U.S. patent application number 15/554849 was filed with the patent office on 2018-02-08 for cell therapeutic agent for cancer treatment and combination therapy with same.
The applicant listed for this patent is AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION. Invention is credited to Da Young Chang, Woo Sup Hwang, Jin Hwa Jung, Sung Soo Kim, Su Jung Lee, Young Don Lee, Jin Sung Park, Hae Young Suh.
Application Number | 20180037869 15/554849 |
Document ID | / |
Family ID | 56879471 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037869 |
Kind Code |
A1 |
Suh; Hae Young ; et
al. |
February 8, 2018 |
CELL THERAPEUTIC AGENT FOR CANCER TREATMENT AND COMBINATION THERAPY
WITH SAME
Abstract
The present disclosure relates to a method for preparing cells
for cancer treatment and a kit for cancer treatment comprising
cells prepared by the method. The preparation method of the present
disclosure can provide F cells, which, in spite of having no
difference in the proliferative capacity compared with mesenchymal
stem cells expressing cytosine deaminase that are harvested and
used immediately after the culture, exhibit a very excellent tumor
suppressive effect through the treatment together with 5-FC and
induce a remarkable synergistic effect exceeding an effect from
combinative treatment with an existing anticancer drug in cases of
a combinative treatment with another anticancer drug. Therefore,
the present disclosure can be utilized for a kit for cancer
treatment comprising such F cells, and thus can be favorably used
to maximize the effect of existing cancer treatments.
Inventors: |
Suh; Hae Young;
(Gyeonggi-do, KR) ; Kim; Sung Soo; (Seoul, KR)
; Chang; Da Young; (Gyeonggi-do, KR) ; Jung; Jin
Hwa; (Gyeonggi-do, KR) ; Lee; Young Don;
(Gyeonggi-do, KR) ; Hwang; Woo Sup; (Gyeonggi-do,
KR) ; Park; Jin Sung; (Seoul, KR) ; Lee; Su
Jung; (Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION |
Gyeonggi-do |
|
KR |
|
|
Family ID: |
56879471 |
Appl. No.: |
15/554849 |
Filed: |
March 4, 2016 |
PCT Filed: |
March 4, 2016 |
PCT NO: |
PCT/KR2016/002188 |
371 Date: |
August 31, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/675 20130101;
A61P 43/00 20180101; A61K 33/24 20130101; A61P 15/00 20180101; A61P
11/02 20180101; A61P 13/10 20180101; A61P 13/12 20180101; A61K
31/4745 20130101; A61P 1/00 20180101; A61P 1/16 20180101; A61K
31/4188 20130101; A61P 35/00 20180101; C12N 2501/73 20130101; C12N
2523/00 20130101; A61K 31/506 20130101; A61K 45/06 20130101; A61P
1/02 20180101; C12N 5/0665 20130101; A61P 1/04 20180101; C12N
2510/00 20130101; A61K 35/12 20130101; A61K 35/28 20130101; A61P
11/00 20180101; A61P 1/18 20180101; A61K 31/704 20130101; A61P
25/00 20180101; A61P 35/02 20180101; A61P 13/08 20180101; A61P
17/00 20180101; C12N 5/0663 20130101; A61K 31/513 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A61K 31/513 20060101 A61K031/513; A61K 45/06 20060101
A61K045/06; A61K 35/28 20060101 A61K035/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2015 |
KR |
10-2015-0031751 |
Claims
1. A method for preparing F cells comprising: 1) introducing
cytosine deaminase (CD) into a mesenchymal stem cell (MSC) to
prepare mesenchymal stem cell/cytosine deaminase (MSC/CD); 2)
freezing the prepared MSC/CD to prepare frozen MSC/CD; and 3)
thawing and suspending the frozen MSC/CD to prepare F cells.
2. The method according to claim 1, wherein the F cell(s) is not
subjected to cell culturing after freezing.
3. The method according to claim 1, wherein the F cell(s) is used
for cancer treatment.
4. The method according to claim 3, wherein the cancer is at least
one selected from the group consisting of squamous cell cancer,
small cell lung cancer, non-small cell lung cancer, lung cancer,
peritoneal cancer, colorectal cancer, biliary tumor, nasopharyngeal
cancer, laryngeal cancer, bronchial cancer, oral cancer,
osteosarcoma, gallbladder cancer, kidney cancer, leukemia, bladder
cancer, melanoma, brain cancer, glioma, brain tumor, skin cancer,
pancreatic cancer, breast cancer, liver cancer, bone cancer,
esophageal cancer, colon cancer, gastric cancer, cervical cancer,
prostate cancer, ovarian cancer, head and neck cancer, and rectal
cancer.
5. The method according to claim 1, wherein the F cell(s) is an
adjuvant for anti-cancer.
6. An adjuvant for anti-cancer comprising the F cells prepared by
the method according to claim 1.
7. A kit for cancer treatment comprising the F cells prepared by
the method according to claim 1, and 5-fluorocytosine (5-FC).
8. The kit according to claim 7, further comprising an anti-cancer
agent.
9. The kit according to claim 8, wherein the anti-cancer agent is
at least one selected from the group consisting of nitrogen
mustard, imatinib, oxaliplatin, rituximab, elotinib, trastuzumab,
gefitinib, bortezomib, sunitinib, carboplatin, sorafenib,
bevacizumab, cetuximab, viscum album, asparaginase, tretinoin,
hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin,
ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid,
amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate
chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur,
capecitabine, gimeracil, oteracil, azacytidine, cytarabine,
fludarabine, enocitabine, decitabine, mercaptopurine, thioguanine,
cladribine, carmofur, raltitrexed, docetaxel, paclitaxel,
belotecan, topotecan, vinorelbine, etoposide, vincristine,
vinblastine, tenifocide, idarubicin, epirubicin, mitoxantrone,
mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin,
aclarubicin, pepromycin, temozolomide, busulfan, ifosfamide,
cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa,
nimustine, chlorambucil, mitolactol, taxotere, gleevec, taxol,
herceptin, tarceva, avastin, zoladex, adriamycin, irinotecan,
10058-F4, cisplatin, cyclophosphamid, nitrosourea-based anti-cancer
agent, methotrexate, carmustine (BCNU), lomustine (CCNU), and
doxorubicin.
10. A method for preventing or treating cancer comprising: 1)
introducing cytosine deaminase (CD) into a mesenchymal stem cell
(MSC) to prepare mesenchymal stem cell/cytosine deaminase (MSC/CD);
2) freezing the prepared MSC/CD to prepare frozen MSC/CD; 3)
thawing and suspending the frozen MSC/CD to prepare F cells; 4)
administering the F cells to a subject in need of treatment; and 5)
administering 5-fluorocytosine (5-FC) to a subject in need of
treatment.
11. The method according to claim 10, further comprising 6)
administering an anti-cancer agent to a subject in need of
treatment.
12. The method according to claim 10, further comprising
administering an anti-cancer agent to a subject in need of
treatment before administration of the F cells according to step 4)
or simultaneously with administration of the F cells according to
step 4).
13. The method according to claim 11, wherein the anti-cancer agent
is at least one selected from the group consisting of nitrogen
mustard, imatinib, oxaliplatin, rituximab, elotinib, trastuzumab,
gefitinib, bortezomib, sunitinib, carboplatin, sorafenib,
bevacizumab, cetuximab, viscum album, asparaginase, tretinoin,
hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin,
ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid,
amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate
chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur,
capecitabine, gimeracil, oteracil, azacytidine, cytarabine,
fludarabine, enocitabine, decitabine, mercaptopurine, thioguanine,
cladribine, carmofur, raltitrexed, docetaxel, paclitaxel,
belotecan, topotecan, vinorelbine, etoposide, vincristine,
vinblastine, tenifocide, idarubicin, epirubicin, mitoxantrone,
mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin,
aclarubicin, pepromycin, temozolomide, busulfan, ifosfamide,
cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa,
nimustine, chlorambucil, mitolactol, taxotere, gleevec, taxol,
herceptin, tarceva, avastin, zoladex, adriamycin, irinotecan,
10058-F4, cisplatin, cyclophosphamid, nitrosourea-based anti-cancer
agent, methotrexate, carmustine (BCNU), lomustine (CCNU), and
doxorubicin.
14. The method according to claim 10, wherein the F cells of step
4) and the 5-fluorocytosine of step 5) are administered
simultaneously or sequentially.
15. The method according to claim 11, wherein the 5-fluorocytosine
of step 5) and the anti-cancer agent of step 6) are administered
simultaneously or sequentially.
16. The method according to claim 11, wherein the administration of
the F cells according to step 4) is repeated with a cycle of 10 to
30 days.
17. The method according to claim 10, wherein the cancer is at
least one selected from the group consisting of squamous cell
cancer, small cell lung cancer, non-small cell lung cancer, lung
cancer, peritoneal cancer, colorectal cancer, biliary tumor,
nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral
cancer, osteosarcoma, gallbladder cancer, kidney cancer, leukemia,
bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin
cancer, pancreatic cancer, breast cancer, liver cancer, bone
cancer, esophageal cancer, colon cancer, gastric cancer, cervical
cancer, prostate cancer, ovarian cancer, head and neck cancer, and
rectal cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. national phase under the
provisions of 35 U.S.C. .sctn.371 of International Patent
Application No. PCT/KR2016/002188 filed Mar. 4, 2016, which in turn
claims priority of Korean Patent Application No. 10-2015-0031751
filed Mar. 6, 2015. The disclosures of such international patent
application and Korean priority patent application are hereby
incorporated herein by reference in their respective entireties,
for all purposes.
TECHNICAL FIELD
[0002] The present disclosure relates to a method for preparing
cells for cancer treatment, an adjuvant for anti-cancer comprising
cells prepared by the same, a kit for cancer treatment, and a
method for cancer treatment.
