDE (1)..(7) 62 Lys Ser Val Gly Gly Leu Gln 1
5 63 7 PRT Homo sapiens PEPTIDE (1)..(7) 63 Thr Cys Ser Arg Leu Leu
Thr 1 5 64 7 PRT Homo sapiens PEPTIDE (1)..(7) 64 Phe Cys Leu Leu
Cys His Met 1 5 65 7 PRT Homo sapiens PEPTIDE (1)..(7) 65 Asn Arg
Gly Arg Gly Tyr Leu 1 5 66 7 PRT Homo sapiens PEPTIDE (1)..(7) 66
Phe Phe Trp Ser Thr Ala Gln 1 5 67 7 PRT Homo sapiens PEPTIDE
(1)..(7) 67 Phe Leu Phe Trp Gly Arg Thr 1 5 68 7 PRT Homo sapiens
PEPTIDE (1)..(7) 68 Val Met Leu Ser Thr Gly Pro 1 5 69 7 PRT Homo
sapiens PEPTIDE (1)..(7) 69 Gly Ile Val Cys Leu Gly Arg 1 5 70 7
PRT Homo sapiens PEPTIDE (1)..(7) 70 Gly Val His Ser Arg Cys Gly 1
5 71 7 PRT Homo sapiens PEPTIDE (1)..(7) 71 Tyr Arg Gly Phe Pro Pro
Pro 1 5 72 7 PRT Homo sapiens PEPTIDE (1)..(7) 72 Ala Arg Gly Met
Pro Leu Phe 1 5 73 7 PRT Homo sapiens PEPTIDE (1)..(7) 73 Cys Arg
Asp Ser Cys Gly Arg 1 5 74 7 PRT Homo sapiens PEPTIDE (1)..(7) 74
Gly Leu Leu Cys Gly Arg Asp 1 5 75 7 PRT Homo sapiens PEPTIDE
(1)..(7) 75 Ile Arg Val Ser Tyr Gly Arg 1 5 76 7 PRT Homo sapiens
PEPTIDE (1)..(7) 76 Trp Arg Arg Val Gly Asp Leu 1 5 77 7 PRT Homo
sapiens PEPTIDE (1)..(7) 77 Leu Gly Ser Gly Ser Trp Pro 1 5 78 7
PRT Homo sapiens PEPTIDE (1)..(7) 78 Val Phe Ser Pro Val Asn Pro 1
5 79 7 PRT Homo sapiens PEPTIDE (1)..(7) 79 Ser Leu Gln Ser Val Val
Ala 1 5 80 7 PRT Homo sapiens PEPTIDE (1)..(7) 80 Ile Arg Gly Ile
Gly Gly Ala 1 5 81 7 PRT Homo sapiens PEPTIDE (1)..(7) 81 Lys Val
Phe Ala Arg Leu Gly 1 5 82 7 PRT Homo sapiens PEPTIDE (1)..(7) 82
Val Gly Arg Thr Val Ile Gln 1 5 83 7 PRT Homo sapiens PEPTIDE
(1)..(7) 83 Gly Leu Pro Arg Leu Ser Gly 1 5 84 7 PRT Homo sapiens
PEPTIDE (1)..(7) 84 Asp Cys Val Trp Asp Cys Met 1 5 85 7 PRT Homo
sapiens PEPTIDE (1)..(7) 85 Gly Leu Gly Ile Tyr Val Leu 1 5 86 7
PRT Homo sapiens PEPTIDE (1)..(7) 86 Phe Phe Ile Thr Pro Arg Ser 1
5 87 7 PRT Homo sapiens PEPTIDE (1)..(7) 87 Met Gly Gly Ser Leu Phe
Gly 1 5 88 7 PRT Homo sapiens PEPTIDE (1)..(7) 88 Ala Ala Arg Tyr
Gly Ile Asp 1 5 89 7 PRT Homo sapiens PEPTIDE (1)..(7) 89 Trp Arg
Arg Ser Glu Arg Thr 1 5 90 7 PRT Homo sapiens PEPTIDE (1)..(7) 90
Lys Leu Ser Gly Val Ser Leu 1 5 91 7 PRT Homo sapiens PEPTIDE
(1)..(7) 91 Trp Val Gly Gly Ile Arg Gly 1 5 92 7 PRT Homo sapiens
PEPTIDE (1)..(7) 92 Ile Pro Arg Ser Thr Phe Gly 1 5 93 7 PRT Homo
sapiens PEPTIDE (1)..(7) 93 Val Cys Trp Ala Ser Trp Cys 1 5 94 7
PRT Homo sapiens PEPTIDE (1)..(7) 94 Val Arg Ala Ser Pro Ser Leu 1
