U.S. patent application number 15/527717 was filed with the patent office on 2017-11-09 for peptides for targeting gastric cancer, and medical use tehreof.
This patent application is currently assigned to UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERATION. The applicant listed for this patent is UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERRATION. Invention is credited to Eun Kyung CHOI, Seong-Yun JEONG, Eun Jin JU, Kyoung Jin LEE, Seol Hwa SHIN, Si Yeol SONG.
Application Number | 20170322213 15/527717 |
Document ID | / |
Family ID | 56106110 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170322213 |
Kind Code |
A1 |
CHOI; Eun Kyung ; et
al. |
November 9, 2017 |
PEPTIDES FOR TARGETING GASTRIC CANCER, AND MEDICAL USE TEHREOF
Abstract
Provided is a peptide for targeting gastric cancer, a
composition for diagnosing radioresponsiveness-dependent gastric
cancer using the peptide, and a drug delivery use of the peptide,
wherein a functional peptide capable of targeting cancer has been
discovered so as to implement personalized diagnosis and treatment
for individual patients having cancer, consideration of problems
occurring during treatment in which treatment cases of respective
patients differ due to different therapeutic responses resulting
from genetic differences in the individual patients.
Inventors: |
CHOI; Eun Kyung; (Seoul,
KR) ; JEONG; Seong-Yun; (Yongin-si, Gyeonggi-do,
KR) ; SONG; Si Yeol; (Seoul, KR) ; LEE; Kyoung
Jin; (Seoul, KR) ; SHIN; Seol Hwa; (Seoul,
KR) ; JU; Eun Jin; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF ULSAN FOUNDATION FOR INDUSTRY COOPERRATION |
Ulsan |
|
KR |
|
|
Assignee: |
UNIVERSITY OF ULSAN FOUNDATION FOR
INDUSTRY COOPERATION
Ulsan
KR
|
Family ID: |
56106110 |
Appl. No.: |
15/527717 |
Filed: |
July 29, 2015 |
PCT Filed: |
July 29, 2015 |
PCT NO: |
PCT/KR2015/007943 |
371 Date: |
May 18, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/58 20130101;
G01N 33/574 20130101; C07K 7/08 20130101; G01N 33/00 20130101; A61K
49/0008 20130101; A61K 47/00 20130101; G01N 33/57446 20130101; A61K
49/00 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/58 20060101 G01N033/58; C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 2014 |
KR |
10-2014-0160818 |
Jul 28, 2015 |
KR |
10-2015-0106581 |
Claims
1. A peptide for targeting gastric cancer, the peptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 1 to 6.
2. The peptide of claim 1, wherein the peptide comprising an amino
acid selected from the group consisting of SEQ ID NOs: 1 to 3 is
specific to an irradiated gastric cancer tissue.
3. A polynucleotide encoding the peptide of claim 1.
4. A method for diagnosing gastric cancer, comprising: obtaining a
gastric cancer tissue sample from a patient; transplanting the
tissue sample into a subject; applying the composition comprising
the peptide of claim 1 to the subject; and identifying the presence
of the gastric cancer.
5. A method for diagnosing radio-sensitive gastric cancer,
comprising: obtaining a gastric cancer tissue sample from a
patient; transplanting the tissue sample into a subject; applying
the composition comprising the peptide of claim of 2 to the
subject; and identifying the presence of the radio-sensitive
gastric cancer.
6. The method of claim 4, wherein the peptide is labeled with one
selected from the group consisting of a chromogenic enzyme, a
radioactive isotope, a chromopore, and a luminescent or fluorescent
material.
7. A composition for delivering a drug, the composition comprising
the peptide of claim 1.
8. The composition of claim 7, wherein the drug comprises an
anticancer drug.
Description
TECHNICAL FIELD
[0001] The present invention relates to a peptide for targeting
gastric cancer, a composition for diagnosing gastric cancer based
on performance of irradiation using the peptide, and drug delivery
use of the peptide.
BACKGROUND ART
[0002] Cells which are the smallest unit of the human body maintain
the balance of cell number by cell division upon intracellular
regulatory functions, cell growth, and cell death and disappear,
when cells are normal. If the cells are damaged by any cause, cells
may be recovered by treatment to thereby serve as normal cells.
However, if cells are not recovered, cells die by themselves. A
condition in which abnormal cells that do not control proliferation
and inhibition thereof for a variety of reasons are excessively
proliferated and also cause tumefaction and destruction of normal
tissues by invading surrounding tissues and organs is defined as
cancer. As such, cancer refers to cell proliferation that is not
inhibited, and cancer destroys the structure and function of normal
cells and organs. In this regard, it is significantly important to
diagnose and treat cancer.
[0003] However, there are problems during treatment in which
treatment cases of respective patients differ due to different
therapeutic responses resulting from genetic differences in the
individual patients having cancer. Thus, in order to effectively
treat cancer patients, it is required to develop a functional
targeting agent capable of targeting tumor microenvironment, which
depends on radioresponsiveness, and a biomarker. Accordingly, it is
possible to establish personalized diagnosis and treatment for
individual patients.