BACKGROUND ART
[0003] Millions of people around the world die from various types
of cancer including bone cancer, bladder cancer, blood cancer
(leukemia), brain cancer, breast cancer, colorectal cancer,
cervical cancer, esophageal cancer, bowel cancer, kidney cancer,
lung cancer, liver cancer, oral cancer, nasal cancer, neural
cancer, ovarian cancer, pancreatic cancer, skin cancer, stomach
cancer, prostate cancer, neck cancer, uterine cancer, and vaginal
cancer. Over the years, several methods including radiation and
chemotherapy have been used to treat cancer, but the number of
cancer patients is still increasing. It has been widely reported
that radiation and chemotherapy may cause toxicity related diseases
and even some patients die. In addition, in the case of specific
cancer, there is a problem in that malignant cells remain difficult
to treat. As a result, it is required to extensively study
physiology or phenotype of cancer cells to find a treatment method
that selectively kills the cancer cells without causing adverse
effects on the patient's healthy cells.
[0004] In particular, a brain tumor is a type of cancer that causes
great damage to the brain and has a very low survival rate. Among
conventional treatment methods for the brain tumor, extraction by
surgical operation is the most effective treatment, but there are a
number of cases in that the surgery is not possible depending on
the type and location of the brain tumor, and the risk of
postoperative complications is very high at the time of complete
extraction. Further, since a brain-blood barrier (BBB) that
inhibits drug penetration is present in the brain, in order to
treat brain tumor by chemotherapy using an anti-cancer agent, it is
required to administer a high concentration of anti-cancer agent as
compared to other types of cancer, which causes serious side
effects in other organs of the body. Therefore, there is a need for
a novel adjuvant method, a therapeutic agent, and an adjuvant that
are able to lower the dose of the anti-cancer agent by enhancing
effects of the anti-cancer agent.
[0005] In addition, a gene therapy method is a method of directly
introducing a gene that inhibits proliferation of cancer cells into
the cancer cells. For gene introduction, a virus is mainly used,
but since the virus itself does not have an ability to move to
cancer, it is required to inject the virus surgically. However, it
is practically impossible to inject the viruses into every
microscopic tumor or cancer cells, and thus, there is a limitation
in targeting the cancer cells.
[0006] In this connection, Korean Patent Registration No.
10-1022401 is known as a method for introducing a suicide gene into
a mesenchymal stem cell to enhance targeting and treat cancer.
However, KR 10-1022401 does not disclose a combination therapy
adjuvant for improving an effect of an anti-cancer agent by
manipulating the mesenchymal stem cell in a special environment and
administering the mesenchymal stem cell in combination with another
anti-cancer agent, and does not disclose a method for periodic
administration.
[0007] Further, recently, a number of studies on combination
therapies such as combination therapy of two or more kinds of
anti-cancer agents have been conducted. In this regard, not only a
combination therapy of a chemotherapeutic agent, but also a
combination therapy of a chemotherapeutic agent in combination with
different kinds of anti-cancer therapies, etc., has been recently
reported. However, in the case of combination therapy, there are
many cases in which combination administration of each drug shows a
simple additive effect of the same degree of drug efficacy.
Further, unpredictable side effects have been reported, for
example, an effect of the drugs is rather deteriorated by an
interaction of the drugs, etc. Thus, studies on the combination
administration of drugs are still continuing.
[0008] In connection with the combination administration in cancer,
there is also a study on the combination therapy of
chemotherapeutic agents and cell therapeutic agents, such as
mesenchymal stem cells. However, in the combination therapy using
the mesenchymal stem cells, researches aiming at administration of
cells such as autologous stem cells for recovery of cells damaged
after anti-cancer therapy by chemotherapeutic agents have been
intensively conducted, and there are not many attempts to utilize
the stem cells themselves as therapeutic agents for combination
therapy.
[0009] Therefore, there is a need to develop a therapeutic agent
and a treatment method capable of having high targeting so that
cancer or tumor therapeutic agents are able to be accurately
transferred to a cancer tissue, being non-toxic so as not to affect
parts other than the tumor, and being repeatedly administered until
cancer is eliminated because of no immunotoxicity. Further, there
is a need and requirement for a novel therapeutic method,
therapeutic agent, therapeutic adjuvant and combination therapy
that are able to improve or enhance the efficacy of existing
anti-cancer agents.
DISCLOSURE
Technical Problem
[0010] The present inventors studied a novel cancer therapeutic
agent that is capable of overcoming the limitations of the existing
chemotherapy, found that cells prepared by introducing a cytosine
deamine gene into a mesenchymal stem cell to prepare mesenchymal
stem cell/cytosine deaminase (MSC/CD), followed by freezing and
thawing, could exhibit very excellent tumor suppressive effect and
survival rate improvement effect as compared to simply cultivated
MSC/CD cells, and maximize effects of combination therapy with
existing anti-cancer agents, and completed the present
disclosure.
[0011] An object of the present disclosure is to provide a method
for preparing "F cells" which are cells usable as an adjuvant for
cancer treatment, a kit for cancer treatment comprising F cells, a
combined administration agent with the F cells and a
chemotherapeutic agent, and a combined administration method.
Technical Solution
[0012] In order to achieve the foregoing objects, the present
disclosure provides a method for preparing F cells comprising: 1)
introducing cytosine deaminase (CD) into a mesenchymal stem cell
(MSC) to prepare mesenchymal stem cell/cytosine deaminase (MSC/CD);
2) freezing the prepared MSC/CD to prepare frozen MSC/CD; and 3)
thawing and suspending the frozen MSC/CD to prepare F cells.
[0013] In addition, the present disclosure provides an adjuvant for
anti-cancer comprising the F cells prepared by the method.
[0014] Further, the present disclosure provides a kit for cancer
treatment comprising the F cells prepared by the method, and
5-fluorocytosine (5-FC).
[0015] In addition, the present disclosure provides a method for
preventing or treating cancer comprising: 1) introducing cytosine
deaminase (CD) into a mesenchymal stem cell (MSC) to prepare
mesenchymal stem cell/cytosine deaminase (MSC/CD); 2) freezing the
prepared MSC/CD to prepare frozen MSC/CD; 3) thawing and suspending
the frozen MSC/CD to prepare F cells; 4) administering the F cells
to a subject in need of treatment; and 5) administering
5-fluorocytosine (5-FC) to a subject in need of treatment.
Advantageous Effects
[0016] According to the preparation method of the present
disclosure, it is possible to provide the F cells, in spite of
having no difference in the proliferative capacity compared with
mesenchymal stem cells expressing cytosine deaminase, which are
harvested and used immediately after culturing, exhibit a very
excellent tumor suppressive effect through the treatment together
with 5-FC, and induce a remarkable synergistic effect exceeding an
effect from combination therapy with an existing anti-cancer agent,
even in cases of a combination therapy with another anti-cancer
agent. Therefore, the present disclosure may be utilized as a kit
for cancer treatment comprising such F cells, and thus may be
favorably used to maximize the effect of existing cancer
treatments.
DESCRIPTION OF DRAWINGS
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0018] FIG. 1 is a schematic diagram of a mesenchymal stem cell
line (MSC/CD) that stably expresses cytosine deaminase, a suicide
effect thereof, and a bystander effect.
[0019] FIG. 2 shows treatment process of MSC or MSC/CD with 5-FC in
graph (A) and results of the suicide effect in graph (B) derived
therefrom.
[0020] FIG. 3 shows treatment of MSC or MSC/CD with 5-FC in graph
(A) and results of the bystander effect in fluorescent images (B)
and graph (C) derived therefrom.
[0021] FIG. 4 shows process of cell preparation and treatment of I
cell, F cell (DMSO), and F cell (CS10) in graph (A) for comparing
bystander effect on respective cells and bystander effects in graph
(B) on respective cells.
[0022] FIG. 5 shows a microscopic observation of a colony forming
ability of the I cell and the F cell in images (A), and a graph for
comparing quantitative analysis results using CFU-A (colony forming
unit assay) in graph (B).
[0023] FIG. 6 shows a process of treating the I-cell or the F-cell
with 5-FC in a brain tumor model in graph (A), results of brain
tumor changes following the treatment observed under a dissecting
microscope obtained by observing brain tumor changes by the
treatment in images (B), and tumor volume changes in graph (C).
[0024] FIG. 7 shows a graph for tumor volume changes according to
the I cell or F cell treatment in graph (A) in subcutaneous tumor
models in which liver cancer cells are implanted subcutaneously,
and representative tumor models showing an external size of the
tumor in images (B).
[0025] FIG. 8 shows tumor volume changes according to the I cell or
F cell treatment, in subcutaneous tumor models in which pancreatic
cancer cells are implanted subcutaneously in graph (A), in
subcutaneous tumor models in which lung cancer cells are implanted
subcutaneously in graph (B), and in subcutaneous tumor models in
which colon cancer cells are implanted subcutaneously in graph
(C).
[0026] FIG. 9 shows F cells+5-FC+TMZ combination therapy in graph
(A), the bystander effects derived therefrom in graphs (B) and (C),
confirmation on results of cell death through fluorescence images
of U87MG expressing GFP in images (D), and isobologram results of
ICso values in graph (E).
[0027] FIG. 10 shows F cells+5-FC+anti-cancer agent combination
therapy in graph (A), isobologram results of ICso values of
carmustine in graph (B), and isobologram results of ICso values of
irinotecan in graph (C), respectively, in anti-cancer agent
combination therapy.
[0028] FIG. 11 shows process of F cells+5-FC+temozolomide (TMZ)
combination therapy in graph (A), the results of brain tumor
changes following the treatment observed under a dissecting
microscope showing brain tumor changes in images (B), tumor volume
changes in graph (C), and synergistic effects in improvement of
survival rates in graph (D) by the combination therapy.