5 95 7 PRT Homo sapiens PEPTIDE (1)..(7) 95 Pro Leu Leu Tyr Arg Asn
Ala 1 5 96 7 PRT Homo sapiens PEPTIDE (1)..(7) 96 Leu Arg Ser Gly
Arg Gly Ser 1 5 97 7 PRT Homo sapiens PEPTIDE (1)..(7) 97 Trp Ala
Leu Thr Thr Ala Leu 1 5 98 7 PRT Homo sapiens PEPTIDE (1)..(7) 98
Ile Val Phe Gly Arg Gly Ser 1 5 99 7 PRT Homo sapiens PEPTIDE
(1)..(7) 99 Met Arg Val Phe Gly Gly Val 1 5 100 7 PRT Homo sapiens
PEPTIDE (1)..(7) 100 Val Leu Gly Ser Leu Gly Ser 1 5 101 7 PRT Homo
sapiens PEPTIDE (1)..(7) 101 Leu Trp Ser Glu Pro Met Val 1 5 102 7
PRT Homo sapiens PEPTIDE (1)..(7) 102 Glu Arg Ala Pro Leu Lys Ala 1
5 103 7 PRT Homo sapiens PEPTIDE (1)..(7) 103 Ile Ser Arg Phe Gly
Tyr Val 1 5 104 7 PRT Homo sapiens PEPTIDE (1)..(7) 104 Gly Leu Lys
Phe Asn Trp Ser 1 5 105 7 PRT Homo sapiens PEPTIDE (1)..(7) 105 Lys
Ser Ser Glu Ile Pro Arg 1 5 106 7 PRT Homo sapiens PEPTIDE (1)..(7)
106 Arg Arg Ala Leu Phe Ala Thr 1 5 107 7 PRT Homo sapiens PEPTIDE
(1)..(7) 107 Gly Trp Arg Gly Leu Arg Thr 1 5 108 7 PRT Homo sapiens
PEPTIDE (1)..(7) 108 Asp Tyr Phe Trp Phe Ala Asp 1 5 109 7 PRT Homo
sapiens PEPTIDE (1)..(7) 109 Ser Arg Tyr Trp Thr Arg Ser 1 5 110 7
PRT Homo sapiens PEPTIDE (1)..(7) 110 Arg Arg Glu Gly Leu Arg Ser 1
5 111 7 PRT Homo sapiens PEPTIDE (1)..(7) 111 Ser Trp Tyr Thr Leu
Arg Ser 1 5 112 7 PRT Homo sapiens PEPTIDE (1)..(7) 112 Val Ser Met
Ser Arg Ser Leu 1 5 113 7 PRT Homo sapiens PEPTIDE (1)..(7) 113 Leu
Ala Tyr Arg Leu Arg Ser 1 5 114 7 PRT Homo sapiens PEPTIDE (1)..(7)
114 Val Tyr Tyr Gly Leu Arg Arg 1 5 115 7 PRT Homo sapiens PEPTIDE
(1)..(7) 115 Leu Thr Tyr Arg Leu Arg Ser 1 5 116 7 PRT Homo sapiens
PEPTIDE (1)..(7) 116 Leu Leu Tyr Gly Leu Glu Trp 1 5 117 7 PRT Homo
sapiens PEPTIDE (1)..(7) 117 Val Arg Pro Gly Leu Arg Ser 1 5 118 7
PRT Homo sapiens PEPTIDE (1)..(7) 118 Ile Arg Ser Gly Phe Gly Ser 1
5 119 7 PRT Homo sapiens PEPTIDE (1)..(7) 119 Leu Arg Ser Gly Ser
Gly Ser 1 5 120 7 PRT Homo sapiens PEPTIDE (1)..(7) 120 Ala Gly Phe
Gly Met Leu Leu 1 5 121 7 PRT Homo sapiens PEPTIDE (1)..(7) 121 Val
Leu Gly Phe Ser Pro Trp 1 5 122 7 PRT Homo sapiens PEPTIDE (1)..(7)
122 His Arg Arg Asp His Pro Glu 1 5 123 7 PRT Homo sapiens PEPTIDE
(1)..(7) 123 Ala Arg Gly Leu Gln Arg Arg 1 5 124 7 PRT Homo sapiens
PEPTIDE (1)..(7) 124 Gly Val Gly Ala Arg Arg Ser 1 5 125 7 PRT Homo
sapiens PEPTIDE (1)..(7) 125 Gly Met Ile Val Val Gly Gly 1 5 126 7
PRT Homo sapiens PEPTIDE (1)..(7) 126 Arg Arg Tyr Ser Ala Asp Ser 1
