U.S. patent application number 12/127743 was filed with the patent office on 2008-11-27 for treatment of graft-versus-host disease with il-10 expressing cells.
Invention is credited to Sun Uk SONG.
Application Number | 20080292601 12/127743 |
Document ID | / |
Family ID | 40072607 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080292601 |
Kind Code |
A1 |
SONG; Sun Uk |
November 27, 2008 |
TREATMENT OF GRAFT-VERSUS-HOST DISEASE WITH IL-10 EXPRESSING
CELLS
Abstract
This present application describes a therapeutic agent for
treating acute or chronic graft-versus-host disease using clonal
marrow stem cells (cMSCs) as active ingredient.
Inventors: |
SONG; Sun Uk; (Incheon,
KR) |
Correspondence
Address: |
JHK LAW
P.O. BOX 1078
LA CANADA
CA
91012-1078
US
|
Family ID: |
40072607 |
Appl. No.: |
12/127743 |
Filed: |
May 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60940349 |
May 25, 2007 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/372 |
Current CPC
Class: |
A61K 38/2066 20130101;
A61K 35/17 20130101; A61P 17/00 20180101; A61P 27/02 20180101; A61P
1/00 20180101; A61K 2035/122 20130101; C12N 5/0663 20130101; A61P
37/06 20180101; A61P 1/16 20180101; A61K 35/28 20130101; A61P 37/02
20180101; A61K 2035/124 20130101 |
Class at
Publication: |
424/93.7 ;
435/372 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/08 20060101 C12N005/08; A61P 37/02 20060101
A61P037/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2007 |
KR |
10-2007-0053298 |
Claims
1. A therapeutic agent for treating acute or chronic
graft-versus-host disease comprising a homogeneous population of
clonal marrow stem cells as the active ingredient.
2. The therapeutic agent according to claim 1, which is isolated
using subfractionation culturing method.
3. The therapeutic agent according to claim 1, wherein the cells
express CD29, CD44, and CD105 cell surface antigens, but not HLA-DR
cell surface antigens.
4. The therapeutic agent according to claim 1, wherein the cells
express CD29, CD44, CD73, CD90, CD105, and CD166 cell surface
antigens but not CD106, CD119, or HLA-DR cell surface antigens.
5. The therapeutic agent according to claim 1, wherein the cells
secrete at least about 5 ng/ml of interleukin-10 (IL-10).
6. The therapeutic agent according to claim 1, wherein the cells
secrete IL-10, which is at least about 5 fold greater than cells
isolated using density centrifugation methods.
7. A homogeneous population of clonal marrow stem cells, which
express IL-10 at least about 5 fold greater than cells isolated
using density centrifugation methods.
8. A homogeneous population of clonal marrow stem cells, which
express IL-10 at least about 5 fold greater than cells isolated
using density centrifugation methods, wherein the homogeneous
population of clonal marrow stem cells is obtained by (i) allowing
a sample of bone marrow cells to settle in a container; (ii)
transferring supernatant from the container to another container;
and (iii) isolating cells from the supernatant, which has
comparatively lower density in the sample.
9. The homogeneous population of clonal marrow stem cells according
to claim 8, wherein the marrow cells are incubated for about 1 to 3
hours at 37.degree. C. and then repeatedly incubated about 2 to 3
times for about 12 to 36 hours at 37.degree. C. and then incubated
for about 24 to 72 hours at C with an upper culture supernatant
being transferred to a new culture container each time.
10. The homogeneous population of clonal marrow stem cells
according to claim 8, wherein the container is treated with a
coating.
11. The homogeneous population of clonal marrow stem cells
according to claim 10, wherein the coating is collagen,
poly-lysine, fibrinogen, or gelatin.
12. The therapeutic agent according to claim 9, wherein the
repeated incubation is carried out about 4 to 5 times.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. Provisional Patent Application No. 60/940,349, filed May 25,
2007, the contents of which are incorporated by reference herein in
their entirety. The present application also claims the benefit of
priority to U.S. patent application Ser. No. 11/471,684, filed Jun.
19, 2006, now pending, the contents of which are incorporated by
reference herein in their entirety. The present application also
claims the benefit of priority to U.S. patent application Ser. No.
11/755,320, filed May 30, 2007, now pending, the contents of which
are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a therapeutic agent for
treating acute or chronic graft-versus-host disease. The present
invention also relates to a therapeutic agent for treating acute or
chronic graft-versus-host disease that contains mesenchymal stem
cells as an active ingredient.
[0004] 2. General Background and State of the Art:
[0005] Graft-versus-host disease (GVHD) refers to a disease wherein
the body of the patient has an immune reaction to the donor's
peripheral blood or the T lymphocytes in the bone marrow that are
injected during homogeneous transplantation. Namely, it is a
disease that is induced by living lymphocytes which were transfused
causing an immune reaction that leads to problems in liver
function, skin lesions, jaundice, diarrhea, fever, pancytopenia,
and so on, and in severe cases patient death.
[0006] Graft-versus-host disease can be classified largely into
acute graft-versus-host disease (aGVHD) and chronic
graft-versus-host disease (cGVHD). cGVHD is the most major and
common side effect, occurring in 20%-70% of patients living past
100 days following blood and marrow progenitor cell
transplantation, and a major cause of death following
transplantation. Because cGVHD and aGVHD are not successive
diseases, aGVHD requires a different approach and cGVHD is becoming
the bigger problem due to developments in blood and marrow
progenitor cell transplantation therapeutic methods.