[0004] In addition, drug delivery systems or targeted therapies
that selectively deliver drugs to cancer cells and cancer tissues
are technologies that have received much attention, because even if
the same amount of an anticancer agent is used, drug efficacy may
be increased while side effects of drugs on normal tissues may be
significantly reduced at the same time. In addition, when such
technologies are applied to gene therapy, selective delivery of
virus to cancer cells can increase treatment efficacy and reduce
severe side effects. For this purpose, antigens that are mainly
specific to tumor cells and antibodies that target such antigens
have been developed up to date. However, in the case of antibodies,
there are problems including concerns of immune response and low
efficiency of penetration into tissues. In the case of peptides, a
molecular weight thereof is so small that there is less concern of
an immune responses and the penetration of peptides into tissues is
easy. Therefore, if cancer-targeting peptides are coupled with
existing anticancer drugs, such resulting products can be utilized
as intelligent drug vehicles that selectively deliver drugs to
tumors.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0005] The present invention, unlike screening methods that have
been studied at the existing cell culture levels, establishes mouse
models transplanted with a cancer tissue of an actual human, to
thereby divide them into an irradiated population and a
non-irradiated population as a control group. In addition, a method
of screening a peptide that specifically binds to each population
above is disclosed to provide a novel peptide for targeting gastric
cancer and a medical use of such a novel peptide.
Technical Solution
[0006] To solve the technical problem above, the present invention
provides a peptide for targeting gastric cancer and a
polynucleotide encoding the peptide, the peptide including an amino
acid sequence selected from the group consisting of SEQ ID NOs: 1
to 6.
[0007] In an embodiment, the present invention provides a peptide
for targeting gastric cancer, the peptide including an amino acid
selected from the group consisting of SEQ ID NOs: 1 to 3.
[0008] In an embodiment, the present invention provides a
composition including the peptide for diagnosing gastric cancer and
a composition including the peptide for diagnosing radio-reactive
gastric cancer.
[0009] the present invention provides a composition including the
peptide for delivering a drug.
Advantageous Effects of the Invention
[0010] present invention relates to a peptide for targeting
gastric, a composition for diagnosing radioresponsiveness-dependent
gastric cancer using the peptide, and a drug delivery use of the
peptide. Considering problems during treatment in which treatment
cases of respective patients differ due to different therapeutic
responses resulting from genetic differences in the individual
patients having cancer, a functional peptide capable of targeting
cancer has been discovered so as to establish personalized
diagnosis and treatment for individual patients. Animal models
similar to cancer microenvironments of actual patients having
cancer are prepared and divided into irradiated populations and
non-irradiated populations as a control group, to thereby test
target efficiency for respective peptides that are selected by
screening peptides specifically binding to the respective
populations. As such, the present invention can be finally utilized
in the technical development of image diagnosis for predicting
responsiveness to radiotherapy, and accordingly, the development of
customized targeted therapeutic agents.
DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is an image showing a method of establishing an
irradiated animal model after transplanting an actual patient's
gastric cancer tissue into a mouse according to Example 1 of the
present invention, and also showing confirmation results of the
established animal model. FIG. 1A shows a patient's gastric cancer
tissue distributed from the Bio Research Center (BRC, Korea), FIG.
1B shows a NOD/SCID mouse that undergoes heterotrophic
transplantation into the flanks of the mouse, FIG. 1C shows a
cancer tissue cut into pieces each having a size of 3.times.3 mm to
be used for subculturing, when the size of the cancer tissue of
FIG. 1B is increased up to 500 mm.sup.3, FIG. 1D shows a mouse
model prepared in a way that a nude mouse is anesthetized via
intraperitoneal injection and undergoes heterotrophic
transplantation of one piece of the cut tissues subcutaneously on
the both thighs, and FIG. 1E shows irradiation of 10 grays (Gy) of
radiation over the thigh portions where the cancer cell is formed,
when the size of the cancer tissue is increased up to 150-200
mm.sup.3. Here, a control group is not subjected to
irradiation.
[0012] FIG. 2 shows a biopanning scheme for identifying a sequence
of a peptide, which targets a gastric cancer tissue of an
irradiated in vivo patient, by using an M13 phage display method
according to embodiments of the present invention.
[0013] FIG. 3 shows results of comparing phage concentrations
obtained by eluting phages after extracting heart, lung, liver,
spleen, kidney, and tumor during biopanning process performed five
times.
[0014] FIG. 4 shows results of a linking proportion between a Cy5.5
fluorescent probe and a phage by calculating phages of the same
concentration after a discovered peptide-expressing phage of the
present invention is amplified in terms of linking with the Cy5.5
fluorescent probe, and by calculating region of interest (ROI)
values in connection with linking between the Cy5.5 fluorescent
probe and the phage.
[0015] FIG. 5 shows results confirming specific binding to in vivo
gastric cancer tissue based on images on the 2.sup.nd day after
injecting a peptide phage labeled with a Cy5.5 fluorescent probe
into each mouse mode.
[0016] FIG. 6 shows results confirming fluorescence intensity of
cancer tissue after only cancer tissue is extracted and also
confirming places where fluorescence is located on cancer tissue
that is equally divided, as in vivo image confirmation is completed
on the 2.sup.nd day.
[0017] FIG. 7 shows a schematic diagram for observing changes in
targeting ability of a peptide phage of the present invention as
being selected depending on irradiation. In detail, after a
patient's gastric cancer tissue is transplanted, mouse models in
which the size of the cancer tissue is increased up to 150-200
mm.sup.3 are divided into 1) irradiated mouse models and 2)
irradiated mouse models with 2 grays (Gy) of radiation. After
having recovery time is provided for no longer than 24 hours to the
mice irradiated with 10 Gy of radiation, a selected peptide phage
sample was injected thereto, and in vivo imaging are examined until
the 2.sup.nd day of the injection.
[0018] FIG. 8 shows results verifying specific binding ability of a
peptide sequence discovered in each population through
immunohistochemistry.