[0029] FIG. 12 shows the analysis process on effects of the I cell
or the F cell+5-FC+temozolomide (TMZ) combination therapy in the
subcutaneous tumor models in graph (A), tumor volume changes in
graph (B), and synergistic effects of the F cell+5-FC+temozolomide
(TMZ) in analysis of survival rates in graph (C).
BEST MODE
[0030] The present disclosure provides a method for preparing F
cells including: 1) introducing cytosine deaminase (CD) into a
mesenchymal stem cell (MSC) to prepare mesenchymal stem
cell/cytosine deaminase (MSC/CD); 2) freezing the prepared MSC/CD
to prepare frozen MSC/CD; and 3) thawing and suspending the frozen
MSC/CD to prepare F cells.
[0031] According to the method for preparing F cells of the present
disclosure, it is possible to provide the F cells, in spite of
having no difference in the proliferative capacity compared with
mesenchymal stem cells expressing cytosine deaminase, which are
harvested and used immediately after culturing I cells, exhibit a
very excellent tumor suppressive effect when treated with 5-FC, and
induce a remarkable synergistic effect exceeding an effect from
combination therapy with an existing anti-cancer agent, even in
cases of a combination therapy with another anti-cancer agent.
[0032] The "MSC/CD" used herein refers to a mesenchymal stem cell
prepared to express cytosine deaminase (CD) in the mesenchymal stem
cell. The mesenchymal stem cell into which the cytosine deaminase
is introduced refers to a cell that moves to the vicinity of cancer
cells with a tropism towards the cancer cells, and continuously
expresses cytosine deaminase to induce suicide effect itself, and
converts an non-toxic prodrug into an active anti-cancer agent to
exhibit a bystander effect in which surrounding cancer cells are
killed. The MSC/CD is advantageous in that since it undergo cell
death, side effects that may be caused by undesired continuous
effects may be reduced, and since it moves to the vicinity of
cancer cells, infiltrative cancer cells may be killed, thereby
acting as an effective anti-cancer agent. In particular, the "F
cell", which is a cell prepared by freezing/thawing the MSC/CD,
exhibits synergistic effect in which an anti-cancer effect is
significantly increased when used in combination with the existing
anti-cancer agent, thereby being utilized as an adjuvant enhancing
the effect of the anti-cancer agent.
[0033] The term "mesenchymal stem cell (MSC)" of the present
disclosure is a kind of stem cells that are able to be collected
from bone marrow and umbilical cord blood, and is referred to a
multipotent stromal cell that is capable of proliferation, and in
particular, that is capable of being differentiated into various
types of cells such as bone cells, cartilage cells, muscle cells,
and fat cells, unlike hematopoietic stem cells. Therefore, the
mesenchymal stem cell of the present disclosure may preferably be
the multipotent mesenchymal stem cell.
[0034] Term "cytosine deaminase" of the present disclosure is a
suicide gene, which functions to convert a prodrug harmless to the
human body into a cytotoxic anticancer material, and specifically,
a gene that converts 5-fluorocytosine (5-FC) which is a prodrug of
5-fluorouracil (5-FU), into 5-FU.
[0035] The suicide gene may be introduced into the mesenchymal stem
cell according to methods for intracellular introduction of genes
known in the art, such as a method of using a non-viral vector or a
virus vector including the same, or a physical method. Examples of
such physical methods for gene transfer may include
electroporation, hydroporation, injection, etc. Examples of gene
transfer using non-viral vectors may include methods of using
cationic lipids, lipid polymers, or nanoparticles. The viral vector
may include, without limitation, a replicable viral vector, a
non-replicable viral vector, such as an adenoviral vector, an
adeno-associated viral vector, a retroviral vector, herpes simplex
virus, a hybrid adenoviral system, pox virus, a lentivirus vector,
and Epstein Barr virus.
[0036] For example, in an exemplary embodiment of the present
disclosure, the suicide gene may be introduced into the retrovirus
vector to construct an expression vector, the vector may be
transfected into packaging cells, the transfected packaging cells
may be cultured and filtered to obtain a retrovirus solution, and
this retrovirus solution may be used to transfect the mesenchymal
stem cell, thereby inserting the suicide gene into the mesenchymal
stem cell. Next, the mesenchymal stem cell that continuously
expresses the suicide gene may be obtained using a selection marker
included in the retrovirus vector.
[0037] In addition, since mesenchymal stem cell is known to have
low immunorejection even during allotransplantation, it is possible
to use either cells isolated from others or cells collected from
patients with cancer themselves, and to further minimize
immunorejection among individuals through separation from bone
marrow having similar HLA (human leukocyte antigen) types using a
database of blood banks.
[0038] The MSC/CD of the present disclosure may be obtained by
introducing a suicide gene into the isolated stem cell at first,
followed by screening, proliferation and differentiation under an
appropriate condition in a test tube, or may be obtained by
sufficiently proliferating the stem cell, introducing a suicide
gene into the stem cell, followed by differentiation into the
mesenchymal stem cell. The MSC/CD is injected into a subject in
need of treatment by the presence of the suicide gene, and kills
themselves after, for example, 2 to 30 days, preferably 5 to 15
days, more preferably 5 to 10 days, but is not limited thereto,
from the injection, thereby reducing side effects that may be
induced by continuous action.
[0039] Term "F cell (frozen cell)" of the present disclosure refers
to a cell that is not undergone an additional culturing step, free
of culturing F cells, after freezing and thawing the MSC/CD, which
is the mesenchymal stem cell into which the cytosine deaminase (CD)
is introduced, and is defined as a cell being thawed and suspended,
or suspended and washed with a purpose of administration of cells
to the subject to be treated. More specifically, the F cell refers
to a cell obtained by introducing cytosine deaminase (CD) into the
mesenchymal stem cell to prepare the MSC/CD, culturing, freezing
the cell in a freezing medium, thawing the cell at room temperature
immediately before use, followed by suspension, for example, in a
Plasma Solution A, or the like, containing human serum albumin,
which is not limited thereto, and centrifugation. As a cell
differentiated from the F cell of the present disclosure, an
immediately harvested cell (I cell)" may be exemplified, which is
obtained by performing the same freezing and thawing step on the
MSC/CD, but maximizing the concentration and activity through the
subsequent culturing process, and is used by directly harvesting
the cell in the culture.
[0040] The F cell, compared to the I cell, of the present
disclosure is characterized in that the anti-cancer effect is
increased without culturing step, which is performed generally to
maximize activation and without increasing a colony forming
ability. In addition, the F cell of the present disclosure may have
an advantage of being immediately administered to a patient in need
of treatment together with the improvement of the anti-cancer
effect.
[0041] In the present disclosure, the cells in a cryopreserved
state or frozen state may be stored by using any means known in the
art for maintaining the cells to be in frozen state, such as a
freezing preservation liquid, a freezing medium, etc. For example,
the means may include media including freezing protectants such as
dimethylsulfoxide (DMSO), dextran, human serum, human serum
albumin, bovine serum, bovine serum albumin, poly-1-lysine,
polyvinylpyrrolidone, hydroxy-ethyl-starch, ethylene glycol,
polyethylene glycol, glycerol, and percoll (for example, cryostor
CS2, cryostor CS10 commercially available from BioLife Solutions
Inc.), and specifically, cryostor CS2, cryostor CS10, and 2 to 10%
DMSO medium, and more specifically, 2, 5, and 10% DMSO media,
etc.
[0042] The F cell of the present disclosure exhibits in vitro
colony forming ability similar to that of the I cell, and also
effectively exhibits the bystander effect of the MSC/CD. Further,
the F cell exhibits an inhibitory effect in vivo on a variety of
tumors including brain tumors and subcutaneous tumors twice or
higher as compared to the I cell or the control group, and exhibits
a highly elevated anti-cancer effect in the combination therapy
with temozolomide (TMZ) which is the existing anti-cancer agent. In
other words, even though the F cell has no difference in the colony
forming ability as compared to the I cell, the F cell exhibits a
remarkable anti-cancer effect that are not able to be expected in
vitro and an effect as an anti-cancer agent adjuvant, as compared
to other I cells, due to the intrinsic activity of the F cell given
according to the preparation method.
[0043] That is, the F cells of the present disclosure may be
differentiated from I cells that are conventionally used, by
preparation methods in which cell culturing is not further
performed after freezing.
[0044] Therefore, the present disclosure provides the method for
preparing F cells characterized in that the F cells are not
subjected to cell culturing after freezing.
[0045] The F cell of the present disclosure is a cell for cancer
treatment that is usable for cancer treatment. The F cells may be
used to treat, without limitation, all carcinomas capable of being
extracted as cancer and carcinoma, and may be administered to a
subject in need of treatment before, simultaneously with, and after
chemotherapy using administration of anti-cancer agent.
[0046] The cancer may be, for example, at least one selected from
the group consisting of squamous cell cancer (for example, squamous
cell cancer of epithelium), small cell lung cancer, non-small cell
lung cancer, lung cancer, peritoneal cancer, colorectal cancer,
biliary tumor, nasopharyngeal cancer, laryngeal cancer, bronchial
cancer, oral cancer, osteosarcoma, gallbladder cancer, kidney
cancer, leukemia, bladder cancer, melanoma, brain cancer, glioma,
brain tumor, skin cancer, pancreatic cancer, breast cancer, liver
cancer, bone cancer, esophageal cancer, colon cancer, gastric
cancer, cervical cancer, prostate cancer, ovarian cancer, head and
neck cancer, and rectal cancer, and more preferably, brain tumor,
pancreatic cancer, liver cancer, lung cancer, colon cancer, and
skin cancer.