[0007] cGVHD in the case of homogenous transplantations, occurs
usually 4-6 months following transplantation and its occurrence
within 80 days or after 1 year is uncommon. Accordingly, it can be
seen that a homogeneous reaction is a major prerequisite for
causing cGVHD and the pathogenesis of cGVHD goes through a long
incubation period or the effect on the target organ shows up
slowly. Various problems in the function of the thymus gland are
being discovered in the cGVHD, and it is thought that if normal
thymus glands are not removed through damage caused by treatment
prior to transplantation or isoantigen/autoantigen through
peripheral mechanisms, pathological graft T cells increase as a
reaction, and this type of pathological CD positive T cells, as a
Th2 immunological reaction, cause immune deficiencies similar to
auto immune diseases including cytolytic attack, secretion of
inflamed fibrosis cytokine, B cell activation, and damage to target
organs through formation of auto antibodies.
[0008] Clinical symptoms of cGVHD include changes in the skin such
as erythema, dryness, itchiness, pigmentation change, and
maculopapular rashes; changes in hair such as thinning of hair and
loss of hair, and changes in the mouth such as inflamed gums,
mucositis, and lip atrophy. Aside from the various lesions
appearing on the eyes, reproductive organs, liver, lungs,
gastrointestinal tracts, fascia, skeletal system, serous membranes,
and so forth.
[0009] While cGVHD is generally defined as GVHD that occurs after
100 days following bone marrow transplantation, the manifested
conditions are more important to the diagnosis than the manifested
time period. In accordance with the manifested time period of the
symptoms, classification can be made between a progressive onset in
which aGVHD not having been cured since occurrence shifts to cGVHD,
a quiescent onset in which cGVHD appears after aGVHD has been fully
cured, and de novo in which it occurs without prior appearance of
aGVHD. The morbidity and death rate is highest in the progressive
onset, next is quiescent onset, and it is lowest in the case of de
novo. As for manifested conditions, in many cases lichenous shaped
rashes in the skin and mucous coat of the mouth are the first
symptoms, and while it may appear on the same parts as in aGVHD,
the lesions are papulous, invasive, and covered with white scales.
When compared with aGVHD from a pathological histology perspective,
while legions of satellite cell necrosis can still be found, the
lymphocyte infiltration shows an over consolidated band. Aside from
that, the gall bladder duct is diminished, and while bile
accumulation can be seen, because there may be cases in which it
may be mixed with legions related to the medication or viral
hepatitis, there may be cases in which it is difficult to
differentiate from cGVHD.
[0010] In the case of cGVHD, because immune functions are already
decreased, there is fear of serious infection during treatment, and
a new effective treatment with little side effect from the
treatment is keenly needed. In relation to this, many researches
are being reported that state there is ability for mesenchymal stem
cells to be differentiated into many organ cells and that
graft-versus-hot reactions can be improved by suppressing T
cells.
[0011] While mesenchymal stem cells can be propagated in an
undifferentiated situation as primordial cells of the original
mesoderm and be separated from various organs such as bone marrow,
fat tissue, liver, tendon, synovial membrane, and umbilical cord, a
single marker that can precisely define it as a mesenchymal stem
cell is not in existence. However, CD14, CD34, and CD45 are well
known as markers for bone marrow and SH-2(CD105), SH-3(CD73), SH-4,
and Th7-1 are well known as markers for mesenchyma. Mesenchymal
stem cells or mesenchymal stromal cells (MSC) express major
histocompatibility complex (MHC) class 1, and MHC class can induce
manifestation through interferon gamma (IFN-.gamma.), and because
it does not manifest FAS or FAS L(CD40) type costimulatory
molecules, it does not induce immunological reactions, and is free
from cytolysis due to cytotoxic T lymphocytes (CTL) and natural
killer (NK) cells. In addition, while mesenchymal stem cells
suppress proliferation of T cells through density reliance at time
of mixed lymphocyte reaction (MLR) and suppress proliferation of B
cells as well as formation of immunoglobulin, it is known that MHC
compatibility is not a necessity for MSC immune suppression. In
addition, it is known that there is no change in the activity of
karyotypes or telomerases in the MSC when split 50 times.
[0012] However, because MSC that exists in the body is very rare,
development of technology that isolates it is important. Currently,
density gradient centrifugal separation, method using monoclonal
antibodies specifically for Sca-1 or STRO-1, and separation method
according to cell size are known methods for separating MSC. The
inventors herein have previously developed an effective MSC
separation method (Republic of Korea Public Patent No. 10-0802011)
that does not require a particular mechanical device or reagent,
and the above method is characterized by the fact that marrow taken
from the individual is cultivated, and the cultured upper liquid is
further cultivated by repeatedly removing to a new container.
[0013] In regards to treatment methods for cGVHD, U.S. Pat. No.
6,544,506 presents a GVHD prevention and treatment method that has
as its distinctive feature the removal of cytotoxic T lymphocytes
by injecting non-alloreactive anti-cytotoxic lymphocytes in organ
transplantation patients. U.S. Pat. No. 6,936,281 describes a GVHD
treatment method using mesenchymal stem cells. U.S. Pat. No.
7,173,016 describes a GVHD treatment method that includes the step
of injecting adenosine deaminase inhibitors. U.S. Pat. No.
6,328,960 describes a GVHD treatment method that has as its
distinctive feature the injection of mesenchymal stem cells in an
amount that can lessen the immunological reaction of effector cells
against the antigens in the target organ transplantation patient in
order to lessen the effector cell's immunological reaction against
the alloantigen in the target transplantation patient. U.S. Pat.