5 127 7 PRT Homo sapiens PEPTIDE (1)..(7) 127 Ser Glu Leu Gly Gly
Gly Asp 1 5 128 7 PRT Homo sapiens PEPTIDE (1)..(7) 128 Ala Gly Leu
Ser Ala Asp Ile 1 5 129 7 PRT Homo sapiens PEPTIDE (1)..(7) 129 Thr
Ser Gly Gly Gly Ile Val 1 5 130 7 PRT Homo sapiens PEPTIDE (1)..(7)
130 Val Leu Phe Gln Val Gln Pro 1 5 131 7 PRT Homo sapiens PEPTIDE
(1)..(7) 131 Asp Arg Val Thr Gly Ala Trp 1 5 132 7 PRT Homo sapiens
PEPTIDE (1)..(7) 132 Val Val Glu Val Ala Ser Thr 1 5 133 7 PRT Homo
sapiens PEPTIDE (1)..(7) 133 Ala Val Gln Asp Pro Arg Arg 1 5 134 7
PRT Homo sapiens PEPTIDE (1)..(7) 134 Gly Pro Val Thr Ile Asp Gly 1
5 135 7 PRT Homo sapiens PEPTIDE (1)..(7) 135 Phe Lys Gly Pro Arg
Leu Met 1 5 136 7 PRT Homo sapiens PEPTIDE (1)..(7) 136 Tyr Arg Met
Ile Ala Asp Trp 1 5 137 7 PRT Homo sapiens PEPTIDE (1)..(7) 137 Phe
Ile Leu Gly Val Arg Asp 1 5 138 7 PRT Homo sapiens PEPTIDE (1)..(7)
138 Gln Thr Thr Tyr Gly Asp Pro 1 5 139 7 PRT Homo sapiens PEPTIDE
(1)..(7) 139 Gly Gly Ala Val Asn Val Tyr 1 5 140 7 PRT Homo sapiens
PEPTIDE (1)..(7) 140 Asp Val Ile Ser Asp Pro Leu 1 5 141 7 PRT Homo
sapiens PEPTIDE (1)..(7) 141 Val Ile Val Gly Val Trp Phe 1 5 142 7
PRT Homo sapiens PEPTIDE (1)..(7) 142 Gly Gly Ile Trp Val Val Ile 1
5 143 7 PRT Homo sapiens PEPTIDE (1)..(7) 143 Val Glu Ala Pro Asp
Gly Thr 1 5 144 7 PRT Homo sapiens PEPTIDE (1)..(7) 144 Leu Arg Phe
Val Gly Pro Arg 1 5 145 7 PRT Homo sapiens PEPTIDE (1)..(7) 145 Phe
Asp Glu Arg Gly Ser Phe 1 5 146 7 PRT Homo sapiens PEPTIDE (1)..(7)
146 Ala Gly Gly Thr Leu Gly Val 1 5 147 7 PRT Homo sapiens PEPTIDE
(1)..(7) 147 Gly Thr Arg Leu Val Leu Ser 1 5 148 7 PRT Homo sapiens
PEPTIDE (1)..(7) 148 Trp Gly Val Leu Val Arg Asp 1 5 149 7 PRT Homo
sapiens PEPTIDE (1)..(7) 149 Lys Arg Ile Glu Asp Glu Pro 1 5 150 7
PRT Homo sapiens PEPTIDE (1)..(7) 150 Arg Arg Thr Ser Ile Met Ala 1
5 151 7 PRT Homo sapiens PEPTIDE (1)..(7) 151 Glu Glu Phe Gln Ser
Pro Asp 1 5 152 7 PRT Homo sapiens PEPTIDE (1)..(7) 152 Leu Pro Arg
Ala Val Val Glu 1 5 153 8 PRT Homo sapiens PEPTIDE (1)..(8) 153 Pro
Tyr Glu Gly Pro Met Pro Trp 1 5 154 8 PRT Homo sapiens PEPTIDE
(1)..(8) 154 Gln Gly Gly Glu Thr Gly Tyr Glu 1 5 155 8 PRT Homo
sapiens PEPTIDE (1)..(8) 155 Asn Gln Ser Leu Pro Ser Gly Asn 1 5
156 8 PRT Homo sapiens PEPTIDE (1)..(8) 156 Gly Ala Gln Ser Thr Ser
Ser Gln 1 5 157 8 PRT Homo sapiens PEPTIDE (1)..(8) 157 Pro Ser Ser
Asn Arg Trp Phe Pro 1 5 158 8 PRT Homo sapiens PEPTIDE (1)..(8) 158