[0047] Further, the F cell of the present disclosure may also be an
adjuvant for anti-cancer. The adjuvant for anti-cancer refers to an
adjuvant that is capable of inducing faster action of a primary
therapy and strongly forming an anti-cancer action thereof by
combination therapy with the primary therapy, for example, cancer
treatment by chemical therapy or surgical operation. The adjuvant
for anti-cancer may be used to enhance the effect of anti-cancer
agent, which is a main therapeutic agent primarily used in
chemotherapy using the anti-cancer agent, and may be used to
maximize cancer treatment effect by treating residual cancer cells
after surgery.
[0048] The F cells may be present in a form of a composition for
use in cancer therapy or for use in the adjuvant for anti-cancer,
and such a composition may include pharmaceutically acceptable
excipients, carriers and diluents. A particularly preferred
injection form is preferably formulated into an injection form
suitable for injection into a tissue or organ.
[0049] Accordingly, the present disclosure provides an adjuvant for
anti-cancer including the F cells.
[0050] The F cell may be injected into the patient's body according
to the physician's prescription or by a method well known in the
art, and a single dose will be determined in consideration of
various related factors such as a disease to be treated, the
severity of the disease, the route of administration, the body
weight, age, and sex of the patient, etc.
[0051] Further, the present disclosure provides a kit for cancer
treatment including the F cells, and 5-fluorocytosine (5-FC).
[0052] The kit for cancer treatment is prepared for the purpose of
the combination therapy of F cells and 5-fluorocytosine (5-FC), and
means a kit prepared so that the F cells are able to be injected
before, simultaneously with, and after administration of
5-fluorocytosine (5-FC) which is a prodrug, to a subject in need of
treatment.
[0053] The kit for cancer treatment may comprise a first
compartment and a second compartment, wherein the first compartment
may comprise the F cells for cancer treatment or the F cells acting
as an adjuvant for anti-cancer, and the second compartment may
include 5-fluorocytosine (5-FC). The kit may be contained in one
container or in several different small containers with each
divided dosage in order to improve convenience and portability.
Therefore, several containers may exist in each container according
to an arrangement method. The kit of the present disclosure may
include, if necessary, equipment necessary for use, instructions
including descriptions of administration methods for each
component, etc.
[0054] In addition, a kit for cancer treatment of the present
disclosure may further include an anti-cancer agent. The
anti-cancer agent may include, but is not limited to, anti-cancer
agents known in the art, and preferably may be at least one
selected from the group consisting of nitrogen mustard, imatinib,
oxaliplatin, rituximab, elotinib, trastuzumab, gefitinib,
bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab,
cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide,
dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab
tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine,
alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan,
gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine,
gimeracil, oteracil, azacytidine, cytarabine, fludarabine,
enocitabine, decitabine, mercaptopurine, thioguanine, cladribine,
carmofur, raltitrexed, docetaxel, paclitaxel, belotecan, topotecan,
vinorelbine, etoposide, vincristine, vinblastine, tenifocide,
idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin,
daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin,
temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan,
altretamine, dacarbazine, thiotepa, nimustine, chlorambucil,
mitolactol, taxotere, gleevec, taxol, herceptin, tarceva, avastin,
zoladex, adriamycin, irinotecan, 10058-F4, cisplatin,
cyclophosphamid, nitrosourea-based anti-cancer agent, methotrexate,
and doxorubicin. The nitrosourea includes carmustine, lomustine,
and the like. The anti-cancer agent may be included in the third
compartment of the kit for cancer treatment.
[0055] In addition, the present disclosure provides a method for
preventing or treating cancer including: 1) introducing cytosine
deaminase (CD) into a mesenchymal stem cell (MSC) to prepare
mesenchymal stem cell/cytosine deaminase (MSC/CD); 2) freezing the
prepared MSC/CD to prepare frozen MSC/CD; 3) thawing and suspending
the frozen MSC/CD to prepare F cells; 4) administering the F cells
to a subject in need of treatment; and 5) administering
5-fluorocytosine (5-FC) to a subject in need of treatment.
[0056] The subject is preferably a mammal, including a human, and
may include all of a patient in need of cancer treatment, a patient
undergoing cancer treatment, a patient experienced with cancer
treatment, and a patient who need to be treated for cancer, and may
also include a patient who underwent surgery to extract the cancer
for cancer treatment.
[0057] Further, the present disclosure provides the method for
preventing or treating cancer, further including: 6) administering
an anti-cancer agent to a subject in need of treatment.
[0058] Further, the present disclosure provides a method for
preventing or treating cancer including: 4) administering an
anti-cancer agent to a subject in need of treatment before,
simultaneously with administration of the F cells of step 4).
[0059] That is, the present disclosure provides the method for
preventing or treating cancer including both of simultaneous
administration of the F cells and the anti-cancer agent to a
subject in need of treatment, and sequential administration, such
as administration of the anti-cancer agent after administration of
the F cells, and administration of the F cells after administration
of the anti-cancer agent to a subject in need of treatment.
[0060] The anti-cancer agent may include, but is not limited to,
anti-cancer agents known in the art, and preferably may be at least
one selected from the group consisting of nitrogen mustard,
imatinib, oxaliplatin, rituximab, elotinib, trastuzumab, gefitinib,
bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab,
cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide,
dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab
tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine,
alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan,
gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine,
gimeracil, oteracil, azacytidine, cytarabine, fludarabine,
enocitabine, decitabine, mercaptopurine, thioguanine, cladribine,
carmofur, raltitrexed, docetaxel, paclitaxel, belotecan, topotecan,
vinorelbine, etoposide, vincristine, vinblastine, tenifocide,
idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin,
daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin,
temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan,
altretamine, dacarbazine, thiotepa, nimustine, chlorambucil,
mitolactol, taxotere, gleevec, taxol, herceptin, tarceva, avastin,
zoladex, adriamycin, irinotecan, 10058-F4, cisplatin,
cyclophosphamid, nitrosourea-based anti-cancer agent, methotrexate,
and doxorubicin. The nitrosourea includes carmustine (BCNU),
lomustine (CCNU), etc. The anti-cancer agent may be appropriately
and selectively prescribed according to a prescription dose of the
anticancer agent which is widely known in the art, but may be
prescribed at a dose smaller than an average prescription dose in
consideration of the synergistic effect with the F cells of the
present disclosure.
[0061] The F cell of the present disclosure and 5-fluorocytosine
may be administered simultaneously or sequentially with time
differences, which may be selected depending on appropriate time
and period for conversion of 5-fluorocytosine to 5-fluorouracil by
the F cell.
[0062] Therefore, the present disclosure provides the method for
preventing or treating cancer characterized in that the F cells of
step 4) and the 5-fluorocytosine of step 5) are administered
simultaneously or sequentially.
[0063] Further, the anti-cancer agent used in combination with the
5-fluorocytosine of the present disclosure may be administered
simultaneously or sequentially with time differences, and may be
selected according to appropriate time and period.
[0064] Therefore, the present disclosure provides the method for
preventing or treating cancer characterized in that the
5-fluorocytosine of step 5) and the anti-cancer agent of step 6)
are administered simultaneously or sequentially.
[0065] The sequential administration in the present disclosure
means that multiple components to be administered are administered
to a subject with time differences, and the order of each of active
components may be appropriately adjusted.
[0066] The F cells may be delivered in a pharmaceutically effective
amount to a tumor site of the subject, and the delivery is
preferably performed by an injection form, but is not limited
thereto. In particular, it is more preferable that the injection is
administered for mammals, preferably a human patient.
[0067] The F cells are the MSC/CD cells that are immediately used
by freezing, thawing, or thawing and washing, and have an cytotoxic
effect, which may be killed in a subject body for a predetermined
period of time by a suicide effect, preferably 5 to 15 days after
injection. Thus, the F cells may be repeatedly administered for
continuous treatment. Further, the 5-FC, the anti-cancer agent may
be repeatedly administered depending on the therapeutic purpose and
the condition of the patient. The cycle of the repeated
administration of the F cells, 5-FC, and the anti-cancer agent may
be appropriately adjusted according to the therapeutic condition of
the patient, and the cycle may be preferably every 10 to 30 days.
For example, in consideration of the cell death of the MSC/CD, the
repeated administration is preferable after at least 10 days, and
the repeated administration every 30 days is also preferable
considering that the cells are injected with a syringe.
[0068] Therefore, the present disclosure provides the method for
preventing or treating cancer characterized in that the
administration of the F cells of step 4) is repeated every 10 to 30
days.
[0069] Depending on the repetition of the F cell administration
cycle, the 5-fluorocytosine may also be repeatedly administered
periodically, and optionally, the anti-cancer agent for combined
administration may also be repeatedly administered
periodically.
[0070] In the method for preventing or treating cancer of the
present disclosure, F cells may be used to treat, without
limitation, all carcinomas capable of being extracted as cancer and
carcinoma, and may be administered to a subject in need of
treatment before, simultaneously with, and after chemotherapy using
administration of anti-cancer agent. The cancer may be, for
example, at least one selected from the group consisting of
squamous cell cancer (for example, squamous cell cancer of
epithelium), small cell lung cancer, non-small cell lung cancer,
lung cancer, peritoneal cancer, colorectal cancer, biliary tumor,
nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral
cancer, osteosarcoma, gallbladder cancer, kidney cancer, leukemia,
bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin
cancer, pancreatic cancer, breast cancer, liver cancer, bone
cancer, esophageal cancer, colon cancer, gastric cancer, cervical
cancer, prostate cancer, ovarian cancer, head and neck cancer, and
rectal cancer, and more preferably, brain tumor, pancreatic cancer,
liver cancer, lung cancer, colon cancer, and skin cancer.