No. 6,368,636 describes a method of lessening the immunological
reaction caused by effector cells, which includes the step of
contacting the effector cells with the upper liquid of the
mesenchymal stem cells in an injection amount that can reduce the
immunological reaction against alloantigen.
[0014] Until now, there is no report of a case in which cGVHD has
been treated successfully using mesenchymal or marrow stem cells.
Accordingly, the inventors herein through attempting to treat cGVHD
completed this invention by verifying clinically that mesenchymal
or marrow stem cells that were separated using a subfractionation
culturing method effectively treats cGVHD.
SUMMARY OF THE INVENTION
[0015] A goal of this invention is to provide a therapeutic agent
for acute or chronic graft-versus-host disease.
[0016] Another goal of this invention is to provide a treatment
method for acute or chronic graft-versus-host disease.
[0017] In order to achieve the above mentioned goals, this
invention provides a therapeutic agent that includes mesenchymal or
marrow stem cells for treating acute or chronic graft-versus-host
disease.
[0018] In addition, this invention also includes a method for
treating graft-versus-host disease that includes the step of
injecting an effective dose of mesenchymal or marrow stem cells in
patients with acute or chronic graft-versus-host disease.
[0019] In one aspect, the present invention is directed to a method
of inhibiting activity of T-cell from donor marrow in a subject
identified as suffering from graft-versus-host disease, comprising
administering to the subject in need thereof a therapeutically
effective amount of a population of homogeneous clonal marrow stem
cells. A dosage of the stem cells per each administration may be
between 1.times.10.sup.4 cells/kg body weight to 1.times.10.sup.8
cells/kg weight. The stem cells may be isolated using a
subfractionation culturing method. The stem cells may express CD29,
CD44, and CD105 cell surface antigens, but not HLA-DR cell surface
antigen. The stem cells may express CD29, CD44, CD73 CD90, CD105,
and CD166 cell surface antigens but not CD106, CD119, or HLA-DR
cell surface antigens. The homogeneous clonal marrow stem cells may
secrete interleukin-10 at a concentration of at least about 5
ng/ml.
[0020] In another aspect, the invention is directed to a method of
treating symptoms of graft-versus-host disease in a subject
identified as suffering from graft-versus-host disease, comprising
administering to the subject in need thereof a therapeutically
effective amount of a population of homogeneous clonal marrow stem
cells. The symptom of graft-versus-host disease may be in the
gastrointestinal tract, sclerotic skin, limitation of oral intake,
dryness of eyes, liver symptoms, shortness of breath, or tightness
of arms or legs. The gastrointestinal symptom may be elevated daily
volume of stool or inflamed colon. The liver symptom may be raised
alkaline phosphatase level in blood serum.
[0021] In another aspect, the invention is directed to a method of
inhibiting activity of T-cell from donor marrow in a subject
identified as suffering from graft-versus-host disease,
comprising:
(A) manipulating a biological sample of bone marrow cells,
comprising:
[0022] (i) allowing the sample of cells to settle in a
container;
[0023] (ii) transferring supernatant from the container to another
container; and
[0024] (iii) isolating cells from the supernatant, which has
comparatively lower density in the sample to obtain a population of
homogeneous clonal marrow stem cells; and
(B) administering to the subject in need thereof a therapeutically
effective amount of the population of homogeneous clonal marrow
stem cells obtained in (A). In the above method, the container may
be treated with a coating. The coating may be collagen,
poly-lysine, fibrinogen, or gelatin.
[0025] In still another aspect, the invention is directed to a
method of treating symptoms of graft-versus-host disease in a
subject identified as suffering from graft-versus-host disease,
comprising:
(A) manipulating a biological sample of bone marrow cells,
comprising:
[0026] (i) allowing the sample of cells to settle in a
container;
[0027] (ii) transferring supernatant from the container to another
container; and
[0028] (iii) isolating cells from the supernatant, which has
comparatively lower density in the sample to obtain a population of
homogeneous clonal marrow stem cells; and
(B) administering to the subject in need thereof a therapeutically
effective amount of the population of homogeneous clonal marrow
stem cells obtained in (A).
[0029] A dosage of the cells may be between 1.times.10.sup.4
cell/kg to 1.times.10.sup.8 cell/kg. The cells may express CD29,
CD44, and CD105 cell surface antigens, but not HLA-DR cell surface
antigens. The cells may express CD29, CD44, CD73 CD90, CD105, and
CD166 cell surface antigens but not CD106, CD119, or HLA-DR cell
surface antigens. The population of cells may secrete
interleukin-10 at a concentration of at least about 5 ng/ml.
[0030] These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present invention will become more fully understood from
the detailed description given herein below, and the accompanying
drawings which are given by way of illustration only, and thus are
not limitative of the present invention, and wherein;
[0032] FIG. 1 shows a diagram of the procedure of isolating clonal
marrow stem cells or mesenchymal stromal cells by subfractionation
culturing method used in this invention.
[0033] FIG. 2 shows a FACS phenotyping result of cell surface
epitopes on the clonal marrow stem cells isolated by
subfractionation culturing method and used for the treatment of a
GVHD patient.
[0034] FIGS. 3A-3B show graphs of the amount of (A) TGF-.beta. and
(B) IL-10 secreted from the clonal marrow stem cells (cMSC-15) used
in the treatment of the GVHD patient and control mesenchymal stem
cells isolated by the conventional density gradient centrifugation
method.
[0035] FIG. 4 shows a graph of the changes in the volume of daily
stool amount and activity of alkaline phosphatase after the
administration of clonal marrow stem cells in the GVHD patient.