Ala Leu Lys Ala Tyr His Leu Pro 1 5 159 8 PRT Homo sapiens PEPTIDE
(1)..(8) 159 Gly Glu Ser Ala Ala Arg Val His 1 5 160 8 PRT Homo
sapiens PEPTIDE (1)..(8) 160 Gln Pro Asp Asn Lys His Leu Phe 1 5
161 8 PRT Homo sapiens PEPTIDE (1)..(8) 161 Thr Ala Leu Lys Pro Ser
Phe His 1 5 162 8 PRT Homo sapiens PEPTIDE (1)..(8) 162 Tyr Asn Arg
Asp Thr Ser Leu Met 1 5 163 8 PRT Homo sapiens PEPTIDE (1)..(8) 163
Thr Ser Ala Pro Thr Tyr Glu Ser 1 5 164 8 PRT Homo sapiens PEPTIDE
(1)..(8) 164 Leu His His Arg Tyr Gln Lys Gln 1 5 165 8 PRT Homo
sapiens PEPTIDE (1)..(8) 165 Pro Tyr Ser Arg Asn Thr Leu Cys 1 5
166 8 PRT Homo sapiens PEPTIDE (1)..(8) 166 Asn Cys Ala Lys Leu Pro
Cys Val 1 5 167 8 PRT Homo sapiens PEPTIDE (1)..(8) 167 Tyr Ala Leu
Thr Val Asn Leu Gly 1 5 168 8 PRT Homo sapiens PEPTIDE (1)..(8) 168
Gly Leu Ser Pro Ser Gly Glu Gln 1 5 169 8 PRT Homo sapiens PEPTIDE
(1)..(8) 169 Lys Asn Ser Glu Ala Met Phe Thr 1 5 170 8 PRT Homo
sapiens PEPTIDE (1)..(8) 170 Lys Trp Ala Asp Cys Arg Arg Pro 1 5
171 11 PRT Homo sapiens PEPTIDE (1)..(11) 171 Trp Pro Pro Cys Gly
Trp Gly Cys Arg Gly Arg 1 5 10 172 11 PRT Homo sapiens PEPTIDE
(1)..(11) 172 Ser Ile Ser Cys Leu Trp Gly Cys Gly Ser Trp 1 5 10
173 11 PRT Homo sapiens PEPTIDE (1)..(11) 173 Gly Met Gly Cys Leu
Gly Leu Cys Gly Gly Ser 1 5 10 174 11 PRT Homo sapiens PEPTIDE
(1)..(11) 174 Gly Asp Gly Cys Pro Glu Val Cys Val Phe Pro 1 5 10
175 11 PRT Homo sapiens PEPTIDE (1)..(11) 175 Tyr Glu Met Cys Asp
Leu Ser Cys Val Tyr Trp 1 5 10 176 11 PRT Homo sapiens PEPTIDE
(1)..(11) 176 Arg Met Pro Cys Ser Val Ser Cys Asp Leu Met 1 5 10
177 11 PRT Homo sapiens PEPTIDE (1)..(11) 177 Gly Asn Ser Cys Ser
Leu His Cys Tyr Ile Trp 1 5 10 178 11 PRT Homo sapiens PEPTIDE
(1)..(11) 178 Ala Arg Leu Cys Gly Gly Ala Cys Arg Gly Leu 1 5 10
179 11 PRT Homo sapiens PEPTIDE (1)..(11) 179 Gly Glu Glu Cys Ala
Pro Gly Cys Thr Arg Gly 1 5 10 180 11 PRT Homo sapiens PEPTIDE
(1)..(11) 180 Asp Val Asp Cys Arg His Leu Cys Asn Val His 1 5 10
181 11 PRT Homo sapiens PEPTIDE (1)..(11) 181 Pro Gln Leu Cys Gly
Gly Thr Cys Arg Gly Leu 1 5 10 182 11 PRT Homo sapiens PEPTIDE
(1)..(11) 182 Val Ala Gly Cys Pro Val Gly Cys Ile Arg Gly 1 5 10
183 11 PRT Homo sapiens PEPTIDE (1)..(11) 183 Leu Gly Tyr Cys Ser
Trp Gly Cys Ala Arg Glu 1 5 10 184 11 PRT Homo sapiens PEPTIDE
(1)..(11) 184 Trp Pro Ala Cys Ser Pro Glu Cys Arg Trp Pro 1 5 10
185 11 PRT Homo sapiens PEPTIDE (1)..(11) 185 Thr Ala Gly Cys Gly