[0019] FIG. 9 shows preparation of a liposome, a drug encapsulation
process and optimization thereof. As a result of verifying a
liposome manufacturing process, a drug encapsulation process, and
size distribution of drug, it is confirmed that there is no
difference in size before and after drug encapsulation and that
drugs are evenly distributed.
[0020] FIG. 10 shows results of linking a peptide to a liposome
including to a liposome including a drug encapsulated therein. FIG.
10A is a schematic diagram showing linking of a peptide to a
liposome including a drug encapsulated therein and also shows a
chemical constitutional formula representing actually linked
residues, and FIG. 10B shows results of a reduction test to
calculate the number of --SH residues in a liposome before being
linked to a peptide and also shows results confirming stability
through verification of the size distribution after being linked to
a peptide.
[0021] FIG. 11 shows in vivo imaging results for verifying
targeting ability of a material in which a peptide is linked to a
liposome including a drug encapsulated therein. It is confirmed
that only a liposome linked to a peptide is targeted in an
irradiated mouse.
[0022] FIG. 12 shows in vivo tumor growth delay results for
verifying possibility of a material in which a peptide is linked to
a liposome including a drug encapsulated therein to be used as an
anticancer drug. In this regard, only a liposome linked to a
peptide and including a drug encapsulated therein is proved to be
effective in treating tumors in an irradiated group.
[0023] FIG. 13 shows results confirming targeting ability of a
selected peptide upon irradiation to mice transplanted with other
patient's gastric cancer tissue according to Example 8 of the
present invention.
BEST MODE
[0024] The present invention provides a peptide for targeting
gastric cancer, the peptide including an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1 TO 6. The
above-mentioned amino acid sequences are shown in Table 1.
[0025] The peptide of the present invention is a low-molecular
weight peptide consisting of 7 amino acids. Such a low-molecular
weight peptide is small in size so that it can be stabilized
three-dimensionally. In addition, a low-molecular weight peptide
has the advantage of being able to easily pass through a membrane
and to recognize a target molecule deep in tissues. Since the
stability of the low-molecular weight peptide of the present
invention is secured through local injection and the
immunoreactivity can be minimized, there is an advantage that
cancer can be diagnosed early. In addition, the mass production of
the low-molecular weight peptide of the present invention is
relatively easy compared that of an antibody, and the toxicity of
the low-molecular weight peptide of the present invention is
weak.
[0026] In addition, the low-molecular weight peptide of the present
invention is has an advantage of a strong binding force to a target
material compared to an antibody, and do not undergo denaturation
during thermal/chemical treatment. In addition, due to a small
molecular size, the low-molecular weight peptide can be used as a
fused protein as being attached to other proteins. In detail, the
low-molecular weight peptide can be also used as being attached to
a high-molecular weight protein chain, and accordingly, can be used
as a diagnosis kit and a drug delivery carrier.
[0027] The low-molecular weight peptide of the present invention
can be easily prepared according to the chemical process known in
the art (Creighton, Proteins; Structures and Molecular Principles,
W. H. Freeman and Co., NY, 1983). As representative methods, liquid
or solid phase synthesis, fractional condensation, F-MOC or T-BOC
chemical method, or the like may be used (Chemical Approaches to
the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC
Press, Boca Raton Fla., 1997; A Practical Approach, Athert on &
Sheppard, Eds., IRL Press, Oxford, England, 1989), but the method
is not limited thereto.
[0028] In addition, the low-molecular weight peptide of the present
invention can be prepared according to a genetic engineering
method. First, according to a conventional method, a DNA sequence
encoding the sequence low-molecular weight peptide is prepared.
Here, a DNA sequence can be prepared by PCR amplification using an
appropriate primer. Alternatively, according to a standard method
known in the art, a DNA sequence can be synthesized using, for
example, an automatic DNA synthesizer (manufactured by Biosearch or
AppliedBiosystems). Such a synthesized DNA sequence is inserted to
a vector including one or more expression control sequences (for
example: a promoter, an enhancer, or the like) that are operatively
linked with the DNA sequence to control expression of the DNA
sequence, and then, a host cell is transformed with a recombinant
expression vector prepared therefrom. A resulting transformant is
cultured in an appropriate medium under suitable conditions to
allow the expression of the DNA sequence, so that substantially
pure peptides that are encoded by the DAN sequence are recovered
from the culture. Such recovery may be performed according to a
method known in the art (for example, chromatography). The term
`substantially pure peptides` used herein refers to peptides that
do not substantially include any protein derived from the host.
[0029] In addition, the present invention provides a peptide for
targeting gastric cancer, the peptide being specific to an
irradiated gastric cancer tissue and including an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1 to
3.
[0030] In detail, a peptide including an amino acid sequence
selected from the group consisting of SEQ ID NOs: 1 to 3
specifically binds to a gastric cancer tissue, in particular, an
irradiated gastric cancer tissue.
[0031] The term "target" or "specific" used herein refers to
ability to specifically bind only to a gastric cancer tissue,
especially an irradiated gastric cancer tissue, without binding to
other normal tissues. A gastric cancer-specific peptide can
specifically bind to the inside or outside of a gastric cancer
tissue.
[0032] In addition, the present invention provides a polynucleotide
encoding an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1 to 3.
[0033] The term "polynucleotide" used herein refers to a
single-stranded or double-stranded polymer of deoxyribonucleotides
or ribonucleotides. Such a polynucleotide includes a RNA genome
sequence, a DNA sequence (for example, gDNA and cDNA), and a RNA
sequence transcribed from the DNA sequence. Unless otherwise
mentioned, a polynucleotide includes an analog of a natural
polynucleotide.