[0036] FIGS. 5A-5D show colonoscopy pictures of the GVHD patient
who was injected with the clonal marrow stem cells. (A) is a
picture prior to treatment, (B) is a picture taken 10 days after
treatment, (C) is a picture taken 1 month after treatment, and (D)
is a picture taken 3 months after treatment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] In the present application, "a" and "an" are used to refer
to both single and a plurality of objects.
[0038] As used herein, "bodily sample" refers to any sample
obtained from a mammal from which is desired to isolate a single
type of cell. Such bodily sample includes bone marrow sample,
peripheral blood, cord blood, fatty tissue sample, and
cytokine-activated peripheral blood.
[0039] As used herein, "clonal marrow stem cells" refers to cells
that are derived from a single stem cell. This phrase is used
interchangeably with "multi-lineage stem cell", which are obtained
by subfractionation culturing methods.
[0040] As used herein, "homogeneous" population of cells generally
indicates that the same type of cells are present within the
population. Substantially homogeneous may mean about 80%
homogeneity, or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%. 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
or 99.9% homogeneity. In particular, homogeneity of the cells is
attributed to the expansion of the cells from a single cell origin.
No MSC-specific antigen is currently available and therefore no
MSC-specific antibody is available. Theoretically, the only way to
obtain 100% homogeneous population of MSCs is to expand a single
cell which is identified as a MSC later by characterizing their
differentiation and proliferation potentials. The "homogeneous
population of stem cells" refers to stem cells that are derived
from a single cell identified as a stem cell later by such
characterization studies.
[0041] As used herein, "lower density cell" refers to cells that
have lower density than others in the sample, and are the object of
isolation. The lower density cell includes without limitation,
multi-lineage stem cells, progenitor cells, other marrow stromal
cells.
[0042] As used herein, "mammal" for purposes of discussing the
source of the cells and treatment refers to any animal classified
as a mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, cats, cattle, horses, sheep,
pigs, rats, mice, rabbits, and so on. Preferably, the mammal is
human.
[0043] As used herein, "MLSC" refers to multi-lineage stem
cell.
[0044] As used herein, "MLSC/PC" refers to multi-lineage stem cell
or progenitor cell.
[0045] As used herein, "MSC" refers to marrow stromal cells or
mesenchymal stem cells, or marrow stem cells which terms are used
interchangeably.
[0046] As used herein, "sample of cells" refers to any sample in
which is contained a mixture of different types of cells, including
bone marrow sample, peripheral blood, cord blood, fatty tissue
sample, and cytokine-activated peripheral blood.
[0047] Subfractionation Culturing Technique
[0048] While not being bound by any particular method of isolating
MSC, MLSC or MLSC/PC, a preferred method of obtaining the cells for
use in treating Graft Versus Host Disease is by a "subfractionation
culturing method", which method is used to isolate a highly
homogeneous population of clonal marrow stem cells or multi-lineage
stem cells (MLSCs) from a bodily sample or source such as human
bone marrow. The procedure is described in U.S. Patent Application
Publication US2006/0286669 (Ser. No. 11/471,684, filed Jun. 19,
2006), "Isolation of Multi-Lineage Stem Cells", the contents of
which are incorporated herein by reference in their entirety.
[0049] Bone marrow MSCs have been known to be difficult to isolate
without contamination by hematopoietic cells. For application in
clinical settings, it is important to have a homogeneous population
of MSCs in order to prevent immunogenic problems and to evaluate
the clinical effects correctly. Conventionally, isolation of
homogeneous populations of MSCs was carried out by MSC-specific
antibody column purification. However, even this method is not
adequate as no such perfect MSC-specific antibody is yet
available.
[0050] In the practice of the subfractionation culturing method, it
is not necessary to employ centrifugation of any type to pre-remove
any type of cells such as red or white blood cells from the sample
because most of the heavier or more dense cells can be removed
within the first two, 2-hour incubation steps. Thus, one advantage
of the inventive system is that conventionally used density
gradient centrifugation and mononuclear cell fractionation steps,
which may introduce contamination such as Picoll, Ficoll or
Ficoll-hypaque into the cell culture may be avoided. Accordingly,
the inventive subfractionation culturing method is a simple,
effective, and economic protocol to isolate highly homogeneous
MLSCs from a bodily sample, preferably a bone marrow sample.
[0051] Alternatively, mononuclear cells isolated/fractionated by
conventional density gradient centrifugation method of MSC
isolation can also be subjected to the D1 dish to obtain single
cell-derived colonies and then to isolate homogeneous populations
of stem or progenitor cells (FIG. 1). Therefore, the
subfractionation culturing method can be used with the mononuclear
cells fractionated by the conventional density gradient
centrifugation method.
[0052] The present application describes diversity of
characteristics in cell surface protein expression of the isolated
single-cell derived stem cell lines, which indicates that there are
several different types of multi-lineage stem or progenitor cells
that exist in biological samples, and in particular bone marrow
samples, which are exemplified. The isolated MLSCs were generally
negative or dimly positive for CD34, HLA-DR, CD31, CD166, HLA Class
I and highly positive for CD44, CD29, CD105. However, some cell
lines from D4 and D5 dishes exhibited distinctive levels of surface
proteins, which indicates that there could be several different
types of multi-lineage stem or progenitor cells in bone marrow
(FIG. 1). These MSCs having different surface markers may represent
different differentiation potential of the cells. Therefore,
isolation of single-cell derived homogeneous stem cells by the
subfractionation culturing method makes it possible to isolate
tissue-specific stem or committed progenitor cells, as long as
these groups of cells exist in the bone marrow or other
specifically isolated bodily sample, and culture conditions do not
change their potential during cell expansion. The safety and
efficacy of MSC treatment and cell engraftment process is improved
by being able to characterize subpopulations of cells with specific
properties, as shown in the present application.