Ser Met Cys Leu His Val 1 5 10 186 11 PRT Homo sapiens PEPTIDE
(1)..(11) 186 Leu Phe Leu Cys Val Phe Gly Cys Ala Leu Val 1 5 10
187 11 PRT Homo sapiens PEPTIDE (1)..(11) 187 Asp Val Gln Cys Tyr
Val Arg Cys Ser Pro Asp 1 5 10 188 11 PRT Homo sapiens PEPTIDE
(1)..(11) 188 Gly Gly Val Cys Leu Gly Arg Cys Leu Gly Gly 1 5 10
189 11 PRT Homo sapiens PEPTIDE (1)..(11) 189 Trp Arg Val Cys Gly
Ala Leu Cys Gly Pro Ala 1 5 10 190 11 PRT Homo sapiens PEPTIDE
(1)..(11) 190 Ser Gly Arg Cys Leu Gly Val Cys Gly Trp Ala 1 5 10
191 11 PRT Homo sapiens PEPTIDE (1)..(11) 191 Ala Glu Arg Cys Arg
Met Asn Cys Met Lys Pro 1 5 10 192 11 PRT Homo sapiens PEPTIDE
(1)..(11) 192 Arg Lys Ser Cys Ser Gly Ala Cys Val Trp Gly 1 5
10 193 11 PRT Homo sapiens PEPTIDE (1)..(11) 193 Gly Ala Ala Cys
Gly Ser Gly Cys Leu His Val 1 5 10 194 11 PRT Homo sapiens PEPTIDE
(1)..(11) 194 Thr Gly Ala Cys Ile Pro Gly Cys Gly Gly Trp 1 5 10
195 11 PRT Homo sapiens PEPTIDE (1)..(11) 195 Gln Ala Pro Cys Val
Ser Gly Cys Gly Val Asp 1 5 10 196 11 PRT Homo sapiens PEPTIDE
(1)..(11) 196 Arg Arg Trp Cys Gly Thr Leu Cys Leu Cys Trp 1 5 10
197 11 PRT Homo sapiens PEPTIDE (1)..(11) 197 Tyr Ile Thr Cys Gly
His Asp Cys Val Thr Phe 1 5 10 198 11 PRT Homo sapiens PEPTIDE
(1)..(11) 198 Arg Arg Ser Cys Gly Phe Ser Cys Val Ala Gly 1 5 10
199 11 PRT Homo sapiens PEPTIDE (1)..(11) 199 Leu Arg Val Cys Asn
Val Asp Cys Met Thr Gly 1 5 10 200 11 PRT Homo sapiens PEPTIDE
(1)..(11) 200 Ser Leu Phe Cys Gln Ile Asp Cys Val Met Trp 1 5 10
201 11 PRT Homo sapiens PEPTIDE (1)..(11) 201 Trp Asp Val Cys Leu
Ser Asp Cys Val Phe Asn 1 5 10 202 20 DNA Homo sapiens Primer 202
ccctcatagt tagcgtaacg 20 203 8 PRT Homo sapiens MISC_FEATURE
(1)..(8) RGD-4C 203 Cys Asp Arg Gly Asp Cys Phe Cys 1 5 204 6 PRT
Homo sapiens MISC_FEATURE (1)..(6) From CX6C library 204 Leu Arg
Ser Gly Arg Gly 1 5 205 4 PRT Homo sapiens MISC_FEATURE (1)..(4)
Family-identifying motif from CX9 collection 205 His Trp Gly Phe 1
206 14 PRT Homo sapiens MISC_FEATURE (1)..(14) One of a family of
peptides which result in destruction of mitochondrial membranes 206
Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10 207
5 PRT Homo sapiens MISC_FEATURE (1)..(5) Conjugate peptide used in
chemotherapy 207 Cys Asn Gly Arg Cys 1 5 208 4 PRT Homo sapiens
MISC_FEATURE (1)..(4) Shared sequence motif 208 Gly Ser Gly Ser
1
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