[0071] Hereinafter, preferred Preparation Examples and Examples of
the present disclosure will be described to assist in understanding
the present disclosure. However, the following Preparation Examples
and Examples are provided only to more easily understand the
present disclosure, and therefore, the present disclosure is not
limited thereto.
Preparation Example 1: Preparation of Mesenchymal Stem Cell
Expressing Cytosine Deaminase (CD)
[0072] 1.1 Isolation and Culture of Mesenchymal Stem Cell
[0073] 4 ml of human bone marrow donated after the IRB examination
of Ajou University Medical Center was overlaid on 4 ml of
HISTOPAQUE 1077 (Sigma-Aldrich) in a sterilized 15 ml test tube,
and centrifuged at 400.times.g for 30 minutes at room temperature
using a centrifuge. After centrifugation, 0.5 ml of the
intermediate buffy coat was carefully collected using a Pasteur
pipette, and transferred to a test tube containing 10 ml of
sterilized phosphate buffer solution (pH 7.4) After centrifugation
at 250.times.g for 10 minutes, the supernatant was discarded, and
10 ml of phosphate buffer solution was added and gently suspended,
followed by centrifugation at 250.times.g for 10 minutes. This
procedure was repeated twice, and the final precipitate was added
to a DMEM medium (Gibco) supplemented with 10% FBS (Hyclone) and
dispensed in a 100 mm animal cell culture dish to obtain
1.times.10.sup.9 cells. The culture container was placed in an
incubator and cultured at 37.degree. C. for 4 hours while supplying
5% carbon dioxide and 95% air. Subsequently, in order to remove
cells that did not stick to the bottom of the culture container,
the supernatant was removed. A fresh medium was added thereto, and
cultured in an incubator.
[0074] The isolated mesenchymal stem cells were maintained in a
CO.sub.2 incubator at 37.degree. C., and cultured in a mesenchymal
stem cell culture medium [(10% FBS (Gibco)+10 ng/ml bFGF
(Sigma-Aldrich)+1% penicillin/streptomycin (Gibco)+89% DMEM
(Gibco)]. The cells were cultured while replacing the medium every
two days. When the cells reached about 80% in the culture
container, the cells were isolated using 0.25% trypsin/0.1 mM EDTA
(Gibco), diluted so as to satisfy about 800 to 1,1000
cells/cm.sup.2, or 1:10 to 1:20 with the medium, and subcultured in
a new culture container. Some of the cells were stored frozen by a
freezing medium supplemented with 2-10% DMSO (Sigma-Aldrich, 2, 5,
10%).
[0075] 1.2 Preparation of Retrovirus Including Cytosine Deaminase
(CD)
[0076] A retrovirus expressing cytosine deaminase (CD), a suicide
gene, was constructed. Detailed description thereof is as
follows:
[0077] DNA was isolated from E. coli K-12 MG1655 (Korea Research
Institute of Bioscience and Biotechnology), and then PCR was
performed under conditions of 5 minutes at 94.degree. C., 30
seconds at 94.degree. C., 40 seconds at 60.degree. C., 1 minute at
72.degree. C., 27 cycles; 7 minutes at 72.degree. C. using primers
CD-F(5'-GAA TTC AGG CTA GCA ATG TCT CGA ATA ACG CTT TAC AAA C-3')
and CD-R (5'-GGATTC TCT AGC TGG CAG ACA GCC GC-3'). The PCR product
was using a pGEM-T Easy vector cloning kit (Promega), and then a
PGEM-CD vector containing the CD gene was constructed through
blue-white colony screening using X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside,
Sigma-Aldrich) and IPTG (Isopropyl 13-D-1-thiogalactopyranoside,
Sigma-Aldrich). It was confirmed that the base sequence of the
obtained CD gene was identical to that of gi: 298594 of GeneBank
through sequence analysis. The prepared pGEM-CD plasmid and
pcDNA3.1 (Clontech) were digested with EcoRI and Notl restriction
enzymes, respectively, and the CD gene isolated from the plasmid
pGEM-CD and the cut plasmid pcDNA3.1 were ligated with T4 DNA
ligase. Then, the obtained product was used to transform E. coli
DH5a, a competent cell, and cultured on an LB plate containing 50
.mu.g/ml of ampicillin and selected to obtain a plasmid
pcDNA3.1-CD. This plasmid was digested with BamH I restriction
enzyme and introduced into a retroviral vector capable of producing
retrovirus. The obtained plasmid was isolated by a CsCl-ultra
high-speed centrifuge, and transfected into FLYRD18 cell, which is
a retrovirus packaging cell line, by calcium phosphate
precipitation (Jordan, Nucleic Acid Research, 24, 596-601 (1996)).
Then, the cells were cultured at 37.degree. C. in an incubator
while supplying 5% carbon dioxide and 95% air. After 48 hours, only
the culture solution was obtained and filtered through a 0.45 IA
filter membrane to obtain a retrovirus solution. The retrovirus
solution was dispensed and used while storing at -70.degree. C.
[0078] 1.3 Preparation of Mesenchymal Stem Cell into which CD Gene
is Introduced
[0079] The CD gene was introduced into the mesenchymal stem cell
isolated and cultured in the description of 1.1, using the
CD-containing retrovirus prepared in the description of 1.2. More
specifically, the mesenchymal stem cell was cultured to reach about
70% in a 100 mm culture dish. Then, 3 ml of the retrovirus
solution, 3 ml of the fresh mesenchymal stem cell culture medium,
and 4 .mu.g/ml of polybrene (Sigma-Aldrich) were added and cultured
for 8 hours. Then, the virus solution was removed, 10 ml of
mesenchymal stem cell culture medium was added and cultured for 16
hours, and then infected again with retrovirus. After this
procedure was performed 1 to 3 times, the mesenchymal stem cell was
finally taken off with trypsin and subcultured by 1:20 dilution
with the medium. In the subculture, puromycin (Sigma-Aldrich) was
added to the medium to a concentration of 2 .mu.g/ml. The cells
were screened for 2 weeks so that only cells infected with
retrovirus could survive. Finally, the mesenchymal stem cell line
(hereinafter, referred to as MSC/CD) continuously expressing CD,
which is a suicide gene, was constructed.
Example 1. Suicide Effect and Bystander Effect of MSC/CD
[0080] 1.1 Confirmation of Suicide Effect
[0081] The cytosine deaminase (CD) is a suicide gene. The MSC/CD,
which is a mesenchymal stem cell expressing the cytosine deaminase
(CD), may convert 5-fluorocytosine (5-FC) which is a prodrug into
5-fluorouracil (5-FU) and may have a suicide effect (the cells kill
themselves) and a bystander effect (peripheral cells are killed) as
shown in FIG. 1. Therefore, the suicide effect and the bystander
effect of the MSC/CD prepared in the description of 1.3 were
confirmed. First, 10,000 MSC/CD or mesenchymal stem cells (MSCs)
were cultured in a 12-well plate. From the next day, the cells were
treated with the prodrug 5-FC at a concentration of 0 to 1,000
.mu.M, and replaced with a new medium containing the drug once
every two days. The medium was changed to a cell culture medium
containing 0.5 mg/ml of MTT
(3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide,
Sigma-Aldrich) capable of measuring living cells on the 6.sup.th
day after treatment with 5-FC, and reacted for 2 hours at
37.degree. C. Then, the MTT solution was removed. 500 .mu.l of DMSO
was added to each well to perform a color reaction. Then, the
lysates were transferred to a 96-well plate and absorbance was
measured at 540 nm using an ELISA reader (E-max, Molecular device).
A schematic diagram of the experimental method and the results
thereof are shown in FIG. 2, in graphs (A) and (B) thereof.
[0082] As shown in FIG. 2, in graphs (A) and (B) thereof, it was
confirmed that the MSC/CD had a suicidal effect in which the living
cells were reduced as the concentration of the prodrug 5-FC was
increased, and ICso inducing 50% cell death was 60.4 .mu.M. In the
case of mesenchymal stem cell, which is the control group, it was
confirmed that even if the concentration of 5-FC was increased, the
cells did not kill themselves, and thus, the cytototoxic effect was
not observed.
[0083] 1.2 Bystander Effect
[0084] 10,000 GFP-expressing U87MG (Korean Cell Line Bank KCLBNo.
30014) glioma cells and 10,000 MSC/CD or mesenchymal stem cells
(MSCs) were cultured on a 12-well plate. From the next day, the
cells were treated with the prodrug 5-FC at a concentration of 0 to
1,000 .mu.M, and replaced with a new medium containing the drug
once every two days, and after 6 days, the cells were obtained.
Such an experimental method is briefly shown in FIG. 3, in graph
(A) thereof.
[0085] Then, fluorescence images of U87MG expressing GFP remaining
in the whole well were taken using an dissecting fluorescence
microscope (Olympus). After the fluorescence images were taken, 200
.mu.l of 1.times. passive lysis buffer (Promega) was added, and the
cell lysate was placed on ice for 10 minutes and transferred to an
E-tube and centrifuged at 12,000 rpm for 5 minutes, and the
supernatant was transferred to a new container. 100 .mu.l of the
supernatant was transferred to a black 96-well plate, and the
degree of fluorescence was measured under conditions of excitation
488 nm and emission 530 nm using a fluorimeter (GEMINI EM,
molecular device). The results are shown in FIG. 3, in fluorescence
images (B) and bystander effects graph (C) thereof.
[0086] As shown in FIG. 3, in fluorescence images (B) and bystander
effects graph (C) thereof, U87MG cultured with MSC/CD showed a
decrease in the number of cells expressing fluorescence and a
remarkable decrease in fluorescence intensity as the concentration
of 5-FC was increased, but U87MG cultured with the mesenchymal stem
cells in which the CD was not expressed did not show a significant
difference in the number of cells expressing fluorescence and
fluorescence intensity according to the concentration of 5-FC.
Herein, IC.sub.50 inducing 50% cell death was 50.48 .mu.M. The
results indicated that the MSC/CD, which is the mesenchymal stem
cell into which the CD was introduced, showed not only the suicide
effect but also the bystander effect in which the peripheral cells
were killed together.
Preparation Example 2. Preparation of "I Cell" and "F Cell"
[0087] In the case of a cell therapy agent, the effect may vary
depending on the change in the cell state under various conditions.
Therefore, in order to confirm whether the cell ability was changed
according to the state of MSC/CD, cells in a thawed state
immediately after freezing (hereinafter, referred to as F cells
(frozen cells)), and cells in a harvested state immediately from
the cells during the culturing (hereinafter, referred to as I cells
(immediately harvested cells)) were prepared.
[0088] In order to prepare the F cells and the I cells,
respectively, the MSC/CD was cultured, and then 2.times.10.sup.6
cells were dispensed in 1 ml of freezing medium containing 2 to 10%
(2, 5, 10%) DMSO or 2.times.10.sup.6 cells were dispensed in 1 ml
of Cryostor CS10 (BioLife Solutions). The cells were frozen and
stored in a liquid nitrogen container. First, in order to obtain "I
cells" of MSC/CD, cells of passage 5 were dissolved at 37.degree.
C., and placed in 9 ml of culture medium. Then, the cells were
centrifuged at 500 g for 5 minutes to remove the supernatant, and
the precipitated cells were suspended in the culture solution, and
dispensed in a 150 mm culture container. The next day,
1.5.times.10.sup.5 cells were transferred to the 150 mm culture
container, and replaced with new medium once every two days. After
6 days, the cells were collected to prepare "I cells" corresponding
to passage 6.
[0089] In order to obtain the "F cells", the MSC/CD frozen and
stored in the passage 6 at -130.degree. C. or below was dissolved
at 37.degree. C. and suspended in a Plasma Solution A (CJ
HealthCare Corp.) containing 9 ml of human serum albumin, followed
by centrifugation at 500.times.g for 5 minutes, or suspended in
physiological saline containing a final 7.5% dextran-40 (Daihan
Pharm Co., Ltd.) and 5% human serum albumin (Green Cross Corp.),
followed by centrifugation at 800.times.g for 10 minutes. Then, the
supernatant was removed, and the precipitated cells were suspended
in the culture medium and used. In other words, the F cells are
cells that were not subjected to the culturing step after freezing
and thawing. The I cells and the F cells were washed twice,
respectively, before transplantation into the brain.
Example 1. Bystander Effect and Colony Formation of F Cells
[0090] 1.1 Bystander Effect of F Cells
[0091] Experiments were performed to determine whether the F cells
could normally exhibit the bystander effect of MSC/CD and whether
the bystander effect appeared effectively regardless of the
preparation method such as the type of medium. 10,000 I cells
prepared by the method of Preparation Example 2, 10,000 F cells
prepared with 10% DMSO freezing medium (DMSO), and 10,000 F cells
prepared with Cryostor CS10 (CS10) were washed with a Plasma
Solution A, and were co-cultured in 12-well plates together with
glioma U87/GFP cells in which GFP gene was transduced into U87MG
(KCLB No. 30014) donated from Korean Cell Line Bank so as to
express fluorescence. From the next day, the cells were treated
with the prodrug 5-FC at a concentration of 0 to 1,000 .mu.M, and
replaced with a new medium containing the drug once every two days,
and after 6 days, the cells were obtained. 200 .mu.l of 1.times.
passive lysis buffer (Promega) was added, and the cells were placed
on ice for 10 minutes. The cell lysate was transferred to an E-tube
and centrifuged at 12,000 rpm for 5 minutes, and the supernatant
was transferred to a new container. 100 .mu.l of the supernatant
was transferred to a black 96-well plate in which light is blocked,
and the degree of fluorescence was measured under conditions of
excitation 488 nm and emission 530 nm using a fluorimeter (GEMINI
EM, molecular device). A schematic diagram of the experimental
method and the bystander effect are shown in FIG. 4, in graphs (A)
and (B) thereof.
[0092] As shown in FIG. 4, in graphs (A) and (B), the IC.sub.50
values indicating concentration at which 50% of apoptosis is
caused, was 60.4 .mu.M for I cells, 56.2 .mu.M for F cells prepared
with 10% DMSO freezing medium (DMSO), and 55.4 .mu.M for F cells
prepared with Cryostor CS10 (CS10). These results indicated that
the F cell was a cell that could effectively exhibit the bystander
effect as compared to the I cell, and thus it was confirmed that
the F cell exhibited the bystander effect more excellently as
compared to the I cell regardless of the preparation method using
the DMSO medium or the CS10 medium.
[0093] Therefore, hereinafter, the F cells prepared with the 10%
DMSO freezing medium were used among the F cells prepared in
Preparation Example 2.
[0094] 1.2. Colony Forming Effect of F Cells
[0095] A colony forming unit assay was performed to confirm whether
the F cells prepared by freezing and thawing as in Preparation
Example 2 exhibited a difference in proliferative capacity as
compared to the I cells. The I cells prepared as in Preparation
Example 2 and corresponding the same passage and the F cells
prepared with 10% DMSO freezing medium were counted with Countess
(Invitrogen), and then 100 cells were placed in a 100 mm culture
container, respectively, and cultured while replacing the medium
with a new medium once every two days. After 2 weeks, the cells
were fixed with 10% formalin for 10 minutes, washed with phosphate
buffer solution (Gibco), stained with a crystal violet solution
(Sigma-Aldrich) for 10 minutes, and washed three times with water
to obtain images by Versa doc (BioRad). The number of colonies
formed by growing one cell for 2 weeks was visually counted, and
shown in FIG. 5, in image (A) and graph (B) thereof.
[0096] As shown in FIG. 5, in image (A) and graph (B) thereof, it
was confirmed that the I and F cells had no difference in the
colony forming ability. These results indicated that the increased
bystander effect of the F cells as compared to that of the I cells
was not caused by an increase in the number of cells such as an
increase in colony formation, and that the F cells themselves
exhibited more excellent bystander effect as compared to that of
the I cells.
Example 2. Anti-Cancer Effect of F Cell
[0097] 2.1. Anti-Cancer Effect of F Cell on Brain Tumor
[0098] In order to confirm the anti-cancer effect of the F cells on
brain tumor, an orthotopic glioma model was constructed. First,
7-week-old immunodeficient nude mice (central experimental animals)
were anesthetized and their mouths and ears were fixed in a
sterotaxic frame. The head to be incised was sterilized with 70%
ethanol, and the crown of head was vertically incised to about 0.5
cm. 3.times.10.sup.5/3 .mu.l of U87MG glioma cells expressing LacZ
were transplanted at a rate of 0.3 .mu.l/min at the brain
coordinates of AP=+0.5 mm, ML=-1.8 mm, and DV=-3 mm, thereby
constructing a brain tumor orthotopic glioma model. On the 3rd day
after transplantation of U87MG glioma cells expressing LacZ, the F
cells prepared in Preparation Example 2 were suspended in a Plasma
Solution A, followed by centrifugation at 500.times.g for 5
minutes. The supernatant was discarded, and the washing procedure
was repeated once or twice. Then, the cell suspension was prepared
to have a concentration of 3.times.10.sup.5/6 .mu.l. The cell
suspension was transplanted at a rate of 0.3 .mu.l/min at the brain
coordinates of AP=+0.5 mm, ML=-1.8 mm, and DV=-3 mm. From the next
day after the cell transplantation, 5-FC (15 mg/ml physiological
saline) was intraperitoneally injected at a dose of 500 mg/kg for
one week. To compare the anti-cancer effect of the F cells, the I
cells prepared in Preparation Example 2 were transplanted in the
same manner, and 5-FC (15 mg/ml physiological saline) was
intraperitoneally injected at a dose of 500 mg/kg for one week from
the next day after the cell transplantation.
[0099] On the 28.sup.th day after glioma cell transplantation, the
animals were anesthetized, and perfused with physiological saline
by inserting an injection needle into the left ventricle of the
animals, and then, perfused with 10% formalin solution. The brain
was extracted and fixed on a mouse brain matrix (Stoelting). Then,
the brain was cut horizontally at intervals of 1 mm, and put in the
same fixing fluid for 30 minutes, thereby fixing the brain slice.
The brain slice was washed three times with phosphate buffered
saline, immersed in phosphate buffered saline containing 20 mg/ml
of X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside,
Koma Biotech), 5 mM potassium ferrocyanide/ferricyanide
(Sigma-Aldrich), 2 mM MgCl.sub.2, and 0.02% NP40, and reacted at
37.degree. C. for 16 hours. An image of the brain tumor was
obtained through an anatomic microscope. A size of the tumor was
calculated using the image j (NIH) program and the following
Equation, and the results are shown in FIG. 6, in graph (A), images
(B), and graph (C) thereof.
Tumor volume ( mm 3 ) = Number of pixels of tumor area Number of
pixels per unit area .times. 0.5 mm ##EQU00001##
[0100] As shown in FIG. 6, in images (B) thereof, in the F cell
transplantation group, the volume of the brain tumor was remarkably
decreased, and more excellent effect was exhibited as compared to
the I cell. The computed mean size of the tumor was 91.3 mm.sup.3
in the vehicle control group, 1.5 mm.sup.3 in the I cell
transplantation comparison group, but 0.6 mm.sup.3 in the F cell
transplantation animal group. That is, according to the in vivo
results, it was confirmed that the F cell had a tumor-reducing
effect twice more than that of the I cell among the MSC/CD, and the
F cell could be a more effective anti-cancer agent.
[0101] 2.2. Anti-Cancer Effect of F Cell on Liver Cancer
[0102] 1.times.10.sup.5 Huh7 liver cancer cells (KCLB No. 60104)
were suspended in 100 .mu.l of a phosphate buffer solution
containing 20% Matrigel (BD), and transplanted subcutaneously in
7-week-old immunodeficient nude mice (Balb/c nude). After 14 days
from transplantation, when the tumor size reached 10 to 100
mm.sup.3, the I cells and the F cells prepared in Preparation
Example 2 were injected. More specifically, the I cells and F cells
prepared in Preparation Example 2 were suspended in the phosphate
buffer solution, followed by centrifugation at 500.times.g for 5
minutes. This procedure was repeated twice, and then the I cells or
the F cells were prepared to have concentration of
1.times.10.sup.6/100 .mu.l in the phosphate buffer solution and
injected directly into the tumor using a syringe. From the next day
after the I cell and F cell injection, 5-FC (15 mg/ml physiological
saline) was intraperitoneally injected at a dose of 500 mg/kg for
one week. The size of the tumor was measured once or twice weekly
using a digital caliper, and the tumor volume was calculated using
the following Equation, and the results are shown in FIG. 7, in
graph (A) and animal images (B) thereof.
Subcutaneous tumor volume (mm.sup.3)=Width (mm).times.Length
(mm).times.Height (mm)/6
[0103] [For measurement of tumor volume, see Cancer Chemother.
Pharmacol. (1989) 24: 148-154]
[0104] As shown in graph (A) of FIG. 7, the tumor volume was more
effectively decreased in the experimental group treated with the F
cell, confirming that the F cell had more excellent anti-cancer
effect than the I cell. As shown in animal images (B) of FIG. 7,
the increased anti-cancer effect was visually confirmed in the
subcutaneous tumor model transplanted with the liver cancer cells,
and thus, it was confirmed that the therapeutic effect of F cells
was excellent (*, p<0.05 upon comparison with the PBS
group).
[0105] 2.3. Anti-Cancer Effect of F Cell on Pancreatic Cancer
[0106] BxPC3 pancreatic cancer cells (ATCC No. CRL-1687.TM.) had a
rapid growth rate and severe ulceration, and thus, 1.times.10.sup.6
BxPC3 pancreatic cancer cells, the I cells, and the F cells were
injected simultaneously. More specifically, the I cells and F cells
prepared in Preparation Example 2 were suspended in the phosphate
buffer solution, followed by centrifugation at 500.times.g for 5
minutes. This procedure was repeated twice, and then the I cells or
the F cells were prepared to have concentration of
1.times.10.sup.6/100 .mu.l in the phosphate buffer solution.
1.times.10.sup.6 I cells or F cells were suspended in 100 .mu.l of
a phosphate buffer solution containing 20% Matrigel (BD), and
simultaneously transplanted subcutaneously in 7-week-old
immunodeficient nude mice (Balb/c nude). From the next day after
the cell injection, 5-FC (15 mg/ml physiological saline) was
intraperitoneally injected at a dose of 500 mg/kg for one week.
[0107] The tumor volume was measured in the same manner as in
Example 2.2, and the measured tumor volume change was shown in FIG.
8, in graph (A) thereof.
[0108] As shown in graph (A) of FIG. 8, the tumor volume was more
effectively decreased in the experimental group treated with the F
cell, confirming that the F cell had more excellent anti-cancer
effect than the I cell, and had excellent therapeutic effect in the
pancreatic cancer subcutaneous tumor model (*, p<0.05 upon
comparison with the PBS group).
[0109] 2.4. Anti-Cancer Effect of F Cell on Lung Cancer
[0110] 5.times.10.sup.6 A549 lung cancer cells (ATCC No.
CCL-185.TM.) were suspended in 100 .mu.l of a phosphate buffer
solution containing 20% Matrigel (BD), and transplanted
subcutaneously in 7-week-old immunodeficient nude mice (Balb/c
nude). After 8 days from transplantation, when the tumor size
reached 20 to 50 mm.sup.3, the I cells and the F cells prepared in
Preparation Example 2 were injected. More specifically, the I cells
and F cells prepared in Preparation Example 2 were suspended in the
phosphate buffer solution, followed by centrifugation at
500.times.g for 5 minutes.
[0111] This procedure was repeated twice, and then the I cells or
the F cells were prepared to have concentration of
1.times.10.sup.6/100 .mu.l in the phosphate buffer solution and
injected directly into the tumor using a syringe. From the next day
after the I cell and F cell injection, 5-FC (15 mg/ml physiological
saline) was intraperitoneally injected at a dose of 500 mg/kg for
one week. The tumor volume was measured in the same manner as in
Example 2.2, and the measured tumor volume change was shown in FIG.
8, in graph (B) thereof.
[0112] As shown in graph (B) of FIG. 8, the tumor volume was more
effectively decreased in the experimental group treated with the F
cell, confirming that the F cell had more excellent anti-cancer
effect than the I cell, and had excellent therapeutic effect in the
lung cancer subcutaneous tumor model (*, p<0.05 upon comparison
with the PBS group).
[0113] 2.5. Anti-Cancer Effect of F Cell on Colon Cancer
[0114] 5.times.10.sup.6 HT29 colon cancer cells (ATCC No.
HTB-38.TM.) were suspended in 100 .mu.l of a phosphate buffer
solution containing 20% Matrigel (BD), and transplanted
subcutaneously in 7-week-old immunodeficient nude mice (Balb/c
nude). After the transplantation, when the tumor size reached 40 to
100 mm.sup.3, the I cells and the F cells prepared in Preparation
Example 2 were injected. More specifically, the I cells and F cells
prepared in Preparation Example 2 were suspended in the phosphate
buffer solution, followed by centrifugation at 500.times.g for 5
minutes.
[0115] This procedure was repeated twice, and then the I cells or
the F cells were prepared to have concentration of
1.times.10.sup.6/100 .mu.l in the phosphate buffer solution and
injected directly into the tumor using a syringe. From the next day
after the I cell and F cell injection, 5-FC (15 mg/ml physiological
saline) was intraperitoneally injected at a dose of 500 mg/kg for
one week. The tumor volume was measured in the same manner as in
Example 2.2, and the measured tumor volume change was shown in FIG.
8, in graph (C) thereof.
[0116] As shown in graph (C) of FIG. 8, the tumor volume was more
effectively decreased in the experimental group treated with the F
cell, confirming that the F cell had more excellent anti-cancer
effect than the I cell, and had excellent therapeutic effect in the
colon cancer subcutaneous tumor model (*, p<0.05 upon comparison
with the PBS group, and #, p<0.05 upon comparison with the I
cell group).
[0117] From the above results, it was confirmed that the F cell had
an excellent anti-cancer effect in various types of cancer such as
brain tumor, liver cancer, lung cancer, colon cancer, and exhibited
a more excellent anti-cancer effect in all types of cancer as
compared to the I cell.
Example 3. Synergistic Effect of Combination Therapy of F Cells (In
Vitro)
[0118] 3.1 In Vitro Combination Therapy with Temozolomide
[0119] 10,000 GFP-expressing U87MG (Korean Cell Line Bank KCLBNo.
30014) glioma cells and 10,000 F cells or mesenchymal stem cells
(MSCs) were cultured in a 12-well plate. The next day, the cells
were treated with temozolomide (TMZ) at a concentration of 0 to
1000 .mu.M and the prodrug 5-FC at a concentration of 0 to 1,000
.mu.M, and replaced with a new medium containing the drug once
every two days, and after 6 days, the cells were obtained. A
schematic diagram of this experiment is shown in graph (A) of FIG.
9. Fluorescence images of U87MG expressing GFP remaining in the
whole well were taken using a dissecting fluorescence microscope
(Olympus), and cell death was confirmed. The results are shown in
the fluorescence images (D) of FIG. 9.
[0120] As shown in the fluorescence images (D) of FIG. 9, it was
confirmed that the fluorescence expression was decreased according
to an increase in the temozolomide treatment concentration and an
increase in the 5-FC treatment concentration, and the bystander
effect of the F cell was elevated by the combined administration
with temozolomide.
[0121] After the fluorescence images were taken, 200 .mu.l of
1.times. passive lysis buffer (Promega) was added, and the cells
were placed on ice for 10 minutes. The cell lysate was transferred
to an E-tube and centrifuged at 12,000 rpm for 5 minutes, and the
supernatant was transferred to a new container. 100 .mu.l of the
supernatant was transferred to a black 96-well plate, and the
intensity of fluorescence was measured under conditions of
excitation 488 nm and emission 530 nm using a fluorimeter (GEMINI
EM, molecular device) (graphs (B) and (C) of FIG. 9). The actual
ICso values obtained by treating the two types of anti-cancer
agents together were expressed as isobologram. The solid line
connecting the ICso values when the anti-cancer agent was treated
alone in the isobologram is a theoretical additive line, which
means that when the values obtained by simultaneously treating two
anti-cancer agents are on the solid line, there is simply an
additive effect. On the other hand, it means that when the value is
lower than the solid line, there is a synergistic effect, and
conversely, when the value is above the solid line, there is an
antagonistic effect. The results are shown in FIG. 9, in graph (E)
thereof.
[0122] As shown in graph (E) of FIG. 9, all of the marked points
when the MSC/CD was treated with 5-FC and temozolomide in
combination were present at the lower side as compared to the ICso
values when the anti-cancer agent was treated alone, which was
confirmed that there was a synergistic effect which is more
excellent than the additive effect obtained by simple treatment
with the two anti-cancer agents together.
[0123] 3.2 In Vitro Combination Therapy with Carmustine (BCNU)
[0124] 10,000 GFP-expressing U87MG glioma cells, i.e., U87/GFP
(Korean Cell Line Bank KCLBNo. 30014) and 10,000 F cells (MSC/CD)
were cultured in a 12-well plate. From the next day, the cells were
treated with Carmustine (BCNU, Sigma) at a concentration of 0 to
300 .mu.M and the prodrug 5-FC at a concentration of 0 to 300
.mu.M, and cultured for 6 days while replacing with a new medium
containing the drug once every two days. On the 7th day, the
culture solution was removed, 200 .mu.l of 1.times. passive lysis
buffer (Promega) was then added to each well, and each well was
allowed to stand at 4.degree. C. for 10 minutes. The cell lysate
was collected and centrifuged at 12,000 rpm for 5 minutes to obtain
a supernatant. 100 .mu.l of the supernatant was transferred to a
black 96-well plate in which light is blocked, and the intensity of
fluorescence was measured under conditions of excitation 488 nm and
emission 530 nm using a fluorimeter (GEMINI EM, molecular device).
A schematic diagram of this experiment is shown in graph (A) of
FIG. 10.
[0125] The actual ICso values obtained by treatment with the two
types of anti-cancer agents together were expressed as isobologram,
and the results were shown in FIG. 10, in graph (B) thereof.
[0126] As shown in graph (B) of FIG. 10, all of the marked points
when U87/GFP was treated with [F cell (MSC/CD)+5-FC] and carmustine
in combination were present at the lower side of the solid line as
compared to the ICso values when the anti-cancer agent was treated
alone, which was confirmed that there was a synergistic effect
which is more excellent than the additive effect obtained by simple
treatment with the two anti-cancer agents together.
[0127] 3.3 In Vitro Combination Therapy with Irinotecan
[0128] 10,000 U87/GFP and 10,000 F cells (MSC/CD) were cultured in
a 12-well plate. From the next day, the cells were treated with
Irinotecan (Sigma) at a concentration of 0 to 30 .mu.M and the
prodrug 5-FC at a concentration of 0 to 300 .mu.M, and cultured for
6 days while replacing with a new medium containing the drug once
every two days. On the 7th day, the culture solution was removed,
200 .mu.l of 1.times. passive lysis buffer was then added to each
well, and each well was allowed to stand at 4.degree. C. for 10
minutes. The cell lysate was collected and centrifuged at 12,000
rpm for 5 minutes to obtain supernatant. 100 .mu.l of the
supernatant was transferred to a black 96-well plate in which light
is blocked, and the intensity of fluorescence was measured under
conditions of excitation 488 nm and emission 530 nm using a
fluorimeter (GEMINI EM). The actual ICso values obtained by
treatment with the two types of anti-cancer agents together were
expressed as isobologram, and the results were shown in graph (C)
of FIG. 10.
[0129] As shown in graph (C) of FIG. 10, the ICso values obtained
by treatment with irinotecan alone in the isobologram was 3.2 .mu.M
and the ICso values obtained by treatment with 5-FC was 73.6 .mu.M,
and these ICso values were connected to obtain a theoretical
additive line representing a simple additive effect. The ICso
values obtained when U87/GFP was treated with [F cell
(MSC/CD)+5-FC] and irinotecan in combination were present at the
lower side of the solid line as compared to the ICso values when
the anti-cancer agent was treated alone, which was confirmed that
there was a synergistic effect which is more excellent than the
effect obtained by simple treatment with the two anti-cancer agents
together.
[0130] Since all of temozolomide, carmustine, and irinotecan are
known as anti-cancer agents that act on proliferating cells, if
they affect the survival of not only U87/GFP cells but also
proliferating F cells, the anti-cancer effect of [F cell
(MSC/CD)+5-FC] might be reduced. However, contrary to the
expectation, it was confirmed from graph (E) of FIG. 9 that the
synergistic effect could be obtained which was rather elevated as
compared to the administration of the anti-cancer agent alone or in
combination, when [F cell (MSC/CD)+5-FC] was used in combination
with temozolomide. That is, the synergistic effect is an unexpected
effect that is not capable of being expected in the existing
combination therapies.
[0131] 3.4 In Vivo Combination Therapy with Temozolomide
[0132] The synergistic effects confirmed in the above-descriptions
of 3.1 to 3.3 were further confirmed and verified in vivo. On the
6th days after transplantation of U87MG glioma cells expressing
LacZ, the F cells prepared in Preparation Example 2 were suspended
in a Plasma Solution A, followed by centrifugation at 500.times.g
for 5 minutes. The supernatant was discarded, and the washing
procedure was repeated twice. Then, the cell suspension was
prepared to have a concentration of 3.times.10.sup.5/6 .mu.l. The
cell suspension was transplanted at a rate of 0.3 .mu.l/min at the
brain coordinates of AP=+0.5 mm, ML=-1.8 mm, and DV=-3 mm. From the
next day after the cell transplantation, 5-FC (15 mg/ml
physiological saline) was intraperitoneally injected at a dose of
500 mg/kg for one week. After 4 days, temozolomide (1 mg/ml in
DMSO:physiological saline=1:1) was intraperitoneally injected at a
dose of 5 mg/kg for 5 days. On 28th day, brain tissues were
obtained from 8 animals in each animal group and X-gal staining was
performed in the same manner as in Example 2 to measure the size of
brain tumor. The experimental procedure is shown in graph (A) of
FIG. 11, and the changes in measured brain tumor size are shown in
images (B) and graph (C) of FIG. 11.
[0133] As shown in images (B) and graph (C) of FIG. 11, the average
size of vehicle control tumors was 59.8 mm.sup.3, whereas the
average size of the animal group into which the F cells were
transplanted was 11.8 mm.sup.3, the average size of the animal
group treated with temozolomide alone was 9.9 mm.sup.3, and the
average size of the animal group into which the F
cells+temozolomide were transplanted was 2.7 mm.sup.3. The animal
group into which the F cells and temozolomide combination therapy
were transplanted had the most reduced average size of the tumor,
and showed the most excellent anti-cancer effect.
[0134] In addition, the survival rate according to the treatment of
brain tumor was measured up to 90 days, and the results were
compared in each experimental group and showed in graph (D) of FIG.
11.
[0135] As shown in graph (D) of FIG. 11, the median survival in the
control group was 32 days, but prolonged to 42 days in the F cell
treatment group and 42 days in the temozolomide treatment group. In
particular, the median survival was prolonged to 70 days in the
group with the F cell and temozolomide combination therapy, and a
remarkable improvement in the survival rate was observed even in
comparison with the group treated with the anti-cancer agent alone
as well as the control group.
[0136] This suggests that the combination therapy with F cell
showed a remarkably excellent effect even in the combination
therapy with the existing anti-cancer agents, and thus, the
combination therapy with F cell is able to be utilized as a remedy
for improving the existing chemotherapy.
Example 4. Subcutaneous Tumor Treatment Effect of F Cells
[0137] To determine whether the F-cell could exhibit the
anti-cancer effect in tumors other than brain tumor, U87MG glioma
cells were transplanted into subcutaneous tissue to construct a
non-brain cancer model. 1.times.10.sup.6 U87MG glioma cells were
suspended in 100 .mu.l of a phosphate buffer solution containing
20% Matrigel (BD), and transplanted subcutaneously in 7-week-old
immunodeficient nude mice. The size of the tumor was measured twice
weekly using a digital caliper, and the tumor volume was calculated
using the following Equation.
Subcutaneous tumor volume (mm.sup.3)=Width (mm).times.Length
(mm).times.Height (mm)/6
[0138] When the size of the tumor reached 30 mm.sup.3 or more on
20.sup.th day after the U87MG glioma cells were transplanted into
the subcutaneous tissue, the I cells and F cells prepared in
Preparation Example 2 were suspended in a Plasma Solution A, and
centrifuged at 500.times.g for 5 minutes. This procedure was
repeated twice, and then the I cells or the F cells were prepared
to have concentration of 1.times.10.sup.6/100 .mu.l in the Plasma
Solution A and injected directly into the tumor using a syringe.
From the next day after the I cell and F cell injection, 5-FC (15
mg/ml physiological saline) was intraperitoneally injected at a
dose of 500 mg/kg for one week. After one week from the
administration of 5-FC was discontinued, temozolomide
(Sigma-Aldrich) was intraperitoneally injected at a dose of 5 mg/kg
for 5 days. Such an experimental method is briefly shown in graph
(A) of FIG. 12. The measured tumor volume change is shown in graph
(B) of FIG. 12. The Kaplan Meier survival graph obtained by
measuring the lifespan of nude mice in the subcutaneous tumor model
is shown in graph (C) of FIG. 12.
[0139] As shown in graph (B) of FIG. 12, it was confirmed that the
tumor volume was more effectively decreased in the experimental
group treated with F cells until the 11.sup.th day before the
administration of temozolomide, and the F cells showed more
excellent anti-cancer effect than the I cells. Further, on the
21.sup.st day after the administration of temozolomide, the tumor
volume inhibitory effect in the F cell treatment group was
confirmed to be very remarkable as compared to the non-treatment
group (the control group) and the I cell treatment group. In
addition, as shown in graph (C) of FIG. 12, the median survival of
the control group was 23 days, and the median survival of the I
cell treatment group was 30 days, whereas the F cell treatment
group showed the median survival of 58 days, and thus, the life
extension effect was also the greatest in the animal group treated
with the F cells. Therefore, it may be appreciated that the F cells
have excellent therapeutic effect in the subcutaneous tumor model,
and in particular, exhibit a synergistic therapeutic effect through
combination therapy with temozolomide, which demonstrates
superiority of the F cell combination therapy in other organs,
which is difficult to be expected from in vitro experiment
alone.
* * * * *