[0053] By eliminating density gradient centrifugation and
mononuclear cell fractionation steps and without requiring the use
of antibodies to separate stem cells, or particular enzymes, the
subfractionation culturing method generates more homogeneous
populations of MSCs or MLSCs in a simple, effective, and economic
procedure and safer applications for therapeutic settings.
[0054] In carrying out the present invention, preferably and
without limitation, marrow stem cells may be obtained using the
subfractionation culturing method as described above. Further, it
is preferable that the obtained MSC's express any or all of CD29,
CD44, CD105 cell surface antigens. It is also preferable that any
or all of HLA-DR cell surface antigens be not expressed in the
MSC's. More preferably, any and all of CD29, CD44, CD90, CD105, and
CD166 cell surface antigens are expressed on the MSC's, any or all
of the CD106, CD119, and HLA-DR cell surface antigens are not
expressed on the MSC's.
[0055] Preferably, the inventively used MSC's express
Interleukin-10 (IL-10). Preferably, IL-10 is expressed at over 5
ng/ml or over 10 ng/ml after culturing at the time of
treatment.
[0056] In one aspect, the invention is directed to using cell
obtained by the subfractionation culturing method, which includes:
1) the step of obtaining bone marrow from an individual; 2) the
step of cultivating the bone marrow; 3) the step of moving only the
upper liquid in 2) to a new container and culturing; and 4) the
step of separating only the upper liquid of 3) and repeatedly
culturing in a culture container that has been optionally treated
with coating.
[0057] In regards to the above mentioned subfractionation culturing
method, while not limited to any particular amount of time, the
repeated culturing in the above step 4) may be carried out for
about 1 to 4 hours at 37.degree. C. and then repeatedly cultured
for about 2 to 3 times for about 12 to 36 hours at 37.degree. C.
and then cultured for about 24 to 72 hours at 37.degree. C., and
for the upper liquid to be moved to a new culture container each
time.
[0058] In another aspect, collagen, gelatin, fibrinogen or
polylysine-coated culture dishes were used in order to obtain more
adherent stem cells. Applicant has discovered that any charged
culture surface, either positive or negative, helps the attachment
of stem cells to it, compared to the surface of an uncoated dish.
More cells were attached to a collagen or polylysine-coated culture
dish than uncoated dish, approximately by about two to three fold
respectively (data not shown).
[0059] Thus, in one embodiment, the bottom of a culture dish can be
coated by either positively charged amino acids, such as
polylysine, polyarginine, or negatively charged amino acids, such
as polyaspartate, polyglutamate, or a combination thereof to help
stem or progenitor cells adhere better to the bottom of the
dish.
[0060] It is preferable that the culture container is treated with
a coating, and while any material than can improve the attachment
of cells to the container may be used, it is particularly preferred
that collagen, poly-lysine, fibrinogen, or gelatin be used. More
preferably, collagen or poly-lysine may be used. Even more
preferably, collagen may be used. In addition, it is desirable for
the cells to be repeatedly cultured for about 3 to 6 times in a
culture container that has been treated with collagen and even more
desirable for the cells to be repeatedly cultivated about 4 to 5
times.
[0061] Pharmaceutical preparation of the therapeutic agent may be
made using conventional knowledge in the industry. For example, it
can be used in a non oral form of water or a sterilized liquid
solution pharmaceutically permissible as well as a suspended
injection. For example, pharmaceutical preparation by combining it
with a carrier or media that is pharmaceutically permissible,
specifically sterile water or a physiological saline solution,
vegetable oil, emulsifier, suspensions, surfactant, stabilizer,
excipient, vehicle, preservative, binder, and so on, and blending
it in a unit capacity format that is generally accepted as being
required in a pharmaceutical application can be considered. In
addition, sterilized composites for injection can be prescribed
based on known pharmaceutical applications using supporting liquids
such as injectible distilled water.
[0062] For aqueous solutions for injection that can be used
jointly, an example can be physiological saline solution, glucose,
or isotonic solutions including supportive medications such as
D-sorbitol, D-mannose, D-manitol, chloride, or natrium. As for
adequate liquefaction supportive agents, an example can be alcohol,
specifically ethanol or poly alcohol such as propylene glycol, or a
non-ionized surfactant such as polysorbate 80.TM. or HCO-50.
[0063] As an oil agent, sesame oil or soybean oil can be considered
and can be used jointly with a benzyl benzoate or benzyl alcohol.
In addition, it can be combined with a buffering agent such as
phosphate buffering solution, sodium acetate buffering solution, or
analgesic solution such as Novocain or stabilizer such as benzyl
alcohol, phenol, or antioxidant. The prepared injection liquid is
to be charged in a commonly accepted adequate ampoule.
[0064] It is desirable for the administration into the body of the
patient to be non-oral, and more specifically while it is basic to
administer into the vein 1 or 3 times, greater injection is also
allowable. Additionally, the administration length can be short or
long. More specifically, injection type or transdermal type can be
considered. As an example of injection type, while it may be
administered through intravenous injection, arterial injection,
selectable arterial injection, intramuscular injection,
intraperitoneal injection, hypodermic injection, intracerebral
injection, cerebral injection, or bone marrow injection, and
intravenous injection is desirable. In the case of intravenous
injection, because transplantation methods using common blood
transfusion have become possible, the patient does not require
surgery and furthermore because topical anesthesia is not required,
the burden is light on both patient and doctor. When future
development of emergency medicine is considered, administration
during emergency transportation or at the critical site can be
considered.