[0034] The polynucleotide includes not only a nucleotide sequence
that encodes the peptide for targeting gastric cancer, but also a
sequence complementary to the nucleotide sequence, wherein such a
complementary sequence includes not only a perfectly complementary
sequence, but also a substantially complementary sequence.
[0035] In addition, the polynucleotide may be subjected to
modifications. Such modifications include addition, deletion,
non-conservative substitution, or conservative substitution of a
nucleotide. The polynucleotide encoding the amino acid sequence is
also interpreted to include a nucleotide sequence that exhibits
substantial identity to the nucleotide sequence. The substantial
identity is obtained by aligning the nucleotide sequence with any
other sequences to the greatest extent and by analyzing the aligned
sequence using algorithms commonly used in the art, and in this
regard, the substantial identity may indicate a sequence having at
least 80% homology, at least 90% homology, or at least 95% homology
with the aligned sequence.
[0036] In addition, the present invention provides a composition
for diagnosing gastric cancer, the composition including a peptide
including an amino acid sequence selected from the group consisting
of SEQ ID NOs: 1 to 6.
[0037] In addition, the present invention provides a composition
for radio-sensitive diagnosing gastric cancer, the composition
including a peptide being specific to an irradiated gastric cancer
tissue and including an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1 to 3.
[0038] The term "diagnosis" used herein refers to identification of
the presence or characteristic of a pathological condition. For the
purpose of the present invention, the diagnosis is to identify the
presence or characteristic of gastric cancer.
[0039] The diagnosis of gastric cancer using the peptide of the
present invention may be performed by detecting binding of the
peptide of the present invention to a corresponding tissue or cell
directly obtained from blood, urine, or biopsy.
[0040] addition, to easily confirm, detect, and quantify binding of
the peptide of the present invention to the gastric cancer tissue,
the peptide of the present invention can be provided in a labeled
state. That is, the peptide provided herein may be linked to a
detectable label (for example, via covalent binding or
cross-linking). The detectable label may be a chromogenic enzyme
(for example, peroxidase and alkaline phosphatase), a radioactive
isotope (for example, .sup.124I, .sup.125I, .sup.111In, .sup.99mTc,
.sup.32P, and .sup.35S), a chromophore, or a luminescent material
or a fluorescent material (for example, FITC, RITC, rhodamine,
cyanine, Texas Red, fluorescein, phycoerythrin, or quantum
dots).
[0041] Similarly, the detectable label may be an antibody-epitope,
a substrate, a cofactor, an inhibitor, or a affinity ligand. Such
labeling may be performed during the synthesis of the peptide of
the present invention, or may be additionally performed on a
peptide that is already synthesized. When using a fluorescent
material is used as a detectable label, cancer may be diagnosed
according to fluorescence mediated tomography (FMT). For example,
the peptide of the present invention labeled with a fluorescent
material may be circulated into the blood, and the fluorescence by
the peptide may be observed by FMT. If fluorescent is observed, it
is diagnosed as cancer.
[0042] In addition, the present invention provides a composition
for delivering a drug, the composition including the peptide for
targeting gastric cancer.
[0043] The peptide of the present invention may be used as an
intelligent drug delivery vehicle that selectively delivers a drug
to a cancer tissue. If the peptide of the present invention is used
in combination with drugs of the related art in terms of treatment
of cancer, the peptide of the present invention selectively
delivers a drug only to a cancer tissue and a cancer cell, so that
drug efficacy may be increased while drug side effects on a normal
tissue may be significantly reduced at the same time.
[0044] For use as the drug, any anticancer drug that is
conventionally used in the treatment of cancer can be used so long
as the anticancer drug is able to be linked to the peptide of the
present invention. Examples of the drug include cisplatin,
5-fluorouracil, adriamycin, methotrexate, vinblastine, busulfan,
chlorambucil, cyclophosphamide, melphalan, nitrogen mustard,
nitreosourea, taxol, paclitaxel, docetaxel, 6-mercapropurine,
6-thioguanine, bleomycin, daunorubicin, doxorubicin, epirubicin,
idarubicin, mitomycin-C, and hydroxyurea. In addition, the linking
of the anticancer drug to the peptide of the present invention may
be performed by a method known in the art, for example, covalent
bonding, cross linking, or the like. For this purpose, the peptide
of the present invention may be, if necessary, subjected to
chemical modifications to the extent that the activity thereof is
not lost.
MODE OF THE INVENTION
[0045] Hereinafter, to promote understanding of one or more
exemplary embodiments, reference has been made in detail to
embodiments. The present invention, however, may be embodied in
many different forms and should not be construed as being limited
to the exemplary embodiments set forth herein. Rather, these
embodiments are provided so that this disclosure will be thorough
and complete, and will fully convey the scope of the present
invention to one of ordinary skill in the art.
<Example 1> Establishment of a Mouse Model Transplanted with
a Patient's Gastric Cancer Tissue
[0046] Considering overcoming limitations that existing animal
models transplanted with cultured cancer cells had, an ideal animal
model of cancer similar to actual patient's cancer
microenvironments is prepared. Then, to establish an animal model
that can confirm influence of an irradiation-dependent cancer
tissue, first, a mouse model transplanted with a cancer tissue that
was extracted from an actual patient having gastric cancer was
established. Regarding the establishment of such an animal model,
the cancer tissue extracted from a patient was cultured in an
NOD/SCID mouse. Once the cancer tissue was found in the NOD/SCID
mouse, subculturing was carried out by using a Balb/c nude mouse,
beginning from the next subculturing. In detail, after an NOD/SCID
mouse was anesthetized via intraperitoneal injection of
anesthetics, the cancer tissue was cut into pieces each having a
size of 3.times.3 mm. Next, each of both flanks of the NOD/SCID
mouse was transplanted with a piece of the cut cancer tissues,
treated with a mixed solution of 100 .mu.l of 100 IU/ml penicillin,
100 .mu.g/ml streptomycin, 100 .mu.g/ml gentamicin, and 2.5
.mu.g/ml amphotericin B antibiotics, and then, sutured. The
NOD/SCID mouse was recovered on a heating pad (for about 2 hours).