[0065] In addition, this invention provides a method for treating
patients with graft-versus-host disease that includes the step of
administering mesenchymal or marrow stem cells, in an effective
dose for treatment, to the above mentioned patient suffering from
graft-versus-host disease.
[0066] The effective dose per injection of the clonal marrow cells
for treatment of GVHD or the symptoms of GVHD in a mammal and in
particular human being may be between 1.times.10.sup.4 cells/kg
body weight and 1.times.10.sup.8 cells/kg body weight; between
1.times.10.sup.4 cells/kg body weight and 1.times.10.sup.8 cells/kg
body weight; between 2.times.10.sup.4 cells/kg body weight and
1.times.10.sup.8 cells/kg body weight; between 2.5.times.10.sup.4
cells/kg body weight and 1.times.10.sup.8 cells/kg body weight;
between 2.times.10.sup.4 cells/kg body weight and 1.times.10.sup.7
cells/kg body weight; between 2.5.times.10.sup.4 cells/kg body
weight and 1.times.10.sup.7 cells/kg body weight; between
2.times.10.sup.4 cells/kg body weight and 3.times.10.sup.6 cells/kg
body weight; between 2.5.times.10.sup.4 cells/kg body weight and
3.times.10.sup.6 cells/kg body weight; between 2.times.10.sup.4
cells/kg body weight and 2.times.10.sup.6 cells/kg body weight;
between 2.5.times.10.sup.4 cells/kg body weight and
2.times.10.sup.6 cells/kg body weight; between 2.times.10.sup.4
cells/kg body weight and 1.times.10.sup.6 cells/kg body weight;
between 2.5.times.10.sup.4 cells/kg body weight and
1.times.10.sup.6 cells/kg body weight; between 2.times.10.sup.4
cells/kg body weight and 1.times.10.sup.5 cells/kg body weight; or
between 2.5.times.10.sup.4 cells/kg body weight and
1.times.10.sup.5 cells/kg body weight;
[0067] Without being limited to any particular administration
method, it non oral administration method is preferred. While whole
body or partial body administration is possible, whole body
administration is preferred and intravenous injection is most
preferred.
[0068] Treatment of Graft Versus Host Disease
[0069] The inventors herein were able to improve the condition of
acute or chronic graft-versus-host disease patients who were
unresponsive to treatment by administering mesenchymal or marrow
stem cells separated using the methods (FIG. 1) described in U.S.
Patent Application Publication US2006/0286669, filed Jun. 19, 2006,
"Isolation of Multi-Lineage Stem Cells", the contents of which are
incorporated herein by reference in their entirety.
[0070] As regards the manifestations or symptoms of GVHD, they
include sclerotic skin, limitation of oral intake, dryness of eyes,
gastrointestinal (GI) tract symptoms such as dysphagia, anorexia,
nausea, vomiting, abdominal pain, or diarrhea, liver symptoms as
manifested by elevated bilirubin, elevated alkaline phosphatase,
and elevated alanine aminotranferease (ALT)/aspartate
aminotransferase (AST) (AST/ALT) ratio, shortness of breath, and/or
tightness of arms or legs.
[0071] A subject may exhibit multiple symptoms depending on the
tissue that is affected by the graft-versus-host disease. Some
patients have 4-5 symptoms others may have 1-2 symptoms. Therefore,
the present invention is directed to treating any and all of the
symptoms associated with GVHD as manifested any tissue in the
subject. As the manifestations or symptoms are treated, it is
believed that GVHD disease itself is also treated thereby.
[0072] The following description provides details of the
application of the subfractionation culturing technology and using
the homogeneous clonal marrow stem cells obtained thereby to
administer to an individual identified as suffering from the
symptoms of GVHD. Without being bound by theory, it is believed
that the administered stem cells secrete IL-10 in the subject,
which counteracts or inactivates the ill effects of the donor's
T-cells, thereby treating GVHD and the symptoms of GVHD.
[0073] After separating the mesenchymal or marrow stem cell from
the mother of the above patient suffering from chronic
graft-versus-host disease using the subfractionation culturing
method and establishing it as the cell line of monoclonal origin,
it was named cMSC-15. A cell surface antigen analysis was carried
out through parenchyma cell analysis to determine whether the above
cell line was an actual mesenchymal or marrow stem cell (FIG. 2).
As a result, because it was shown that CD29, CD44, CD90, CD105, and
CD166 cell surface antigens were expressed and CD106, CD119, and
HLA-DR cell surface antigens were not expressed, it was confirmed
that it was a mesenchymal or marrow stem cell and not a
hematopoietic stem cell. CD133 and STRO-1 expression was weakly
positive. Accordingly, in order to more specifically verify the
characteristics of the above cell line, the degree of expression of
TGF-.beta. and IL-10 were verified using the ELISA method (FIG. 3).
In the case of TGF-.beta. expression, there was no big difference
with the mesenchymal stem cell obtained through conventional
density gradient centrifugation method, which is the control.
However, IL-10 showed an increase in expression at least 5 times
compared with the control.
[0074] Accordingly, the inventors herein, after cultivating the
above established mesenchymal or marrow stem cell to an amount
sufficient for treatment, applied it in the treatment of a patient
whose life was in a critical situation due to the onset of chronic
graft-versus-host disease. Specifically, the above patient was an
18 year old woman who was diagnosed with acute myelogenous leukemia
and after reaching remission through induction therapy, received
allogeneic bone marrow transplantation. One month following the
stoppage of a six-month administration of immunological
suppressants for 6 months, chronic graft-versus-host disease
started and while it was treated using Cyclosporine A (CsA),
Mycophenolate mofetil (MMF), and steroids, the condition of the
patient deteriorated due to continuing hematochezia, increased
bilirubin, and dryness of skin, mouth, and eyes, and because of the
activation of cytomegalovirus (CMV) due to treatment side effects
and BK virus being found even in the urine and blood due to
infection of the BK virus, a treatment using Cideforvir was
started.
[0075] The inventors herein administered the above mentioned
cultivated mesenchymal or marrow stem cells through intravenous
injection one time after receiving an emergency clinical permit
from the Korean Food and Drug Administration. No adverse/negative
reactions were observed during or after the administration, and the
symptoms of the patient slowly improved after the 1.sup.st
administration, and a 2.sup.nd administration was given after 3
weeks. Afterwards, the patient's symptoms improved and the patient
was discharged 34 days after the 2.sup.nd administration in a state
of having stopped taking steroids and only taking immunological
suppressants.
[0076] In order to verify the pathological effect of the treatment,
the inventors herein measured the amount of stool and activity of
alkaline phosphatase in the blood, which are major indicators of
Graft-Versus-Host-Disease (FIG. 4). The amount of stool had
considerably reduced, and the activity of alkaline phosphatase in
the blood, which is a serological index had also fallen to normal
levels (60-220 ng/ml). In addition, a colonoscopy analysis was
carried out to verify whether symptoms had improved (FIG. 5). As
can be seen in FIG. 5, with the passage of time following
administration of mesenchymal or marrow stem cells in this
invention, ulcers had much improved from a colonoscopy data.
[0077] As was seen above, the inventors herein verified through
clinical trial that mesenchymal or marrow stem cells separated
using the subfractionation culturing method of this invention is
effective in treating chronic graft-versus-host disease.
[0078] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims. The
following examples are offered by way of illustration of the
present invention, and not by way of limitation.
EXAMPLES
Example 1
Separation of Mesenchymal or Marrow Stem Cells Using
Subfractionation Culturing Method
[0079] After applying local anesthesia to a section of the marrow
provider's (mother of treatment target chronic graft-versus-host
disease patient) hip, bone marrow was extracted by inserting an
injection needle into the hip bone. 15 ml of DMEM (Dulbecco's
modified Eagle's Medium, GIBCO-BRL, Life-technologies, MD, USA)
which included 20% FBS and 1% penicillin/streptomycin and 2 ml of
the marrow extracted from the above mentioned marrow provider was
put into a 100 mm culture container and cultivated for 2 hours in a
37.degree. C., 5% CO.sub.2 cell cultivator. After cultivation, the
culture container was slightly leaned so that the cells attached to
the bottom would not fall out and the maximum amount of the upper
layer culture liquid in the culture container was moved to a new
container.
[0080] After repeating the same procedure once more the culture
liquid that was taken was moved to a culture container (Becton
Dickinson) and cultivated for 2 hours at 37.degree. C. The culture
liquid was again moved to a new container and after 24 hours was
moved to another new container and after 24 hours was moved to a
new container again. Lastly, after 48 hours, it was verified by
sight that the cells that were left after moving to a new container
were attached and growing on the bottom of the culture container.
It can be inferred that the cells that can come to this step having
gone through several layered separations are cells that are smaller
than the other cells. Once about 3 to 5 hours have passed, the
cells form a single clone. This single clone was treated with
trypsin, separated, and moved to a 6-well culture container with a
cell number of 10.sup.2 to 6.times.10.sup.2 per well. After
cultivating for 4 to 5 days in a 37.degree. C., 5% CO.sub.2 cell
cultivator when it had grown 80%, it was treated with 0.25%
trypsin/1 mM EDTA (GIBCO-BRL) and after gathering was moved and
successively cultivated in a 75 cm.sup.2 culture container. Cell
lines with monoclonal origins were acquired as above and named
cMSC-15.
[0081] As a result of observing the shape of the above cells
through a microscope, it was seen that the cells in the initial
stage had a shape similar to fibroblast cells and no big changes in
the shape were found up to the successive cultivation in stage 5.
The time it took for the cells to double was observed to be 24-36
hours, not much different from fibroblast cells.
Example 2
Verification of Separated Mesenchymal or Marrow Stem Cells
Example 2.1
Analysis of Mesenchymal or Marrow Stem Cell Characteristics Using
Flow Cytometry
[0082] In order to verify whether the cMSC-15 cells that were
separated from the marrow using the method in the above Example 1
were mesenchymal or marrow stem cells, a flow cytometry (BD
Biosciences) was used to find out if cell surface antigens with
stem cell characteristics existed.
[0083] Stem cells that were successively cultivated for 6 to 7 days
in a 75 cm.sup.2 culture container was treated with 0.25% trypsin
and the cells were gathered. The cells were washed 2 times with a
1.times.PBS/0.4% BSA to remove trypsin as well as culture liquids.