Afterwards, the formation and growth of tumors were observed every
week. When the cancer tissues grew to a size of 400-500 mm.sup.3,
the cancer tissues were separated and cut into pieces each having a
size of 3.times.3 mm for subculturing. Next, for next subculturing,
a nude mouse was anesthetized via intraperitoneal injection of
anesthetics, and a piece of the cut cancer tissues was transplanted
subcutaneously on the right thigh of the nude mouse. A
transplantation site was changed from the flank to the both thighs
so that irradiation can be locally done without affecting other
organs during irradiation. That is, a mouse model in which a cancer
tissue that underwent subculturing and was re-transplanted up to
four times was formed was established. The growth of the cancer
tissue was observed for about a month (4 weeks), and when the size
of the cancer tissue was increased to about 200 mm.sup.3, only the
cancer tissue was locally irradiated with 10 grays (Gy) of
radiation. After having recovery time for no longer than 24 hours,
in vivo peptide screening was performed. Here, as a control group
in an irradiated population, a mouse model that was not irradiated
among the same mouse models was used to screen a peptide. The
results of the establishment of such a mouse model are shown in
FIG. 1.
<Example 2> In Vivo Screening of M13 Phage Peptide
Library--In Vivo Phage Display
[0047] Regarding the mouse model established according to Example
1, i.e., a radio-sensitive xenograft mouse model transplanted with
a patient's gastric cancer tissue, a method for identifying a
peptide having high specificity during in vivo peptide screening
using a random loop peptide library was designed. For use as the
library, a loop peptide library manufactured to have about 2.7
billion different amino acids sequences via random array of 7 amino
acids [(i.e., a library fused with an M13 phage gp3 minor coat
protein)-Ph.D.TM. phage display peptide library kit, New England
Biolabs (NEB)] was purchased. To screen a peptide showing specific
binding to the irradiated gastric cancer tissue in the established
mouse model, a M13 phage peptide library (i.e., a library fused
with an M13 phage gp3 minor coat protein and consisting of 7 amino
acids having about 2.7 billion different amino acids sequences) was
injected into the tail vein of the mouse so that e M13 phage
peptide library was circulated in vivo for 15 minutes (also known
as a method of binding an in vivo cancer tissue with an M13 phage
peptide library). Then, during this process, a peptide-expressing
phage specifically binding to the cancer tissue was selected with
different washing conditions. Such a screening scheme is shown in
FIG. 2. In detail, FIG. 2 an M13 phage screening scheme for
screening a cancer tissue-targeting peptide, wherein (1) a mouse
model in which a cancer tissue was formed on the right hind leg was
established, (2) a phage library in which a loop peptide library
consisting of 7 amino acids was expressed on a surface of a M13
phage was injected into the tail vein of the mouse to allow
circulation of the phage library, (3) phages were washed under a
variety of washing conditions, and (4) phages were obtained by
eluting finally targeted phages. The eluted phages infected
Escherichia coli, and were injected again into the tail vein of the
mouse to allow circulation of the phages. By repeating such cycles
under washing conditions with high intensity, a process of
screening phages having high specificity and a strong binding
strength was repeatedly performed (also known as biopanning).
Biopanning was performed five times per cycle so that a phage
expressing a sequence of a peptide specifically binding to the
patient's in vivo gastric cancer tissue was obtained. To confirm
whether the peptide-expressing phage actually targeted the cancer
tissue only, other in vivo organs were also subjected to
comparison. That is, for every biopanning, phages were eluted from
each of extracted heart, lung, liver, spleen, kidney, and cancer
tissue, and the phage concentration was measured for comparison.
The results of the comparison are shown in FIG. 3. Finally, the
obtained phages infected E. coli ER2738 cells that are host cells,
and were subjected to amplification in an LB medium. Then, 100
phage plaques were selected randomly from each of an irradiated
population (experimental group) and a non-irradiated population
(control group), and the M13 phage genomic DNA (single-stranded
circular DNA) was separated and purified to identify a gene
sequence, thereby identifying an amino acid sequence of a peptide
expressed in a phage-surface protein (e.g., a gp3 minor coat
protein) and targeting the cancer tissue. The results of the
identification are shown in Table 1. Table 1 shows a summary of
sequences discovered from each of the irradiated (experimental
group) and the non-irradiated population (control group) by using
the Clustal X program for sequence analysis.
TABLE-US-00001 TABLE 1 Group No. Peptide sequence Irradiated P1
TVRTSAD (SEQ ID NO: 1) population (10 P2 RYVGTLF (SEQ ID NO: 2) Gy)
P3 NRGDRIL (SEQ ID NO: 3) Non-irradiated W1 NWGDRIL (SEQ ID NO: 4)
population as W2 QRSLPSL (SEQ ID NO: 5) control W3 DVWHSAY (SEQ ID
NO: 6)
<Example 3> In Vivo Imaging for Confirming Targeting Ability
of a Loop Peptide-Expressing Phage Regarding a Patient's Gastric
Cancer Tissue
[0048] To verify, based on in vivo imaging, whether the obtained
phage expressing a loop peptide has exhibited specific binding
ability following amplification and to confirm targeting efficiency
of the obtained phage, a process of linking a fluorescent probe was
performed first. In particular, 1 .mu.g/.mu.l of
N-hydroxysuccinimide esters of Cy5.5 (Amersham) was added to 1 mL
of bicarbonate buffer (pH 8.3) having the phage concentration of
10.sup.11 plaque forming units (pfu), and then, in a condition
where a dark environment was maintained, 3 a phage-surface protein
was linked to the Cy5.5 fluorescent probe at room temperature for 3
hours. That is, loop peptide-expressing phages to which the Cy5.5
fluorescent probe was linked were each obtained by precipitation
with 170 .mu.l of 20% (w/v) PEG 8000/2.5 M NaCl solution and
purification. To determine a proportion of the Cy5.5 fluorescent
probe linked to each of the finally obtained phage samples, an IVIS
spectrum imaging system (Xenogen) was used for measurement, and
region of interest (ROI) values were determined using the software
program of a corresponding device. Accordingly, it was confirmed
that the Cy5.5 fluorescent probe was linked to each of the phage
samples at almost the same linking proportion. The corresponding
results above are shown in FIG. 4.
[0049] After each of the prepared phages expressing the loop
peptide was injected into the ratio-sensitive xenograft mouse model
of Example 1 and the control group through the vein tail of the
mouse, images were measured for 2 days immediately after the
injection, thereby confirming images showing in vivo circulation of
the peptide and the targeting of the peptide only in the cancer
tissue while the targeting to other organs and tissues gradually
disappeared. In this regard, the peptide was proved to completely
target the in vivo gastric cancer tissue. In addition, the
excellent targeting ability of the peptide sequence that was
identified by biopanning according to Example 2 was resulted from
the in vivo imaging and showed in FIG. 5. In addition, to verify
which part of the cancer tissue was targeted by each of the loop
peptide-expressing phages via ex vivo imaging, only the cancer
tissue was separated and extracted, and the whole cancer tissue
itself and the respective cancer tissue were subjected to
fragmentation into several pieces. The imaging results obtained
therefrom are shown in FIG. 6.
[0050] To compare targeting efficiency more accurately based on the
results of FIGS. 5 and 6, the pieces of the extracted cancer tissue
the pieces of the extracted cancer tissue were collected
independently, and phages bound to the cancer tissues were eluted.
The concentration of each of the eluted phages was measured
according to titering, and due to different size and weight of the
extracted cancer tissue, the weight of each cancer tissue was also
measured in terms of establishing numerical standardization. In
this regard, the amount of the identified phages was calculated
relative to the weight of the cancer tissue. In addition, to more
accurately quantify each imaging result, the in vivo imaging and
the ex vivo imaging were confirmed by measuring ROI values that
were determined using the IVIS spectrum (Xenogen) program, and the
results thereof are shown in Table 2.
TABLE-US-00002 TABLE 2 Group Sample Sequence pfu/mg in vivo ROI ex
vivo ROI Irradiated Cy5.5 -- -- 1.00 1.00 population (10 Empty --
-- 7.37 24.58 Gy) P1 TVRTSAD 25.3 16.44 70.66 P2 RYVGTLF 6.3 9.05
38.83 P3 NRGDRIL 18.6 19.83 77.85 Non-irradiated Cy5.5 -- -- 1.00
1.00 population as Empty -- -- 0.95 0.54 control W1 NWGDRIL 11.8
1.86 2.08 W2 QRSLPSL 18.2 2.19 2.68 W3 DVWHSAY 8.1 1.90 3.54
<Example 4> In Vivo Imaging for Confirming Selectively
Binding Peptide Sequence Upon Irradiation
[0051] To confirm whether 3 peptide sequences identified in Example
3 were responsive to cancer microenvironments during irradiation,
the targeting efficiency of these irradiation-dependent peptide
sequences was confirmed. In detail, in the presence of differences
only in irradiation in the same mouse model transplanted with the
patient's gastric cancer, targeting of the peptide which was
dependent upon cancer microenvironments was subjected to
verification. Accordingly, as in the mouse model established in
Example 1, mice in which tumor was formed by transplantation of a
patient's gastric cancer tissue were selected. Among the selected
mice, only some of them were irradiated to thereby establish a
control group and an experimental group. In the same manner as in
Example 3, the selected phages expressing the peptide were
amplified and fluorescent labeling was also performed thereon, The
same sample was injected into an irradiated mouse group and a
control group thereof, thereby obtaining images for the last two
days. Consequently, when comparing targeting in the irradiated
mouse group with that in the control group, the two groups showed
differences in the targeting efficiency. The imaging measurement
results of the present embodiment are shown in FIG. 7. As shown in
FIG. 7, the control group including the mouse model transplanted
with the patient's gastric cancer tissue showed low targeting
efficiency, whereas the irradiated mouse model including the same
mouse model transplanted with the patient's gastric cancer tissue
showed specific binding ability through images.
<Example 5> Histological Verification of Selective Binding
Ability of a Discovered Peptide
[0052] To verify histological targeting ability of the selected
peptide (three sequences per population), each population was
injected via the tail vein. After 24 hours, cancer tissues of each
population were extracted to prepare paraffin blocks and slices. In
detail, (1) the extracted cancer tissues were immersed in a
formaldehyde solution at room temperature for 24 hours in terms of
for immobilization. Then, following a dehydration process, paraffin
was added to the solution through penetration to form paraffin
blocks. Afterwards, microtome was used to manufacture slices having
a thickness of 3 .mu.m. To perform real immunohistological
staining, (2) following a deparaffinization process performed on
the slices, (3) an unmasking process was performed so that
structures of various proteins immobilized to the tissue slices
were recovered to restore sites where antibodies normally bind.
Sequentially, (4) a blocking process was performed using a 5% BSA
solution, primary antibodies were bound (wherein the antibodies
used herein were anti-mouse M13 IgG recognizing M13 phage capsid
proteins), (6) and secondary antibodies were bound while HRP was
bound. Afterwards, (7) sites where phages were present were stained
using DAB development, (7) the nuclei of the phages were stained
with hematoxylin, and (8) a dehydration process was performed
thereon. Once completed, mounting was performed so that the tissue
slices that were immunohistochemically stained were permanently
preserved. The results of immunohistochemical staining performed as
described above are shown in FIG. 8.
<Example 6> Liposome Preparation, Drug Encapsulation, and
Peptide-Liposome Linking Process
[0053] In detail, five lipids constituting a liposome, such as
dipalmitoylphosphatidylcholine (DPPC, concentration of 50 mM),
dipalmitoylphosphatidylglycerol (DPPG-Na, concentration of 50 mM),
N-[3-(2pyridinyldithio)-1-oxopropyl]-L-.alpha.-dipalmitoylphosphatidylcho-
line (DPPE-PDP, concentration of 50 mM), cholesterol (concentration
of 200 mM), and cholesterol--PEG (concentration of 200 mM), were
each dissolved in an organic solvent containing methanol and
chloroform (prepared at a ratio of 1:1). DPPG-Na which does not
melt at room temperature was completely dissolved at 55.degree. C.
for more than 30 minutes. Each of the five dissolved lipids was
added to a round-bottom flask so as to prepare a mixed solution
containing DPPC:DPPE-PDP:DPPG-Na:cholesterol-PEG:cholesterol at a
ratio of 15:15:30:4:36. The round-bottom flask was rotated at
55.degree. C., and was pressurized for about 2 to 3 hours to
volatilize the organic solvent therefrom. Meanwhile, a thin lipid
film was formed within the round-bottom flask. When a white thin
film was formed within the round-bottom flask, the organic solvent
remained at room temperature was completely volatilized, so that
only a pure lipid film remained. Afterwards, to prepare a liposome
using the pure lipid film, 3 ml of HEPES (10 nM, pH 4) buffer was
added thereto, and the round-bottom flask was rotated in a
thermostat (55.degree. C.) for 1 hour to dissolve the pure lipid
film. To dissolve it sufficiently, a vortex was used to strongly
shake the round-bottom flask, so that the pure lipid film was able
to be completely dissolved without leaving any agglomerate. To make
the size of the prepared liposome uniform, a nitrogen gas extruder
and a poly-carbonate filter were used to filter the liposome
through a filter with fine holes, wherein the fine holes used
herein had a diameter of 800 nm, 400 nm, 200 nm, and 100 nm in the
stated order. Considering accurate size and uniformity of the
liposome, a filter having a diameter of 200 nm and a filter having
a diameter of 100 nm were used twice or several times for
extrusion. To load a drug into the extruded liposome, the buffer
containing the liposome dissolved therein was replaced with PBS (pH
7.0) using a Sephadex column. Accordingly, the resulting liposome
was dissolved in the buffer such that the inside of the liposome
had pH 4.0 and outside thereof had pH 7.0. The liposome resulting
from the completion of buffer replacement and doxorubicin dissolved
in buffer having pH 7.0 were mixed in a round-bottom flask. Then,
to encapsulate the drug dissolved in the buffer having pH 7.0
within the liposome having pH 4.0 by a concentration gradient, the
round-bottom flask was rotated in a bath at 60.degree. C. for 20
minutes. Then, to isolate the remaining non-encapsulated drug, a
pure liposome including the drug encapsulated therein was purified
using a Sephadex column. According to the Dinamic light scattering;
DLS method, the drug delivery carrier was optimized by measuring
the size and stability of the finally prepared drug-encapsulated
liposome. The results of the drug delivery process and optimization
thereof described above are shown in FIG. 9.
[0054] Among the peptides verified in Example 5, the peptide having
`TVRTSAD` sequence was synthesized by a request, and ligated with
the drug-encapsulated liposome of Example 6. Accordingly, a peptide
in which the C-terminal of the `TVRTSAD` was linked with a Cy7
fluorescent probe and the N-terminal of the `TVRTSAD` was free from
any process to be linked with a liposome was prepared by a request
from AnyGen Inc. (South Korea). The residue of the N-terminal of
the prepared peptide was processed to be linked with a thiol group
(--SH) of a liposome via a disulfide bond. Before performing
linking with the liposome, the number of the thiol group of the
liposome was counted, and a liposomal reduction test was conducted
so as to link the liposome to the peptide depending on the ratio of
the thiol group. DTT 1 mM was added and pyridine 2-thione was
measured at OD.sub.343 nm, to count the number of the thiol group
of the liposome. Afterwards, to link the liposome with the peptide,
the liposome and the peptide were mixed at a molar ratio of 1:1.5
to allow a reaction for 2 hours at room temperature. Then, to
isolate unreacted (unlinked) peptide, the liposome linked with a
pure peptide was purified using a Sephadex column. Afterwards, to
verify that there is no change in the size and stability of the
liposome before and after being linked with the peptide, the
verification was demonstrated according to the Dinamic light
scattering; DLS method, and the results are shown in FIG. 10.
<Example 7> Verification of Targeting Ability of
Peptide-Linked Liposome and Validation of New Concept Anticancer
Drug
[0055] To verify whether the drug carrier of Example 6 in which the
`TVRTSAD` sequence was linked to the drug-encapsulated liposome
actually played a function in the living body, in vivo imaging was
performed. In detail, the radio-sensitive xenograft mouse model of
Example 1 and the control group were each injected through the vein
tail of the mouse, and images were measured for 2 days immediately
after the injection, thereby confirming that images showing in vivo
circulation of the peptide and the targeting of the peptide only in
the cancer tissue while the targeting to other organs and tissues
gradually disappeared. In this regard, the peptide was proved to
completely target the in vivo gastric cancer tissue. The results of
the in vivo imaging are shown in FIG. 11.
[0056] In addition to the in vivo imaging, in vivo tumor growth
delay was also performed to validate the peptide as a target drug
delivery carrier. The radio-sensitive xenograft mouse model of
Example 1 and the control group were established, and once the
tumor size was increased to about 100 mm.sup.3, grouping was
performed thereon. In this regard, a total of 5 groups, i.e., 1;
PBS, 2; irradiation (2 Gy), 3; DOX(2 mg/kg)+irradiation (2 Gy), 4;
LP-DOX (2 mg/kg)+irradiation (2 Gy), 5; P1(peptide)-LP-DOX (2
mg/kg)+irradiation (2 Gy), were prepared. Here, test group was
designated as Group 5 while the control groups were designated as
Groups 1 to 4 for observation. Each group included 5 mice (n=5).
Compared to the control groups, the test group (i.e., Group 5)
showed that the tumor size was significantly small, and
accordingly, the results of validation of a new concept anticancer
drug are shown in FIG. 12.
[0057] As verified in FIGS. 11 and 12, the material linked with the
corresponding peptide and the drug-encapsulated liposome were
verified to be utilized in the in vivo imaging and the in vivo
tumor growth delay, and accordingly, the possibility of the
material as a new concept anticancer drug that can simultaneously
diagnose and treat cancer was proved.
<Example 8> In Vivo Imaging for Verifying Selective Binding
Ability of a Peptide Upon Irradiation on a Gastric Cancer Tissue of
Other Patients
[0058] To verify whether the peptide having selective binding
ability upon irradiation on the patient's gastric cancer tissue
obtained in Examples above also exhibited the same selective
binding ability in cases associated with gastric cancer tissues of
other patients having the same gastric cancer and the same
characteristics upon irradiation, other than the corresponding
gastric cancer case showing selective binding of the peptide upon
irradiation, gastric cancer tissues each extracted from different
patients were prepared to establish a mouse model. In detail, in
addition to the patient's gastric cancer tissue used in Example
above, two gastric cancer tissues of other patients were prepared,
wherein all the gastric cancer tissues used herein were
characterized as adenocarcinoma. In the same manner as in Example
1, mouse models including each of the corresponding gastric cancer
tissues was established, and some of them were irradiated to
thereby establish a control group and an experimental group.
According to the in vivo imaging which is the same method as the
one used for confirming targeting efficiency in Example 4, the
irradiation-dependent targeting efficiency of the peptide was
verified. The amplification of phages expressing the selected
peptide and the fluorescent labeling were performed in the same
manner as in Example 3. Then, the completed sample was injected
into each of the irradiated test mouse group and the control group,
and images thereof were confirmed on the 2.sup.nd day of the
observation. As a result, it was confirmed that the two gastric
cancer tissues of other patients also showed selective accumulation
of peptide-phage only in the tumors of the irradiated test mouse
group, in the same manner as in the existing gastric cancer tissue
of the patient. The results of the imaging measurement of the
corresponding embodiments are shown in FIG. 13. As shown in FIG.
13, it was confirmed that the peptide-expressing phage did not
target the cancer tissue when there is no irradiation applied to
the cancer tissue, whereas the peptide-expressing phage targeted
the cancer tissue when irradiation is applied to the cancer tissue.
In conclusion, it was confirmed that the peptide sequence selected
in the corresponding technology was clearly verified as the peptide
sequence selectively targeting the gastric cancer upon irradiation,
and that the targeting ability of the corresponding peptide is not
limited to the patient's gastric cancer tissue only. That is, as
the peptide sequence exhibiting selective targeting ability only in
the case where the gastric cancer tissue of other patients are
irradiated, it was verified that the scope of application of the
peptide of the present invention is not limited in clinical
applications.
[0059] It should be understood that embodiments described herein
should be considered in a descriptive sense only and not for
purposes of limitation. Descriptions of features or aspects within
each embodiment should typically be considered as available for
other similar features or aspects in other embodiments. While one
or more embodiments have been described with reference to the
figures, it will be understood by those of ordinary skill in the
art that various changes in form and details may be made therein
without departing from the spirit and scope as defined by the
following claims.
Sequence CWU 1
1
617PRTArtificial SequenceSynthetic peptide 1Thr Val Arg Thr Ser Ala
Asp 1 5 27PRTArtificial SequenceSynthetic peptide 2Arg Tyr Val Gly
Thr Leu Phe 1 5 37PRTArtificial SequenceSynthetic peptide 3Asn Arg
Gly Asp Arg Ile Leu 1 5 47PRTArtificial SequenceSynthetic peptide
4Asn Trp Gly Asp Arg Ile Leu 1 5 57PRTArtificial SequenceSynthetic
peptide 5Gln Arg Ser Leu Pro Ser Leu 1 5 67PRTArtificial
SequenceSynthetic peptide 6Asp Val Trp His Ser Ala Tyr 1 5
* * * * *