The cells were collected using centrifugal separation and after
measuring the number of cells, 1.times.10.sup.6 cells were gathered
in a 1.5 ml tube and blocked for 1 hour in room temperature using
goat serum (Vector). After the completion of blocking, the cells
were washed 2 times with a 1.times.PBS/0.4% BSA and treated with a
phycoerythrin (PE) attached anti-CD14, CD29, CD31, CD34, CD44,
CD73, CD90, CD105, CD106, CD119, CD133, CD166, HLA-DR, HLA Class 1
and STRO-1 antibody (Serotec Ltd, Kidington, OX, UK) each and
reacted for 40 minutes at 4.degree. C. After the cells were washed
2 times with a 1.times.PBS/0.4% BSA, they were suspended in 0.5 ml
1.times.PBS/0.4% BSA, loaded in the flow cytometry and
analyzed.
[0084] CD29, which is an integrin antigen specific for mesenchymal
stem cells, as well as CD44 and CD105, which are matrix receptor
antigens also specific for mesenchymal stem cells showed a positive
reaction. CD90, CD166, and HLA-Class 1 cell surface antigens were
expressed, and CD106, CD119, as well as HLA-DR cell surface
antigens were not expressed. Aside from this, in the case of CD31,
CD133, and STRO-1, an expression level was weakly positive (FIG.
2). Expression of such cell surface antigens was maintained in the
cells that has undergone 6 successive cultivations. This indicates
that the separated cells, even if they are successively cultivated,
the antigens specific for mesenchymal stem cells would be
continually expressed.
Example 2.2
Analysis of Cytokine Secretion Condition Related to Mesenchymal
Stem Cell's Immunological Suppression
[0085] The inventors herein, in order to find out more about the
characteristics of the isolated mesenchymal stem cells using the
subfractionation culturing method in this invention, analyzed the
expression levels of immunological suppression related
cytokines.
[0086] Specifically, after cultivating the mesenchymal or marrow
stem cells taken from the same marrow provider and separated using
the existing density gradient centrifugation method (control group)
and the cMSC-15 in this invention separated using the above
mentioned method described in Example 1, the expressed amount of
the TGF-.beta. and IL-10 secreted in the culture liquid was
analyzed using an enzyme-linked immunosorbent assay (ELISA). For
precise analysis, the above two cell lines were cultivated using a
serum free batch, and both of the above ELISA used a kit from
R&D systems (USA), and was carried out in accordance with the
manufacturer's instructions.
[0087] As a result of the analysis, while the TGF-.beta. showed
little difference from the control group, in the case of the IL-10,
the cMSC-15 cells obtained using the subfractionation culturing
method showed increased expression over 5 times compared with the
control group (FIG. 3). This indicates that there is a correlation
between the treatment effectiveness of graft-versus-host disease
and the IL-10 expression level of mesenchymal or marrow stem
cells.
Example 3
Clinical Application of Isolated Mesenchymal or Marrow Stem
Cells
Example 3.1
Administration of Mesenchymal or Marrow Stem Cells
[0088] The patient was an 18 year old woman who was diagnosed with
acute myelogenous leukemia and after reaching remission through
induction therapy, received an allogeneic bone marrow
transplantation. One month following the stoppage of a 6-month
administration of immunological suppressants, chronic
graft-versus-host disease started and while it was treated using
Cyclosporine A (CsA), Mycophenolate mofetil (MMF), and steroids,
the condition of the patient deteriorated due to continuing
hematochezia, increased bilirubin, and dryness of skin, mouth, and
eyes, and because of the activation of CMV due to treatment side
effects and BK virus being found even in the urine and blood due to
infection of the BK virus, a treatment using Cideforvir was
started. After receiving an emergency clinical permit from the
Korean Food and Drug Administration, the mesenchymal or marrow stem
cells of the patient's mother which were isolated as described in
Example 1 was cultivated in the GMP facilities and administered to
the patient using intravenous injection. No adverse/negative
reactions were observed during or after the administration, and the
symptoms of the patient slowly improved after the 1.sup.st
administration, and a 2.sup.nd administration in the same amount as
the 1.sup.st was given after 3 weeks. Afterwards, the patient's
symptoms improved considerably and the patient was discharged 34
days after the 2.sup.nd administration in a state of having stopped
taking steroids, left with only taking MMF 1.5 g/day as an
immunological suppressant.
Example 3.2
Change in Disease Indicators after Treatment
[0089] In order to verify whether the mesenchymal or marrow stem
cells isolated using the subfractionation culturing method in this
invention is effective for treatment, the inventors herein measured
the amount of stool and activity of alkaline phosphatase in the
blood which is a major indicator (FIG. 4). The activation of
alkaline phosphatase in the blood was accomplished using a
commercial kit (Sigma Chemical Company, USA).
[0090] As a result, the amount of stool which is a major indicator
in chronic graft-versus-host disease was considerably reduced, and
the activity of alkaline phosphatase in the blood which is a
serological index had also fallen to normal levels (60-220 ng/ml).
In addition, a colonoscopy analysis was carried out to verify
whether symptoms had improved (FIG. 5). As a result, the
colonoscopical opinion at three months following administration of
mesenchymal stem cells is that ulcers have been much improved. As
has been described, the inventors herein, through clinical trial,
have verified that mesenchymal or marrow stem cells separated using
the subfractionation culturing method in this invention are
effective for treating chronic graft-versus-host disease.
[0091] This invention, as it is in regards to a therapeutic agent
for treating acute or chronic graft-versus-host disease that
includes mesenchymal or marrow stem cells as the active ingredient,
the therapeutic agent in this invention can very effectively treat
host-versus-host disease which has been very difficult to treat,
especially the deadly acute or chronic graft-versus-host disease
which occurs frequently as a side effect after bone marrow
transplantation surgery.
[0092] All of the references cited herein are incorporated by
reference in their entirety.
[0093] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
specifically described herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *