U.S. patent application number 12/556162 was filed with the patent office on 2010-01-21 for method for producing a composition for promoting survival of transplanted hematopoietic stem cell.
This patent application is currently assigned to LYMPHOTEC INC.. Invention is credited to Kenzo Bamba, Yasuyuki Kuroiwa, Tomohiro Morio, Norio Shimizu.
Application Number | 20100015593 12/556162 |
Document ID | / |
Family ID | 33513370 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015593 |
Kind Code |
A1 |
Bamba; Kenzo ; et
al. |
January 21, 2010 |
METHOD FOR PRODUCING A COMPOSITION FOR PROMOTING SURVIVAL OF
TRANSPLANTED HEMATOPOIETIC STEM CELL
Abstract
HLA matched activated lymphocytes in mononuclear cells separated
from peripheral blood or umbilical cord blood are proliferated and
activated. After separating and collecting, the HLA matched
activated lymphocytes are employed as the main component of a
composition for promoting survival of transplanted hematopoietic
stem cells. The obtained composition is widely usable in, for
instance, prevention of survival failure of transplanted
hematopoietic stem cells and therapy for promoting the survival
thereof. Although the dose of the composition varies depending on
the age, conditions, etc. of a patient, a humanized antibody is
administered in a dose of from 0.2 to 20 ml/kg/day to mammals
including humans. The composition is administered by intravenous
injection either once a day (single administration or continuous
administration) or intermittently once to 3 times in a week or once
in 2 or 3 weeks.
Inventors: |
Bamba; Kenzo; (Ibaraki,
JP) ; Kuroiwa; Yasuyuki; (Ibaraki, JP) ;
Morio; Tomohiro; (Tokyo, JP) ; Shimizu; Norio;
(Yamanashi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
LYMPHOTEC INC.
Tokyo
JP
|
Family ID: |
33513370 |
Appl. No.: |
12/556162 |
Filed: |
September 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10559448 |
Jul 5, 2006 |
|
|
|
PCT/JP04/07164 |
May 6, 2004 |
|
|
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12556162 |
|
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Current U.S.
Class: |
435/2 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 31/00 20180101; C12N 5/0636 20130101; C07K 16/28 20130101;
C07K 2317/24 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/2 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2003 |
JP |
2003-157996 |
May 7, 2004 |
JP |
2004-138468 |
Claims
1. A method for producing a composition for promoting survival
after hematopoietic stem cell transplantation, comprising a process
for separating mononuclear cells from peripheral blood or umbilical
cord blood containing HLA matched lymphocytes; a process for
proliferating and activating HLA matched lymphocytes in said
separated mononuclear cells; and a process for separating said
proliferating and activating HLA matched lymphocytes and preparing
them to a formulation in which they constitute a main
component.
2. A method for producing a composition for promoting survival
after hematopoietic stem cell transplantation according to claim 1,
wherein proliferation and activation of said HLA matched
lymphocytes are carried out in any one of interleukin 2 or anti-CD3
antibody or in combination of them.
3. A method for producing a composition for promoting survival
after hematopoietic stem-cell transplantation according to claim 1
or 2, wherein various kinds of cytokines or mitogens are added to
medium for proliferation and activation of said HLA matched
lymphocytes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of application Ser.
No. 10/559,448, filed Jul. 5, 2006, which is a 371 national stage
application of PCT/JP04/07164, filed May 26, 2004, and which is
incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a composition for promoting
survival of transplanted hematopoietic stem cells that is employed
for improving survival failure, which is especially a problem in
transplantation of hematopoietic stem cells, disadvantage of
delaying production of antibodies or immunocompromised condition,
or for decreasing a risk for various types of infectious disease or
relapse of cancers after survival, a kit for obtaining the
composition, a method for promoting survival of the transplanted
hematopoietic stem cells, and a human monoclonal antibody which is
employed in therapy for various human diseases and a method for
producing the human monoclonal antibodies or human polyclonal
antibodies.
TECHNICAL BACKGROUND
[0003] Because cell transplanted patients especially hematopoietic
stem cell transplanted patients are in danger of a crisis of
various types of infection or relapse of cancers, a lot of studies
for corresponding to them have been carried out. Sekine who is one
of the inventors of the present invention previously reported that
lymphocytes could be propagated by using immobilized anti-CD3
antibodies and interleukin 2, and the propagated autologous
lymphocytes thus had an antineoplastic effect (JP 03-80076 A1).
[0004] Besides, it has already been reported that the autologous
lymphocytes propagated with the anti-CD3 antibodies and interleukin
2 are effective to virus infections in congenital immunodeficiency
patients [Kimiya ITO, Teruaki SEKINE; IGAKU NO AYUMI, Volume 181,
NO. 6, Page 426 to 427 (1997)]. Furthermore, in a field for
hematopoietic stem cell transplantation, when some of major HLA
which consist of four loci, A, B, C and DR and further loci DQ and
DP are matched between a patient and donor, a bone marrow
transplantation or a blood transfusion can be carried out.
[0005] Moreover, since Kohler and Milstein developed the cell
fusion technique in 1975, various monoclonal antibodies are
produced and applied to an analysis, measurement, a diagnosis and
therapy. Almost of the monoclonal antibodies reported by now are
derived from animals, and especially mouse-derived, and obtained by
immortalizing antibody producing cells from immunized animals by
cell fusion. The monoclonal antibody is considered as an attractive
drug carrier because it is uniform in an antibody subclass, is
higher antigen specificity and has very small clearance, so that
new Drug Delivery System (DDS) with a monoclonal antibody has been
investigated.
DISCLOSURE OF THE INVENTION
Problems to be Resolved by the Invention
[0006] However, even if autologous lymphocytes propagated with
anti-CD3 antibodies and interleukin 2 are effective to an antitumor
effect or a viral infection in a congenital immunodeficiency
patient, it does not mean a stage in which satisfactory effect can
be necessarily expected with it. Although a bone marrow
transplantation or a blood transfusion is carried out when some of
major HLA loci are matched, a risk to develop a fatal side effect
in a bone marrow transplantation or DLT treated patient according
to mismatch in rest of HLA, namely Post Transfusion-Graft versus
Host Disease (hereinafter, abbreviated as "PT-GVHD") is still
existed.
[0007] Furthermore, also in the monoclonal antibodies, when
animal-derived antibodies which are obtained by sensitizing
antigens to the animals as described above are administrated to
humans, it is difficult to use them regularly as a remedy because
the antibodies themselves indicate immunogenicity. In order to
avoid the immunogenicity, a method that the antigenicity can be
decreased by means of a method such as to fragmentize the
monoclonal antibodies originated from animals to Fab2, or F(ab)'2,
to chimerarize with human antibodies, or to humanize, or a method
that humanized antibody cell lines are established with a antibody
producing cell obtained from the real patient or human lymphocytes
sensitized in vitro and so on are examined.
[0008] However, because the respective methods have problems in the
points of a residual of antigenicity, difficulty of establishing
human antibody producing lines, decrease of capacity of antibody
production during cell culture and so on, it is strongly desired to
develop a method for establishing human antibodies easily and
efficiently by the other procedure. Besides, the polyclonal
antibody also has problems similar to in the monoclonal antibody,
so that various methods are examined in order to medicate them to
human safely and efficiently.
A Means for Resolving the Problem
[0009] The inventors in the present invention succeeded in
preparing HLA matched activated lymphocytes possible to improve
survival of hepatocytes to a transplanted patient with that cells
whose HLA is identical to transplanted hematopoietic stem cells are
stimulated and propagated by interleukin 2 and anti-CD3 antibodies
and then they are given to the patient, resulting from diligent
studies for the purpose of developing a medicament and a method for
improving the survival of transplanted cells, especially
hematopoietic stem cells.
[0010] Furthermore, the inventors succeeded in producing human
monoclonal antibodies efficiently by sensitizing antigens as giving
human activated lymphocytes to immunocompromised mouse in which
human hematopoietic stem cells are transplanted. Namely, if an
embodiment of a primary invention is mentioned, an invention
according to claim 1 relates to a composition for stabilizing
survival of transplanted hematopoietic stem cell whose main
component is HLA matched lymphocytes produced by separating and
collecting them after propagating and activating HLA matched
lymphocytes in mononuclear cells separated from peripheral blood or
umbilical cord blood.
[0011] Besides, an invention according to claim 4 relates to a
method for producing a composition for stabilizing survival of
transplanted hematopoietic stem cells, comprising a process for
separating mononuclear cells from peripheral blood or umbilical
cord blood including HLA matched lymphocytes, a process for
propagating and activating the HLA matched lymphocytes in the
separated mononuclear cells, and a process for separating the
propagated and activated HLA matched lymphocytes and preparing
remedy having them as a main component.
[0012] Furthermore, an invention according to claim 7 relates to a
kit for obtaining the composition for stabilizing survival of
transplanted hematopoietic stem cells comprising an implement for
separating HLA matched lymphocytes from peripheral blood or
umbilical blood, an implement for propagation and activation
comprising either/both culture medium including interleukin 2
or/and an anti-CD3 antibody solidified flask, and an implement for
taking out the HLA matched lymphocytes cultured by the implement
for propagation and activation. Moreover, an invention according to
claim 8 relates to human monoclonal antibody producing cell lines
collected by a method such that HLA matched activated lymphocytes
are given before or after transplanting to mammals, antigens are
sensitized after stabilization of survival, and the antigen
sensitized lymphocytes are established or separated from the
mammals, or a method for gene separation or expression, etc.
[0013] Furthermore, an invention according to claim 9 relates to a
method for producing human monoclonal antibody producing cell lines
collected by a method that human hematopoietic stem cells are
transplanted to mammals which are severe combined immunodeficiency
mice such as SCID mice, and further antigens are sensitized after
the survival by giving HLA matched activated lymphocytes before or
after the transplantation, the antigen sensitized lymphocytes are
established or separated from the mammals, or a method for gene
separation or expression, etc.
EFFECT OF THE INVENTION
[0014] As mentioned above, the present invention relates to remedy
for preventing survival failure and promoting survival after
transplantation of hematopoietic stem cells characterized that it
includes HLA matched activated lymphocytes whose main component is
HLA matched propagated/activated lymphocytes or propagated donor
lymphocytes, so that it can be used widely for promoting the
survival of the stem cells at transplantation. As well as it can be
used for promoting the survival of the stem cells, it can also be
used for promoting survival of various internal organs, and
antibodies which have specificity and high binding affinity with
the antigen can be obtained by transplanting human hematopoietic
stem cells to mammals and giving HLA matched propagated/activated
lymphocytes and immunogens. Besides, the human monoclonal
antibodies can be produced efficiently by using the cells producing
the antibodies. Moreover, it can be used widely as screening of the
remedy such as anti-virus medicament, anti-cancer medicament and
anti-hypertension medicament by transplanting the human
hematopoietic stem cells and using the mammals which are
transplanted with HLA-identical propagated/activated
lymphocytes.
BEST MODE FOR CARRYING OUT THE INVENTION
[0015] Hereinafter, concrete contents of the invention are
explained.
[HLA Matched]
[0016] HLA matched in the present invention is matching by not less
than three loci among several primary HLA components such as 4 loci
A, B, C, DR, and further DQ and DP.
[Collecting Lymphocytes]
[0017] HLA matched lymphocytes for propagating and activating can
be collected by a following method. Namely, HLA matched activated
lymphocytes can be prepared from lymphocytes which are collected
from peripheral blood, lymph node, bone marrow, or various body
fluid of a donor of hematopoietic stem cells. Though HLA matched
activated lymphocytes can be prepared by cultivating the
hematopoietic stem cells, it is extremely desired in respect of
efficiency that lymphocytes prepared from the peripheral blood of
the donor is used as a material. Besides, activated lymphocytes
whose HLA is matched to the donor of hematopoietic stem cells in at
least three loci are prepared, and it is possible to use them.
[0018] It is simple and therefore preferred to collect blood from
vein in upper limb of the donor as a method for collecting blood,
but anything containing lymphocytes can be used as a material.
Besides, umbilical cord blood can be used as a material.
[0019] Only a small quantity of blood in a range between 0.001 ml
and 500 ml is preferred as a quantity of collecting blood, and
especially collecting blood in a range approximately between 10 ml
and 100 ml is preferred. Furthermore, heparin or citric acid can be
added into the collected blood so as to prevent coagulation.
[Incubating, Propagating and Activating of HLA Matched
Lymphocytes]
[0020] The collected HLA matched lymphocytes are incubated,
propagated and activated by following processes.
[0021] Note that the term "incubation" of the collected lymphocytes
means that tissue cells are propagated (incubated) artificially by
placing a small piece of cell tissue in a culture medium containing
necessary nutrients for the cells to survive, and the term
"propagation" of them means that the tissue cells are increased
quantitatively in vitro by the above-mentioned incubation
artificially. Furthermore, the term "activation" of them means that
resting functions are regulated so as to work actively by
stimulating the cells with addition of a propagating agent or an
activating agent into the culture medium, and further concretely,
means that a property of prevention of the failure of the survival
and promoting the survival are given.
[0022] Propagation of HLA matched activated lymphocytes can be
carried out by conventional cultivation of lymphocyte cells, and
the cultivation is not limited especially, but for instance, as
disclosed in JP 03-80076 A1, it can be carried out by using either
interleukin 2 or anti-CD3 antibodies alone or by using them in
combination to cultivate them. In this case, from the point of view
of improving propagation efficiency, it is preferred to incubate
under existence of both interleukin 2 and anti-CD3 antibodies.
[0023] Not only the above mentioned interleukin 2 or anti-CD3
antibodies but also various cytokine, anti-CD28 antibodies or
various mitogens can be used. In this case, any matters which have
a function for propagating and activating lymphocytes can be used.
In addition, interleukin 2 used in this case can be a marketed
product, and preferably used so as to achieve a 1 to 2000 U/ml
concentration in the culture medium solution.
[0024] Interleukin 2 can be dissolved and used in any medium
solution widely used for cell culture, such as water, saline,
Dulbecco's phosphate buffered saline, RPMI-1640, D'MEM, IMDM and
AIM-V. Once interleukin 2 is dissolved, it is preferred that the
solution is stored in a refrigerator so as to prevent decrease of
activity. It is to be noted that any culture medium solutions used
in this case are not especially limited if they are available to
cultivation of the lymphocytes, and for instance, an organism
derived materials such as a blood serum or a synthetic medium
adding amino acids, vitamins and nucleic acid into balanced salt
solution can be used, and further desirable examples of them
include RPMI-1640, AIM-V, D'MEM, IMDM and the like, and especially
RPMI-1640 is preferred.
[0025] As the culture medium used in this case, a culture medium
containing normal human serums is preferred because it is superior
in propagation, and marketed culture mediums can be used. In
addition, instead of the human serum, a fetal calf serum can also
be used. Alternatively, a serum-free medium can be used. The
incubation can be achieved by adopting any of the generally
practiced incubation methods, e.g., inside a CO.sub.2 incubator. It
is preferred that CO.sub.2 concentration is in a range from 1 to
10%, especially in a range from 3 to 7%, and that temperature is in
a range from 30 to 40.degree. C., especially in a range from 35 to
38.degree. C.
[0026] The number of days for this incubation are not limited, but
an stimulation signal from anti-CD3 antibody is assumed to be
transmitted to lymphocytes, it is ideal from the point of view such
as to stimulate stably the cells with anti-CD3 antibody and to
increase the incubation efficiency that this incubation for
approximately 2 to 30 days, especially for 3 to 21 days is
preferably carried out. In order to increase the incubation
efficiency, it is more preferred to observe the condition of cells
under a microscope, to count the number of the cells and to add the
culture medium as necessary. It is to be noted that while no marked
increase in the number of cells is observed for the initial 1 to 2
days after the incubation started, cell propagation is first
observed around the third day and once the cells start to propagate
properly and the color of culture medium changes from orange to
yellow.
[0027] Note that an additional loading volume of the culture medium
is preferably in a range approximately between 0.1 and 5 times
relative to the volume of the culture medium prior to addition.
Furthermore, additional loading is preferably carried out once
every 1 to 7 days, especially once every 3 to 5 days in order to
prevent unfavorable condition of the cultivation and decrease of
activity of interleukin 2. Moreover, after the incubation with
anti-CD3 antibodies, the incubation process can be allowed to
continue without stimulation with anti-CD3 antibodies. Namely, the
incubation can be continued in a culture vessel without solidified
anti-CD3 antibodies, e.g., an incubating flask, a roller bottle, or
a gas permeable bag for cultivation until the administration
[0028] The incubation of the lymphocytes under these conditions is
preferably carried out under the same conditions as that of the
incubation under the presence of anti-CD3 antibodies except that
there is no stimulation by anti-CD3 antibodies, and further it is
more preferred in workability, cost performance and safety to
adjust concentration of the human serum and to use a serum-free
medium as necessary.
[0029] At the incubation, for instance, the umbilical cord blood or
the mononuclear cells are floated in a culture medium containing
interleukin 2, and the resulting solution is placed in a
cultivation vessel solidified anti-CD3 antibodies, so that the
cultivation can be started. Furthermore, in this case, the
efficiency of lymphocytes' propagation or activation is more
improved by adding and using various cytokines or mitogens into the
culture medium as the need arises. It is to be noted that anti-CD3
antibodies used in stimulation of the lymphocytes can be produced
in an animal or in cells by using purified CD3 components, but
marketed OKT-3 antibodies (manufacturer: Ortho Pharmaceutical)
which are outstanding in stability and cost performance can be
used.
[0030] However, besides this, antibodies are not especially limited
if they can promote the propagation and activation of the
lymphocytes, for instance, anti-CD28 antibodies can be used.
Anti-CD3 antibodies are preferably used in solid phase from the
point of view of propagation efficiency and operability of the
lymphocytes. A cultivation vessel which the antibodies are
solidified on its surface is an incubation container constituted of
a material such as glass, polyurethane, polyolefin or polystyrene.
In this case, a marketed plastic sterilized culture flask and the
like can be used because it is easy to obtain it, and further a
size of it can be selected as appropriate.
[0031] In addition, solidification of the antibodies can be carried
by adding diluent of anti-CD3 antibodies into the cultivating
vessel, and for instance, by leaving the vessel in a stationary
state for 2 to 24 hours at between 4 to 37.degree. C. It is
preferred that when anti-CD3 antibodies are solidified, anti-CD3
antibodies are diluted to a concentration of 0.1 to 30 .mu.g/ml in
a physiological buffer solution such as sterilized Dulbecco's
phosphate buffered saline. After the solidification, the vessel is
possible to be stored in a cold room or in a refrigerator
(4.degree. C.) until use and, in this case, the liquid can be
removed at the time of use and if necessary, the vessels can be
used by washing them with a physiological buffer solution such as
Dulbecco's phosphate buffered saline at room temperature.
[0032] The HLA matched peripheral blood-derived activated
lymphocytes obtained by propagation as above mentioned can be
processed or formulized as follows, and this can be used over wide
range of applications as a means for biodefence such as a remedy
for the preventing failure of the survival or the promoting
survival after transplantation.
[0033] Furthermore, in the case that there is a risk for any side
effects such as PT-GVHD (Post Transfusion-Graft versus Host
Disease) at transplanting, it is effective that prepared peripheral
blood originating activated lymphocytes are processed with anti-CD4
antibodies and the like and used with preparing so as to be a cell
group including CD4+ cells mainly.
[0034] When a risk for a side effect such as GVHD is little or when
an antineoplastic effect or an antiviral effect in addition to the
preventing failure of the survival or the promoting survival is
expected, it is effective that cell population including CD8+ cells
is used. Besides, in this case, it can be used in therapy as cell
population preparing a content of the CD8+ cells suitably
[Agents Having HLA Matched Activated Lymphocytes as its Main
Component]
[0035] The HLA matched activated lymphocytes having more propagated
cells as a main component can be prepared by adding various medical
components to them, for instance, so as to use them as a
therapeutic formulation for the preventing survival failure or the
promoting survival by preparing in a mode in which they are
suspended in saline for transfusion containing human albumin. It is
to be noted that "formulation" as described here means every
material having a biodefensive function which contains the active
lymphocytes as its main component, and any forms are not limited if
they include HLA matched activated lymphocytes.
[0036] For instance, it is available that a formulation that HLA
matched activated lymphocytes are suspended in an suitable solution
may be also suitable, and in this case, it is preferred that a
formulation is HLA matched activated lymphocytes, especially
suspended in saline for transfusion containing the human albumin,
but it is not limited by this example.
[0037] In addition, in this case, to prepare formulation containing
HLA matched activated lymphocytes as a main component, peripheral
blood or umbilical cord blood can be cultured in vitro or
mononuclear cells can be propagated in vitro after separating from
peripheral blood or umbilical cord blood. Besides, HLA matched
activated lymphocytes contained in the formulation according to the
present invention may be the cells that various genes are
introduced or that original genes are deleted or modified.
[Preparation Kit of a Formulation Containing HLA Matched Activated
Lymphocytes as a Main Component]
[0038] Besides, HLA matched activated lymphocytes according to the
present invention can be used as a formulation with the component
of it as an independent reagent, a kit combining culture medium
containing interleukin 2 and anti-CD3 antibody solidified flask as
components is made, and it is easy to prepare the formulation
according to the present invention by using this kit.
[0039] It is to be noted that the culture medium used in this case
can be added in advance into anti-CD3 antibody solidified flasks
separately, and further they can be cryopreserved. Thus, by
producing a kit combined at least two components as a plurality of
reagents and by using it when activated lymphocytes are needed, it
can be carried out more easily to prepare the formulation according
to the present invention.
[0040] HLA matched activated lymphocytes possible to be used in any
form of the present invention or formulation including them are
cryopreserved, and then they can be used in preventing survival
failure or improving survival such as promoting survival as the
need arises. Besides, HLA matched activated lymphocytes can be
cryopreserved by a method as follows.
[0041] In HLA matched activated lymphocytes to be preserved, the
concentration of them to suspend in a preservation liquid can be
selected availably due to the sizes of them, preferably they are
suspended in the cell preservation liquid at the concentration in a
range from 1.times.10.sup.3/ml to 1.times.10.sup.10/ml to be
cryopreserved, and more preferably they are suspended in the
preservation liquid at the concentration in a range from
1.times.10.sup.5/ml to 1.times.10.sup.8/ml to be cryopreserved. The
preservation liquid to be used in this case is not to be limited
especially, but it is preferred for the sake of convenience that it
can be used in a range from 0.1 ml to 1,000 ml, especially from 0.5
ml to 100 ml.
[0042] When they are cryopreserved, not only marketed preservation
liquid but also preservation liquid in house can be used. As an
ingredient of the preservation liquid, suitable buffer solution or
solution including serums, proteins, macromolecular substances such
as polysaccharide or dimethyl sulfoxide (may be abbreviated to
DMSO) can be used, but all of the listed materials are not always
used due to the preserved HLA matched activated lymphocytes.
Therefore, if the preservation liquid could preserve HLA matched
activated lymphocytes in it, the compositions of them are not
limited. Thus, HLA matched activated lymphocytes are suspended in
the suitable preservation liquid and cryopreserved at low
temperature.
[Dosage]
[0043] Furthermore, the dose of the formulation containing HLA
matched activated lymphocytes as its main component can be adjusted
suitably in accordance with a condition of the transplanted patient
or the objective of the therapy, the standard dose administered
under standard condition is in a range from 1.times.10.sup.2 to
1.times.10.sup.9 lymphocytes relative to 1 Kg of body weight.
Besides, the dose is preferably not less than 1.times.10.sup.3
lymphocytes/kg in order to increase the effect further, and even if
the dose exceeds 5.times.10.sup.8 lymphocytes/kg, the further
effect is not expected, so that the best dose is in a range between
1.times.10.sup.3 and 5.times.10.sup.8 lymphocytes/kg.
[Forms and Methods of Administration]
[0044] Furthermore, as forms of administration of the formulation
as described above, liquid such as an injection and a drip is
preferred, and it is more preferred to suspend the cells in saline
containing 0.01 to 5% of human blood serum albumin as the form of
the injection or the drip. An intravenous drip or an intravenous,
intraarterial or local injetion is preferred as a way for
administration. As a volume of solution to be administered depends
on the way for administration or the administered region, it is
preferred that the volume is usually in a range between 1 and 500
ml and that the solution includes cells of the above described
lymphocytes dosage. Furthermore, the frequency of such
administration is preferably once a day to once a month, and
further it is necessary that the number of administration is at
least more than once.
[Manufacturing Process of Human Monoclonal Antibodies]
[0045] Next, we explain about an effective manufacturing process
for human monoclonal antibodies by using animals, which antibodies
are safe and have good survival in animal after the cell
transplantation. Concretely, HLA matched activated lymphocytes are
administered before or after transplantation to an animal under
immunocomprimised condition, the antigens are immunized after
stabilizing the survival, and then antigen sensitized lymphocytes
are established or collected from the animal by a method such as
the gene selection or gene expression.
[0046] As explaining about an animal under immunocomprimised
condition, a cow, a house, a sheep, a goat, a rabbit, a guinea pig,
a rat, a chicken, a duck and the like are listed up as an example,
especially a mouse is preferred in management or handling easily. A
SCID (severe combined immunodeficiency: hereinafter saying SCID)
mouse among the mice is known as a mouse with extremely lower blood
concentration of immunoglobulin than C.B-17 mouse which is an
allotype congenic lines of BALB/c mouse in immunoglobulin (Ig)
heavy chain gene, and presents severe combined
immunodeficiency.
[0047] Thus, because of deficiency of mature T and B cells in the
SCID mouse, it is known that SCID mouse has some of abnormal
recombinase concerning gene rearrangement indispensable for
expression of immunoglobulin molecules, and especially that there
is abnormality in substrate specificity of the recombinase.
Therefore, self-antibodies in the SCID mouse are hardly produced.
Although no antibody-dependent cell mediated cytotoxicity (ADCC) is
observed in this mouse, the function of antigen presenting cells or
NK cells are normal.
[0048] By applying the properties of the SCID mouse different from
a nude mouse, a TC mouse (Transchromo mouse) or a mouse in which
immune function are disrupted by irradiation, SCID mouse is used in
the analysis of effect of anticancer drug against xenogenically
transplanted various tissue or tumors, or in production of models
for infectious disease specific to human such as acquired
immunodeficiency syndrome (AIDS) or Epstein-Barr virus (EBV). It is
known as SCID-hu mouse which human foetal thymus and a small piece
of embryonic liver is transplanted intrasubcapusular of kidney with
maintaining organ structure and hu-PBL-SCID mouse which human
peripheral blood lymphocytes are transplanted into
intraperitoneal.
[0049] The latter can induce human antibodies specific to tetanus
toxin or C-antigens of hepatitis B virus by immunizing the mouse.
Besides, because of expectable application to an autoimmune disease
model and so on, SCID mice can be model animals indicating human
disease pathology, which can not be obtained from the other
animals, by transplantation of various xenogenic tissue, especially
human tissue.
[0050] Accordingly, in the present invention, these SCID mice are
used as a mass production means of human monoclonal antibodies. The
SCID mice used in the present invention are usually 5- to
15-week-old, preferably 8- to 12-week-old.
[0051] Concretely, the human lymphocytes from 1.times.10.sup.7 to
5.times.10.sup.8, preferably approximately 1.times.10.sup.8, such
that human lymphocyte fractions produced from peripheral blood,
cerebrospinal fluid, umbilical cord blood or immune system tissue
and the like such as the spleen including human stem cells by a
conventional process are suspended in solution which does not
influence to cells and a living body such as physiological saline
and phosphate buffer physiological saline (PBS) are transplanted to
the SCID mouse intraperitoneally or intravenously.
[0052] In this case, as one of transplanting procedure, a method
that transplanting cells are encapsulated in an immunologically
isolated membrane and then transplanted into intraperitoneal may be
adopted. In this method, because the transplanted cells are not in
contact with host cells by the membrane after transplanting, GVH
(graft-versus-host) reaction is prevented, so that efficient
production of antibodies against targeted antigens are
increased.
[0053] Recovering antigen specific human antibody producing cells
is usually carried out in 7 days through 60 days after the
transplantation, preferably 7 days through 28 days, and especially
preferably 25 days through 28 days. Especially from the point of
collection efficiency, it is preferred that increasing
concentration of a human antibody and increasing titer of antigen
specific human antibody are examined by collecting a proper volume
of blood from an orbit of the SCID mouse every week from about
seventh day after the transplantation.
[0054] In measurement of the human antibody concentration and the
antigen specific human antibody titer, there are various methods
such as an enzyme immunoassay (hereinafter, abbreviating EIA), a
passive hemagglutination reaction technique, a fluorescent antibody
technique, an Ouchterlony method, a dot immunoassay method and the
like, but in the case that the antigen is a soluble form, the EIA
using an immuno plate (for instance, Nunc Co. Ltd, made in Denmark)
that conventional anti-human immunoglobulin or the aimed antigens
are solidified is suitable. According to these methods, the human
antibody producing cells can be recovered from the SCID mouse
indicating a higher antibody quantity and a higher antibody
titer.
[0055] Though the antibody producing cells can be collected from
each of the SCID mouse's organs, above all, it is very profitable
in the points of a recovery factor and a purity to collect from the
lymph node. The human monoclonal antibody producing cells obtained
in the present invention are very excellent in comparison with
conventional induction of human antibody producing cells in vitro
in the point of the antibody titer measurement, Plaque method or an
immunofluorescent assay, and further it is very excellent to obtain
the human monoclonal antibodies.
[0056] As a concrete method for collecting and purifying of the
human antibody producing cells according to the present invention,
the following method is used. Firstly, each of the internal organs
is minced into a single cell physically. If the content of the
antibody producing cells in cells collected from the internal
organs is larger, the collected cells are used to establish the
antibody producing cells as it is. However, when the purity of the
antibody producing cells is lower, for instance when cells of the
SCID mouse are contaminated into considerably, the purity of the
antibody producing cells is increased due to purifying by a density
gradient centrifugation, a panning method using the antigens, a
cytolytic method using anti-mouse cell antibodies and complements
for removing the SCID mouse derived cells or an affinity column
method using conventional anti-human B cell antibodies.
[0057] Next, the purified human antibody producing cells are
established by the following method. As the method for established
cell line, an Epstein-Barr virus transformation method and a cell
fusion method by fusing with self-proliferative cells can be mainly
listed up. In the transformation by the Epstein-Barr virus, the
Epstein-Barr virus prepared by a conventional method, for instance
0.5 through 5.times.10.sup.7/ml of the human antibody producing
cells are suspended into a propagation medium containing 1:10 to
10:1 of culture supernatant fluid of the cell line B95.8 and the
like, and are incubated at 37.degree. C. for 1 hour through 18
hours.
[0058] After the incubation, the cells are centrifuged at room
temperature, suspended to culture medium and added 0.1 ml each of
cell suspension at concentration of 1.times.10.sup.6/ml through
1.times.10.sup.5/ml to each well of a 96 wells plate. Furthermore,
the cells are cultivated in an incubator such as a CO.sub.2
incubator at 37.degree. C., the culture supernatant fluid in a well
in which the transformed cells are cultivated for 7 days through 30
days is collected, and then the only transformed cells producing
the targeted antibodies are selected by using a suitable screening
system such as the above mentioned EIA.
[0059] The cell fusion is performed by a conventional method and is
that polyethylene glycol (hereinafter, abbreviated PEG), Sendai
virus and the like are used as a fusion agent. Besides, an
electrofusion method may be used, but PEG is preferably used. PEG
with mean molecular weight of 1000 to 9000 is used, and especially
it is preferred to use PEG 4000 or PEG 6000.
[0060] The concentration in this case is used in a range from 10 to
80%, preferably from 40 to 50%. As a parent cell line using in cell
fusion, every parent cell line can be used, but a cell line with
drug selection marker is preferred. Besides, preferably human
myeloma and human lymphoma cell lines, especially preferably human
lymphoma AC-33 cell, and LICR-2 cell or SKO-007 cell as a human
lymphoblastic cell line are listed up. The antibody producing cells
obtained from SCID mice and suitable cell lines which are
preferably human myeloma and human lymphoma cell lines and
especially preferably human lymphoma AC-33 cell are washed with a
serum-free culture medium. The antibody producing cells and the
parent cells are mixed usually in the ratio of 1:1 through 10:1 are
centrifuged at 700 rpm at room temperature for 5 minutes and the
cells are recovered as pellets.
[0061] Furthermore, the cell pellets are loosened as warming in an
incubator at 37.degree. C., and then pre-warmed PEG solution is
added gradually into and mixed with the cell pellets. Usually, the
PEG solution is added at 1 ml per 10.sup.8 cells, but the volume of
it may be adjusted as the need arises. Next, pre-warmed culture
medium is added dropwise in the cell suspension to decrease PEG
concentration. Usually, 1 through 30 ml of the culture medium is
added in approximately 10 minutes.
[0062] Furthermore, the cells are collected by centrifugation at
room temperature, they are left in a culture medium containing 10%
fetal calf serum (hereinafter, abbreviated FCS) as the parent cells
at cell concentration of 1 through 5.times.10.sup.5/ml overnight,
and then a culture medium (HAT culture medium) containing HAT and
10% FCS is added to them. Besides, for omitting this step of
operation, the cells may be suspended directly in HAT culture
medium after the cell fusion. Then, the culture medium is changed
several times during two through three weeks.
[0063] A method for changing the culture medium is to remove a part
of culture medium ranging from 0.1 to 0.2 ml and to add the same
volume of freshly prepared HAT medium to the culture medium. If
appearance of hybridoma is observed, the hybridoma producing the
targeted antibodies are selected by a suitable screening system
such as the above mentioned EIA as soon as possible. Cloning is
carried out in the obtained hybridoma by a suitable method
immediately.
[0064] As a liquid culture medium using in the cell culture,
Daigo's T medium (NIHON PHARMACEUTICAL Co. LTD.), Dulbecco's
modified Eagle medium, RPMI-1640 medium, Iscove's modified
Dulbecco's medium and the like can be selected. In the case of the
transformation method by Epstein-Barr virus, the culture medium
which contains about 20% FCS is used. In the case of the cell
fusion method, in which contains 10 through 15% FCS. The human
monoclonal antibodies can be obtained from the antigen specific
human antibody producing cell lines by a conventional method.
[0065] For instance, by inoculating the human antibody producing
cell line into intraperitoneal of SCID mouse, ascites fluid
containing the monoclonal antibodies similar to mouse hybridoma at
high concentration can be obtained. The monoclonal antibodies
producing cells can be cultivated in large volume by using a
suitable serum-free culture medium such as GIT medium (NIHON
PHARMACEUTICAL CO., LTD.), HB104 (HANAMEDIA CO., LTD.) and Hybrity
1 (Nihon Yakuhin Kaihatu Co., Ltd). Large amount of highly purified
human monoclonal antibodies can be obtained from the above ascites
or culture supernatant by combining suitable conventional
purification methods.
[0066] In this case, for instance the solution including the
antibodies is centrifuged and then the supernatant fluid of the
solution is salted out. Usually, ammonium sulfate is used on this
occasion. After dissolving the obtained protein sediment to a
suitable buffer solution and dialyzing it, the targeted Human
monoclonal antibodies can be separated and purified by a column
chromatography (a DEAE ion exchange column, a hydroxyl apatite
column, a gel filtration, a protein A column, a protein G column,
and the like) or an immuno affinity chromatography and the like.
The purity of the purified human monoclonal antibodies by these
methods is over 99.9%, so that they are administrable to the human
as a medicament.
[0067] Besides, there are IgA, IgM, IgG, IgD and IgE in a class of
the antibody, and further the IgG class has 4 subclasses of IgG1,
IgG2a, Ig2b, IgG3 in the case of the mouse (IgG1, IgG2, IgG3, IgG4
in the case of the human). When the antigen is given to the animal,
IgM or IgG classes of antibodies are produced in most cases. IgG
whose molecular weight is approximately 160,000 has dimeric
structure and is relatively easy to handle. Compare to IgG, IgM is
a larger molecule with approximately 900,000 molecular weight and
exists in complicated pentameric structure joined with J-chain, so
that there are defects such that purification is difficult, that
preservation is difficult because of its easily aggregating
character, that it is difficult to produce Fab because it is easy
to inactivate due to partial degradation by a proteolytic enzyme,
and that there are many cases that binding activity is lost when
chemical modification such as to conjugate with anticancer drug or
a toxin chemically are carried out.
[0068] About which is superior in a curative effect to the cancer,
the monoclonal antibodies in IgG class or the monoclonal antibodies
in IgM class, Bernstein et al examine in detail by using the
monoclonal antibodies in IgG class and IgM class to Thy-1 antigens
of the lymphocytes. [Monoclonal Antibody, R. H. Kennet, T. J.
McKearnand, K. B. Bechtol editing, Plenum Press 1980, P. 275]
[0069] According to it, as a result of comparing monoclonal
antibodies in IgG class with monoclonal antibodies in IgM class
with reactivity of the same strength to Thy-1 antigen positive
lymphocytes, though the monoclonal antibodies in IgM class was
superior in a complement dependent anti-tumor effect in vitro, the
significant anti-tumor effect was recognized in the monoclonal
antibodies in IgG class in the anti-tumor effect in vivo which was
examined by using a tumor bearing mouse, but the anti-tumor effect
was not recognized in the monoclonal antibodies in IgM class.
[0070] Furthermore, when the isotopes labeled monoclonal antibodies
were injected in the mouse and the half-life in blood was examined,
it was found that the half-life in blood of the monoclonal
antibodies in IgM class was much shorter than the monoclonal
antibodies in IgG class. Thus the result indicates that the
monoclonal antibodies in IgG class are preferred as the monoclonal
antibodies used in human clinical use.
[0071] The humanized antibodies of the present invention can be
used independently or together with at least not more than one kind
of additive allowed upon the preparation. For instance, a suitable
medical composition can be achieved by dissolving the humanized
antibodies in a solution such as saline, glucose, lactose and
mannitol, or powder-injection can be produced by lyophilizing the
humanized antibodies by conventional method and adding sodium
chloride into the lyophilized antibody, and further the present
medical composition can contain additional agents known in a
pharmaceutical field, for instance, pharmaceutically acceptable
salts as the need arises.
[0072] Besides, dosage of the composition according to the present
invention is varied depending on the age, conditions and the like
of a patient, but the humanized antibodies are administered in a
dose of from 0.2 to 20 mg/kg/day to mammals including humans. The
composition is administered by intravenous injection either once a
day (single administration or continuous administration) or
intermittently 1 to 3 times in a week or in 2 or 3 weeks.
[0073] [A Method for Producing Genetically Engineered Human
Monoclonal Antibodies from Human Monoclonal Antibody Producing
Lines]
[0074] Genes coding the human monoclonal antibody are extracted
from the human monoclonal antibody producing cell line of the
present invention by a way in the genetic engineering, they are
inserted into expression vectors, they are introduced in host
cells, and then the human monoclonal antibodies producing
transformant can be obtained.
[0075] The genes used in the present invention are obtained by a
usual method and the other various methods, and can be cloned.
[edited by L. G. Davis et al: Basic methods in Molecular Biology,
Elsevier publication, New York, 1986; and J. Feder et al. American
Journal of Human Genetics, 37, 635 (1985)]
[0076] A mRNA used in the present invention is also prepared by
conventional method used usually. cDNA is prepared from it, and it
can be cloned. [Edited by J. Sambrook et al: Molecular Cloning--A
Laboratory Manual--, Cold Spring Harbor Laboratory Press published,
New York, 1989]
[0077] For instance, the genes can be obtained from genomic
libraries by conventional procedure. Namely, genes of the cells are
prepared by a standard method. For instance, after proteinase K
treatment of the cells under presence of Sodium Dodecyl Sulphate
(SDS), phenol extraction are performed, and further after
DNase-free RNase A treatment, phenol extraction are performed, so
that the genes are obtained. [Edited by Masami Matsumura: Labo
Manual Gene Engineering Maruzen Co., P 59 (1988)]
[0078] Next, if the targeted genes contain immunoglobulin variable
region, the genes are only isolated by a standard method, namely
genomic DNA is fragmentized with restriction endonuclease, inserted
in an appropriate recombinant DNA cloning vector, and screened
against the specified DNA sequence in the present invention by
using a radio labeled probe or enzyme labeled probe. Because DNA
obtained from the genes generally also includes intron, non coding
region, the DNA is modified by deletion or replacement of DNA by a
conventional method. [W. Kramer et al: Nucleic Acids Research, 12,
9441 (1984), and T. A. Kvnkel: Proceedings of National Academy of
Science USA, 82, 488 (1985)]
[0079] DNA that encodes the sequence of the polypeptide of variable
region of immunoglobulin light and heavy chain of the human
monoclonal antibody in this invention can be obtained from cDNA
library by conventional methods. [H. Okayama et al: Molecular and
Cellular Biotechnology, 2, 161 (1982)]. Namely, the cells are
homogenized in guanidine thiocyanate solution, subsequently RNA
pellet is precipitated by ultra-centrifugation on cesium
trifluoroacetate-EDTA density gradient and mRNA as poly
(A).sup.+-RNA further purified on oligo-dT column.
[0080] By using poly (A).sup.+-RNA as a template, a first strand
cDNA is reverse-transcribed by a conventional process using an
oligo-dT primer, a random primer or a specific primer in DNA
sequence of antibody gene, and further a second strand cDNA is
synthesized by using the first strand cDNA as a template. [Edited
by M. Matsumura: labo Manual Gene Engineering; Maruzen Co., P. 70.
P. 77 (1988)]
[0081] The cDNA obtained by thus conventional method is inserted in
a suitable cloning vector, cDNAs coding the variable region are
screened in the obtained clones with an appropriate prove for cDNA
coding the variable region. After isolating the targeted clones
only, cDNA can be handled basically in same way as the genomic DNA.
Besides, DNA coding variable region of immunoglobulin light chain
and heavy chain of the human monoclonal antibody can be obtained by
amplifying specially with polymerase chained reaction by a
conventional method. [R. Orlandi et al: Proceedings of National
Academy of Science USA, 86, 3833 (1989)]
[0082] The DNA coding variable region of light chain and heavy
chain can be chemically synthesized by a conventional process. [N.
D. Shina et al: Nucleic Acids Research, 12, 4359 (1984)] Even if an
original codon is displaced by a degenerate codon, as long as same
amino acid is coded when it is translated, it is not necessary that
these synthesized DNA is the same as DNA obtained by cloning.
[0083] The DNA coding non-variable region of immunoglobulin light
chain and heavy chain in the present invention can be cloned from
genomic DNA and cDNA. Besides, they can be synthesized chemically.
The polypeptide of light and heavy chain with lower immunogenicity
can be obtained by selecting the DNA coding the non-variable region
of human monoclonal antibody. The DNA coding the non-variable
region of human monoclonal antibody can be obtained from human
lymphocytes, for instance peripheral blood lymphocytes.
[0084] The DNA construct obtained thus can be inserted into
suitable recombined DNA cloning vectors and recombined DNA
expression vectors. [Edited by Y. Gluzman: Eukaryotic Viral
Vectors, published by Cold Spring Harbor Laboratories]
[0085] In the present invention, the DNA construct coding the
polypeptide of light and heavy chain is transducted into suitable
host cells as a part of the expression vector. Examples of the host
cell expressing human monoclonal antibodies according to the
present invention are preferably hybridoma, a CHO (Chinese Hamster
Ovary) cell, myeloma, plasmocytoma, lymphoma and the like. Besides,
it may be a plant-derived host cell.
[Preparation of Human Polyclonal Antibody]
[0086] Next, we explain about a method for efficient production of
human polyclonal antibodies by using animals in which antibodies
are safe and have good survival in animal after the cell
transplantation. Concretely, HLA matched activated lymphocytes are
administered before or after transplantation to an animal under
immunocompromised condition, the antigens are immunized after
stabilizing survival, and then antigen sensitized lymphocytes are
collected from the animal by a method such as the gene selection or
gene expression.
[0087] As explaining about an animal under immunocompromised
condition, a cow, a house, a guinea pig, a rat, a chicken, a duck,
and the like are listed up as an example, especially a goat or a
rabbit is advantageous in point of simple management or easy
handling. Besides, there are a large amount of polyclonal
antibodies to be collected. Further, an amount of immunogen can be
also set suitably in accordance with a kind of the animal and
interval of immunization can be also set suitably
[0088] Any method that immunization can be applied to a normal
animal is selectable. For instance, the immunization can be carried
out through any way such as subcutaneous, intraperitoneal,
intravenous, intramuscular, or intradermal. It is desired that the
immunogen is given together with a suitable adjuvant such as a
marketed complete Freund's adjuvant, incomplete Freund's adjuvant,
BCG, aluminium hydroxide gel and pertussis vaccine.
[0089] The human monoclonal antibodies or the human polyclonal
antibodies contained by the present invention is extremely
advantageous in point such that antigenicity is lower or absent
when they are used in the field of various therapies and diagnosis
that is planed to apply a substitute of antiserum against
infectious disease or drug intoxication, as anti-idiotype antibody
vaccine, as anti-adhesion molecules antibody in order to neutralize
inflammation or as catalytic antibody.
[0090] In the cases that the antibodies according to the present
invention are used concretely as a diagnostic reagents, they may be
used as a suitable labeling substance such as enzymes, dyes and
radioisotopes or labeling antibodies conjugated chemically or in a
genetic engineering; and the antibodies according to the present
invention are used as drug, for instance in the case that antigens
are bioactive substance such as toxin, the antibodies which have
preferably a neutralizing capacity to activity of antigens can be
administrated independently or as a admixture with a suitable
combined drug. The antibodies also can be administered for instance
in therapies to the cancer, thrombosis and the like as a targeting
antibody formulation conjugated with suitable agents by chemically
or in genetic engineering.
[0091] Moreover, the human monoclonal antibodies or the human
polyclonal antibodies according to the present invention are
prepared as parenteral agents preferably as injections, according
to the usual ways independently or by mixing with a
pharmacologically acceptable carrier, an excipient or a diluent
after filtration and sterilization with a membrane filter and the
like as the need arises, and then they can be used in therapies for
various diseases by giving to the mammals (mice, rats, cats, dogs,
pigs, cows, monkeys, humans and the like) subcutaneously,
intravenously or intramuscularly and the like.
[0092] Besides, a medicament can be produced by dissolving the
monoclonal antibodies or polyclonal antibodies of the present
invention in a suitable solution such as a phosphate-buffer, a
saline, a Ringer solution and the like, sterilizing according to
the usual way for instance by an aseptic filter and sealing up them
into ampoules. Furthermore, solid-state medicament can be produced
by lyophilization of these liquid-type medicines according to the
usual way. Furthermore, although the dose of the human monoclonal
antibody or the human polyclonal antibody of the present invention
varies depending on weight, age or sexuality of administration
objects, or targeted diseases, symptoms or administration routes,
they can be administered in a dose of from 1 .mu.g to 1 mg/kg
weight/a day as an immunoglobulin quantity once a day or a few
times a day continuously or intermittently.
Embodiment No. 1
[0093] Hereinafter, the present invention is explained due to the
embodiment No. 1.
(1)<Preparation in OKT3 Solidified Flask (Middle) and
(Large)>
[0094] Five ml of OKT3 solution prepared at 5 .mu.g/ml in PBS (-)
was put into a culture flask (middle) and 10 ml of OKT3 solution of
was put into a large sized culture flask (large), so that the
bottom surfaces of the flasks were evenly covered with the
solution. They were preserved in a cold room just before using.
(2)<Collecting Lymphocytes from Umbilical Cord Blood for
Transplantation>
[0095] The OKT3 solution in the OKT3 solidified flask (middle)
prepared in the same way as the above item (1) was aspirated by
suction. Ten ml of PBS (-) was poured into the flask and the flask
was agitated thoroughly with its cap closed. After that, the can
was opened and the solution was discarded. Next, 10 ml of PBS (-)
was poured into the flask in a clean bench, then the flask was
thoroughly agitated with the cap closed, the cap was then opened
and the solution was discarded.
[0096] The liquid remaining inside the flask and on the cap was
aspirated thoroughly by suction, 10 ml of culture medium [44 ml of
medium (RPMI1640+7), 1 ml of IL (interleukin)-2 at the
concentration of 35,000 U/ml and 5 ml of human serum are mixed to
be a culture medium (hereinafter, abbreviated to "culture medium")]
was poured into the flask, stirred lightly, and cell suspension was
transferred to the Flask. Ten .mu.L of cell suspension was sampled
for counting a number of cells and 1500 .mu.L of the cell
suspension was sampled for measuring contents of CD4 and CD8
positive cells (500 .mu.l is sampled to each of three 1.5 ml
tubes). The cultivation of the rest was started in a CO.sub.2
incubator at 37.degree. C. (Cultivation 0 day).
(3)<Counting a Number of Cells by Turk's Solution>
[0097] In the above item (2), 10 .mu.L of collected umbilical cord
blood was mixed with 40 .mu.L of Turk's solution, the resulting
solution of 10 .mu.l was applied to a hemocytometer to count a
number of cells under a microscope. As a result, a number of all
cells in the flasks were 1.3.times.10.sup.6, respectively.
(4)<Analysis of Cell Surface Antigen>
[0098] Three tubes of the suspension prepared in the above item (2)
were centrifuged at 6,000 rpm at 4.degree. C. for 5 minutes to
precipitate the cells. After aspirating the supernatant clearly, 8
.mu.L of PBS (-) and 8 .mu.L of CD3/HLA-DR antibody were added to
the tube No. 1, and 8 .mu.L of PBS (-) and 8 .mu.L of CD4/CD8
antibody were added to the tube No. 2, so that they were reacted
for 30 minutes.
[0099] Besides, only 8 .mu.L of PBS (-) was added to the tube No. 3
as a control for nonspecific reaction. After the incubation, 800
.mu.L of sheath solution (Produced by Coulter Co. Ltd.; Isoton II)
was added to every tube. After mixing with voltex mixer,
centrifugation at 6,000 rpm at 4.degree. C. for 5 minutes was
performed to precipitate the cells. After aspirating the
supernatant of every tube clearly, 800 .mu.L of sheath solution was
added to it. And then cell pellets were loosening by pipetting and
transferred into a tube for FACS measurement.
[0100] In FACS analysis, FACScan (BD Bioscences, NJ USA) was used
and measurement of FACS analysis was performed according to its
manual. Contents of CD3+, HLA-DR+, CD4+ and CD8+ cells resulted
28%, 3%, 46% and 5%, respectively.
(5)<Preparation and Cultivation of the CD4+ Cell by Anti-CD8
Antibody Conjugated Magnetic Beads>
[0101] As a result of having cultivated in the above item (2), 5
days later, the total number of the cells became 3.4.times.10.sup.7
and each number of CD4+ and CD8+ cells became 9.5.times.10.sup.6.
These cultured cells were transferred into a 50 ml tube, and
centrifugation at 1,000 rpm at 20.degree. C. for 10 minutes was
performed. After the centrifugation, the supernatant was discarded
and the cell precipitates were loosened well by vortex mixer.
[0102] On the other hand, 300 .mu.L of the magnetic beads
conjugated with anti-CD8 antibody containing 4 times beads as many
as the number of CD8+ cells was put into a 15 ml tube and installed
to a magnet MPC-1 to incubate for one minute. The resulting
solution was taken out so as not to aspirate the beads and the tube
was removed from the magnet. One ml of PBS (-) was added to the
tube and stirred well, and then the tube was installed to the
magnet. After the incubation for one minute, the resulting solution
was taken out so as not to aspirate the beads and the tube was
removed from the magnet. One ml of D2 PBS(-) was put into the tube
again and stirred well, and then the tube was installed to the
magnet. After the incubation for one minute, the resulting solution
was taken out so as not to suck up the beads and the tube was
removed from the magnet.
[0103] Five ml of medium for reaction prepared by mixing 45 ml of
washing solution and 5 ml of human serum) was added to cell
suspension collected by centrifuging and suspended lightly, then
the cells were transferred into the tube including the magnetic
beads conjugated with anti-CD8 antibodies, and the mixture if cells
and beads were incubated in a cold room for 30 minutes as shaking
lightly with a belly dancer laboratory shaker. After the
incubation, the tube was installed to the magnet and incubated for
one minute. The resulting solution was taken out so as not to
aspirate the beads and transferred to a new 15 ml tube. One ml of
PBS(-) was added to the former tube which contained the magnetic
beads, the tube was installed to the magnet again and incubate for
one minute. The 2nd resulting solution was taken out so as not to
aspirate the beads, transferred into a new tube and centrifuged at
1,000 rpm at 20.degree. C. for 10 minutes. After the
centrifugation, the supernatant was discarded and the cell
precipitates were loosened well by vortex mixer.
[0104] Ten ml of culture medium was added to the recovered CD4+
cells and the resulting matter was suspended lightly. On the other
hand, solution of OKT3 in the OKT3 solidified flask (middle)
produced similarly to the above item (1) was aspirated by suction.
Ten ml of PBS (-) was poured into the flask, then the flask was
thoroughly agitated with a Gap of the flask closed, the cap was
then opened and the solution was discarded. Next, 10 ml of PBS (-)
was poured into the flask in a clean bench, then the flask was
thoroughly agitated with the cap closed, the cap was then opened
and the solution was discarded. The liquid remaining inside the
flask and on the cap was aspirated thoroughly by suction and the
cell suspension was transferred into the flask. Ten .mu.L of
suspension was sampled for counting a number of cells and 1,000
.mu.L of suspension was sampled for measuring content of CD4+ and
CD8+ cells (500 .mu.l is sampled to each of two 1.5 ml tubes). The
cultivation of the rest was started in a CO.sub.2 incubator at
37.degree. C. (Cultivation 0 day)
(6)<Expanding Cultivation of CD4+ Cell>
[0105] 20 ml of culture medium was added to the cultivation medium
in the above item (5) on the third day and the fourth day,
respectively. Five days later, to expand the culture volume more,
culture flask were changed from the flask (middle) to the flask
(large) that was produced similarly to the above mentioned. Namely,
the OKT3 solution in the OKT3 solidified flask (large) prepared in
the same way as the above item (1) was aspirated by suction. 50 ml
of PBS (-) was poured into the flask and the flask was agitated
thoroughly with its cap closed. After that, the cap was opened and
the solution was discarded.
[0106] Next, 50 ml of PBS (-) was poured into the flask in a clean
bench, then the flask was thoroughly agitated with the cap closed,
the cap was then opened and the solution was discarded.
[0107] Next, 50 ml of PBS (-) of 50 ml was poured into the flask
aseptically, then the flask was thoroughly agitated with a cap of
the flask closed, the cap was then opened and the solution was
discarded. The liquid remaining inside the flask and on the lid was
aspirated thoroughly by suction. The cells adhere to a bottom of
the flask (middle) was detached from the bottom by tapping the
flask several times and the cell suspension in the flask (middle)
was transferred to the flask (large). Furthermore, 50 ml of flesh
culture medium was put into the flask (middle) in order to suspend
the remaining cells, and the cell suspension in the flask (middle)
was transferred to a new flask (large) and the flask was incubated
in the CO.sub.2 incubator at 37.degree. C. to continue the
culture.
(7)<Cryopreservation of CD4+ Cell>
[0108] Regarding to cell culture in the above item (6), the cells
were cryopreserved by a following method on the sixth day of the
cultivation. In a method for preservation, the cells adhered to the
bottom of the flask were detached by tapping the flask several
times, then 500 .mu.l of the culture medium in the flask was taken
out and spread onto a tryptic soy agar (TSA) plate, and then the
plate was incubated in the CO.sub.2 incubator at 37.degree. C. to
carry out sterility testing. Furthermore, 10 .mu.l of the culture
medium in the flask was taken out and the number of cells was
counted.
[0109] Next, 40 ml of the cultured cell solution was transferred to
each of two 50 ml tubes, and they were centrifuged at 1,000 rpm at
20.degree. C. for 5 minutes. In every tube, the supernatant was
discarded and the cell pellet was vortexed, the resulting cell
suspension and cell preservation solution [prepared by mixing 5 ml
of human serum, 5 ml of dimethyl sulfoxide (hereinafter,
abbreviated to DMSO) and a 40 ml of medium (RPMI1640+7)] were
cooled down on an ice for 5 minutes, the cell suspension of every
tube was brought together to one tube with 4.5 ml of the cell
preservation solution, the cells in the tube were homogenized by
pipetting lightly, and then the resulting matter was divided into
three cryopreservation tubes (2.0 ml) respectively.
[0110] Furthermore, the tubes were put in a BICELL (Nihon Freezer
co. Ltd., Tokyo Japan), a freezing container, and preserved at
-80.degree. C. for several days, and then preserved in a liquid
nitrogen tank. The cell concentration at the culture was
1.9.times.10.sup.6/ml, and the preserved cell number was
5.1.times.10.sup.7/=tube.
(8)<Counting of a Number of Cells by Trypan Blue Dye
Exclusion>
[0111] 10 .mu.L of the cell suspension sampled for counting cell
number in the above item (7) was mixed with 20 .mu.l trypan blue
solution, the resulting matter was applied to hemocytometer to
count the cell number under the microscope. Consequently, the total
number of the cells was 5.1.times.10.sup.7.
(9)<Course Before Cord Blood Transplantation and Administering
Lymphocytes>
[0112] A patient with acute myelomonocytic leukemia could not be
introduced to remission was carried out bone marrow transplantation
from HLA-identical siblings, but 2 months later the leukemia
relapsed. Then peripheral blood stem cell transplantation from same
donor was carried out twice, blast cells still remained and the
patient developed hematopoietic hypoplasia. GVHD was not observed
in every 3 times transplantation. The same patient received
umbilical cord blood transplantation with 3 loci mismatch and GVHD
grade III was observed 10 days later. The severity of GVHD improved
by mPLS pulse treatment. The three times of stem cell
transplantations ended in failure. Since no GVHD developed and thus
a GVL effect was not induced, it was determined that an activated
CD4+ cell therapy was carried out because of expecting an
anti-tumor effect after the transplantation and promoting of the
donor cells survival.
(10)<Taking Out and Revival of Preserved CD4 Positive
Cells>
[0113] One of the tube containing CD4+ cells cryopreserved in the
above item (7) was taken out and then warmed up by a heat block at
37.degree. C. for 4 minutes. Ten ml of washing medium (RPMI1640+6)
was poured into a 15 ml centrifugation tube aseptically and then
the preserved lymphocyte were suspended the medium with a transfer
pipette. Furthermore, after centrifugation at 1,000 rpm at
20.degree. C. for 5 minutes, the supernatant of it was discarded
and tapped lightly, then the cell pellet was suspended to 50 ml of
culture medium, and then the resulting cell suspension was
transferred to a cloning plate.
[0114] After observing the cells by the microscope, the plate was
incubated in the CO.sub.2 incubator at 37.degree. C. and the
cultivation started again (Cultivation 0 day). On the second day of
the cultivation, the vigorous growth of the cells all over the
plate were confirmed under the microscope and then the cells in the
cloning plate were transferred to a flask (225 cm2, CELL
CULTUREFLASK). Furthermore, after washing the cloning plate with 50
ml of fresh culture medium to recover the remaining cells, the
cells were transferred to a flask and incubated in the CO.sub.2
incubator at 37.degree. C. to continue the cultivation.
[0115] On the fourth day of the cultivation, the OKT3 solution in
the OKT3 solidified flask (large) produced similarly to the above
item (5) was aspirated by suction. Fifty ml of PBS (-) was poured
into the flask, then the flask was thoroughly agitated with a cap
of the flask closed, the cap was then opened and the solution was
discarded. Next, fifty ml of the PBS (-)=was poured into the flask
aseptically, then the flask was thoroughly agitated with a cap of
the flask closed, the can was then opened and the solution was
discarded by decanting.
[0116] The liquid remaining inside the flask and on the cap was
aspirated thoroughly by suction. The cells adhered to the bottom of
the used flask (middle) were detached by tapping the flask several
times, and the cell suspension was transferred to the new flask
(large). Furthermore, 150 ml of fresh culture medium was added to
the used flask (middle) to suspend the remaining cells, and then
the resulting suspension was transferred to the new flask (large).
The new flask (large) was put in the CO.sub.2 incubator at
37.degree. C. for cultivation to continue the cultivation.
(11)<Cultivation of CD4 Positive Cells in a Bag for
Cultivation>
[0117] On the sixth day of the cultivation, the cells adhered to
the bottom of the flask used in the cultivation in the above item
(10) were detached by tapping the flask several times, 1 ml of the
culture medium in the flask was taken out and was spread onto a
sabouraud dextrose agar plate (containing chloramphenicol) or a
sheep blood agar plate equally, and the plates were incubated in
the CO.sub.2 incubator at 37.degree. C. to carry out an sterility
testing.
[0118] Ten .mu. of the culture medium in the flask was taken out to
count the cell number similarly to the above item (7). A culture
bag A-1000 (a bag for cell cultivation) containing 750 ml of medium
in which is 1:1 mixture of AIM-V medium prepared for bag
cultivation [which is that IL-2, human serum and oxaloacetic acid
are added to the medium of 10 L so that the final concentrations of
them become 200 U/ml, 1% and 1 mM, respectively] and CP-2 (LL-7.1)
medium (1 L) was warmed up at 37.degree. C.
[0119] The bag and a 50 ml syringe were connected each other
aseptically and the whole of the culture medium in the flask was
noured into the bag with the syringe. A tube from the bag was
clamped by forceps, an end of the tube was sealed, and the bag was
put in the CO.sub.2 incubator at 37.degree. C. for cultivation to
continue the cultivation.
(12)<Sterility Testing and Endotoxin Assay of CD4 cell>
[0120] On the seventh day of the cultivation (the day before
administration), about 1 ml of the culture medium was sampled from
a sampling port of the culture bag cultivated in the above item (7)
by a 1 ml syringe with 24G injection needle aseptically. The
solution sampled firstly was discarded, about 1 ml of the solution
was sampled by the same syringe again, about 200 .mu.l of the
solution was spread onto the sheep blood agar plate and the rest of
the solution was transferred into a dry heat sterilized conical
test tube.
[0121] After cleaning the sampling port of the bag after sampling
by the forceps clamping an alcohol cotton, the bag was put in the
CO.sub.2 incubator at 37.degree. C. for cultivation to continue the
cultivation. The agar plate was put in the CO.sub.2 incubator at
37.degree. C. for cultivation to carry out sterility testing, and
the dry heat sterilized conical test tube was centrifuged at 1,500
rpm at 20.degree. for 5 minutes.
[0122] One hundred .mu.l of prepared main solution (A kit for
endotoxin testing using a toxicolor systemlS-200 set and a
toxicolor system ET-1 set, Seikagaku Corp., Tokyo Japan) was put
into three dry heat sterilized test tubes with a disposable micro
pipette, respectively. One hundred .mu.l of water for injection is
put into the test tube for negative control and 100 .mu.l of
standard solution was put into the test tube for positive control
with toxipett sampler (Seikagaku Corp.), dry heat sterilized
disposable capillary tip installed micro pipette. Furthermore, 80
.mu.l of water for injection and 20 .mu.l of the centrifuged
culture supernatant were put into the test tube for sample with
toxipett sampler dry heat sterilized disposable capillary tip
installed micro pipette. The three tubes were incubated in a water
bath incubator at 37.degree. C. for 30 minutes.
[0123] Thirty minutes later, 400 .mu.l of 0.8M acetic acid solution
was added to each of three test tubes, the test tubes were stirred
with vortex, and then 400 .mu.l of the reaction solution of each
test tube was transferred to 96 wells plate to measure values in
absorbance at 405 nm (reference 630 nm) with a microplate reader.
It was confirmed that five times as much as the value of the
absorbance of the sample did not exceed to a value of the positive
control.
(13)<FACS Analysis of CD4+ Cell for Administration Before
Harvest>
[0124] Before harvesting the CD4+ cells, a part of the culture
medium cultivated in the bag was taken out as sample, the cell
number was measured by a method similar to the above item (8) and
the content of CD8+ cell was measured by a method similar to the
item (4). Consequently, the total cell number was
3.0.times.10.sup.9 including 0.8% of the CD8 cells. Due to the
total cell number and the ratio that the FACS analysis CD8+ cell
content by percentage, there were 1.5.times.10.sup.6 CD8 cells.
(14)<Harvesting of CD4 Positive Cells>
[0125] Thousand ml of the culture medium cultivated in the bag in
the above item (11) was transferred in a 250 ml centrifugation tube
aseptically, and the tube was centrifuged at 1,500 rpm at
20.degree. C. for 8 minutes. After the centrifugation, the
supernatant of it was discarded, the cell pellet was loosened with
vortex mixer, 200 ml of the cell washing solution was put into the
centrifugation tube, and then it was centrifuged at 1,800 rpm at
20.degree. C. for 8 minutes. After the centrifugation, the
supernatant of it was discarded and the cell Pellet was loosened
with vortex mixer. One hundred ml of the cell washing solution was
put into the centrifugation tube, each of the cell suspension was
transferred into two 250 ml tubes, and then they were centrifuged
at 1,800 rpm at 20.degree. C. for 8 minutes. After the
centrifugation, the supernatant of it was discarded, the cell
pellet was loosened vortex mixer, and then 100 ml of the cell
washing solution was put into the two 250 ml tubes,
respectively.
[0126] After the centrifugation, the supernatant was discarded and
the cell pellet was loosened vortex mixer well. And then the cells
were suspended in a final solution (a solution for administering
lymphocytes was made by putting 100 ml of saline and 5 ml of 20%
human serum albumin solution into a 250 ml centrifugation tube to
be a final solution) sufficiently, it was passed through a filter
with 100 micro-mesh and then fluid volume of the cell suspension
was measured. Besides, the cell suspension was sampled in order to
count the cell number and the content of CD4.
[0127] 50 ml syringe was connected with a separation bag (300 ml)
to pour the cell suspension from the syringe to the separation bag.
When the pouring was completed, liquid remained in the syringe was
pushed out by an inner cylinder, and finally the tube was clamped
by the forceps. The tube was sealed by a tube sealer and syringe
was cut off by scissors. As a result of the measurement, the liquid
volume was 100 ml and the total cell number was
2.5.times.10.sup.9.
(15)<FACS Analysis of CD4+ Cell for Administration After
Harvest>
[0128] FACS analysis of CD4+ cell for administration after harvest
was performed by a method similar to the above item (4).
Consequently, the content by percentage of CD4+ cell was 99.75% and
that of CD8+ cell was 0.25%.
(16)<Administering CD4 Positive Cells to a Patient>
[0129] CD4+ cells prepared in the above item (14) were administered
intravenously to the patient at ratio of 4.5.times.10.sup.7/Kg body
weight over one hour. Administration after that was carried out
three times in every one or two weeks. One dose was
2.5.times.10.sup.6 to 5.9.times.10.sup.7/Kg body weight and was
administered intravenously to the patient during one hour.
Embodiment No. 2
[0130] (1)<Collection of Blood from a Donor>
[0131] After the umbilical cord blood transplantation, the
lymphocytes were infused nine times. Furthermore, to improve
survival, 30 ml of peripheral blood was drawn from the vein of the
patient with a syringe containing heparin on the 87th day after the
transplantation.
(2)<Separation of Peripheral Blood Mononuclear
Leukocytes>
[0132] A needle of an injection syringe collecting blood
aseptically in a clean bench (hereinafter, abbreviated to
"aseptically") was removed so as not to touch an adjacent to a
connected portion of syringe and changed to 19G needle. Fifteen ml
of the washing medium was poured into two 50 ml centrifugation
tubes, respectively and the whole of the collected blood was poured
into them equally slowly. After closing a cap of every
centrifugation tube tightly, the diluted blood was mixed by turning
the tube upside down twice or three times. Three ml of Lymphosepar
I (IBL Co. Ltd., Gunma Japan), Ficoll-Conray solution, was put into
six ml centrifugation tubes with a 10 ml pipette, respectively. Ten
ml of diluted blood with the medium was layered carefully onto
Lymphpsepar I in the every sedimentation tube undisturbed at the
interface so, and then centrifuged at 1,800 rpm at 20.degree. C. in
the condition of braking off for 15 minutes.
[0133] After the centrifugation, the upper layer was drawn off by
the aspirator aseptically to about 1 cm upper a lymphocyte layer so
as not to aspirate the lymphocyte. The lymphocyte layer was taken
out with a 5 ml pipette so as not to suck up a clot layer and
transferred in a 50 ml centrifugation tube in which 25 ml of the
washing medium (RPMI1640+6) was put in advance. After a cap of the
sedimentation tube was closed and mixed by turning the tube upside
down twice or three times, the resulting matter was centrifuged at
1,800 rpm at 20.degree. C. for 10 minutes. After the
centrifugation, the supernatant was discarded and the cell pellet
was loosened well with vortex mixer. Furthermore, after 50 ml of
the washing medium was put into it and mixed by turning the tube
upside down, 10 .mu.l of the solution was sampled for counting cell
number, 500 .mu.l of the solution was sampled to two 1.5 ml micro
tubes for measuring contents of CD4 and CD8, respectively.
(3)<Measurement of Cell Number by Turk's Solution>
[0134] Ten .mu.l of the cell suspension sampled for counting the
cell number in the above item (2) was mixed with 40 .mu.L of Turk's
solution, 10 .mu.l of the resulting matter was applied to a
hemocytometer and the cell number was counted under a microscope,
consequently the total of the cell number was
8.5.times.10.sup.7.
(4)<Analysis of CD4 Positive Cells>
[0135] Two tubes containing the cell suspension prepared in the
above item (2) was centrifuged at 6,000 rpm at 4.degree. C. for 5
minutes to precipitate the cell and then the supernatant was
aspirated clearly. Then, 8 .mu.l of Dulbecco's phosphate-buffered
saline and 8 .mu.l of CD4/CD8 antibodies were put into two tubes,
respectively and stirred well, and then were reacted at 4.degree.
C. for 30 minutes. After the reaction, 800 .mu.l of the sheath
solution was put into every tube and stirred by vortexing, and the
resulting matter was centrifuged at 6,000 rpm at 4.degree. C. for 5
minutes to precipitate the cells.
[0136] After aspirating the supernatant clearly, 800 .mu.l of the
sheath solution was added, the cells were loosened by pipetting,
and then the resulting matter was put into a tube for FACS
measurement. Besides, in FACS, FACScan was used. Measurement of
FACS was performed according to the manual. Consequently,
percentage of CD4+ or CD8+ cells was 28%, respectively. From the
total cell number anf result of FACS analysis the cell number of
CD4+ or CD8+ cells was 2.4.times.10.sup.7, respectively.
(5)<Preparation in OKT3 Solid Phased Flasks (Middle) and
(Large)>
[0137] Five 5 ml of OKT3 solution (5 .mu.g/ml in PBS (-)) was put
into a culture flask (middle) and 10 mnl of the OKT3 solution was
put into a culture flask (large), the solutions immersed bottoms of
the flasks respectively, and they were preserved in a cold room
just before use.
(6)<Separation and Cultivation of CD4+ Cells with Magnetic Bead
Conjugated with Anti-CD8 Antibodies>
[0138] From 50 ml cell suspension prepared in the above item (2),
20 ml of cell suspension (the total cell number was
3.4.times.10.sup.7 and the CD4 and CD8 positive cell number was
9.5.times.10.sup.6 respectively) was transferred into a 50 ml tube,
and centrifuged at 1,000 rpm at 20.degree. C. for 10 minutes. After
the centrifugation, the supernatant was discarded and the cell
pellet was loosened well with vortex mixer.
[0139] Three hundred .mu.L of magnetic bead conjugated with
anti-CD8 antibody solution containing beads 4 times as many as the
CD8+ cell number was put into the 15 ml tube, the tube was
installed to a magnet MPC-1 to be reacted for one minute.
Furthermore, the solution was taken out so as not to aspirate the
beads, the tube was removed from the magnet, then 1 ml of the PBS
(-) solution was put into the tube and stirred well, and then the
tube was installed to the magnet again. After one minute reaction,
the solution was taken out so as not to aspirate the bead, and then
the tube was removed from the magnet. After 1 ml of the PBS (-)
solution was put into the tube and stirred well again, and the tube
was installed to the magnet. After one minute reaction, the
solution was taken out so as not to aspirate the beads and the tube
was removed from the magnet.
[0140] Five ml of medium for reaction prepared by mixing 45 ml of
washing solution and 5 ml of human serum) was added to cell
suspension collected by centrifuging and suspended lightly, then
the cells were transferred into the tube including the magnetic
beads conjugated with anti-CD8 antibodies, and the mixture of cells
and beads were incubated in a cold room for 30 minutes as shaking
lightly with a belly dancer laboratory shaker. After the
incubation, the tube was installed to the magnet and incubated for
one minute. The resulting solution was taken out so as not to
aspirate the beads and transferred to a new 15 ml tube. The tube
was installed to the magnet again, and incubated for one minute.
The resulting solution was taken out so as not to aspirate the
beads, the cells was transferred into a new tube and centrifuged at
1,000 rpm at 20.degree. C. for 10 minutes. After the
centrifugation, the supernatant was discarded and the cell
precipitates were loosened well by vortex mixer.
[0141] Twenty ml of culture medium [44 ml of medium (RPMI1640+7), 1
ml of IL (interleukin)-2 at the concentration of 35,000 U/ml 1 ml
and 5 ml of human serum are mixed to be a culture medium
(hereinafter, abbreviated to "culture medium")] was added to the
recovered CD4+ cells and the resulting matter was suspended
lightly. On the other hand, solution of OKT3 in the OKT3 solidified
flask (middle) produced similarly to the above item (5) was
aspirated by suction. Ten ml of PBS (-) was poured into the flask,
then the flask was thoroughly agitated with a cap of the flask
closed, the cap was then opened and the solution was discarded.
Next, 10 ml of PBS (-) was poured into the flask in a clean bench,
then the flask was thoroughly agitated with the cap closed, the cap
was then opened and the solution was discarded.
[0142] The liquid remaining inside the flask and on the cap was
aspirated thoroughly by suction and the cell suspension was
transferred into the flask. Ten .mu.L of suspension was sampled for
counting a number of cells and 1,000 .mu.L of suspension was
sampled for measuring content of CD4+ and CD8+ cells (500 .mu.l is
sampled to each of two 1.5 ml tubes). The cultivation of the rest
was started in a CO.sub.2 incubator (95% humidity) at 37.degree. C.
(Cultivation 0 day). As a result from the analysis of CD4+ cells
similarly to the above item (4), the content of the CD4+ cell was
46% and the content of CD8+ cell was 5%.
(7)<Counting of Cell Number by Trypan Blue Dye Exclusion>
[0143] Ten .mu.l of the cell suspension sampled for counting cell
number in the above item (6) was mixed with 20 .mu.l of trypan blue
solution, 10 .mu.l of the resulting matter was applied to the
hemocytometer under a microscope., As the result from cell
counting, the total cell number was 2.6.times.10.sup.7.
(8)<Expanding Culture of CD4+ Cell>
[0144] Twenty five ml of culture medium was added to the
cultivation medium in the above item (6) on the third day and the
fourth day, respectively. Five days later, to expand the culture
volume more, culture flask were changed from the flask (middle) to
the flask (large) that was produced similarly to the above
mentioned. Namely, the OKT3 solution in the OKT3 solidified flask
(large) prepared in the same way as the above item (5) was
aspirated by suction. Fifty ml of PBS (-) was poured into the flask
and the flask was agitated thoroughly with its cap closed. After
that, the cap was opened and the solution was discarded.
[0145] Next, 50 ml of PBS (-) was poured into the flask in a clean
bench, then the flask was thoroughly agitated with the cap closed,
then the cap was opened and the solution was discarded. The liquid
remained in the flask and on the lid was aspirated by suction
thoroughly. The cells adhered to a bottom of the flask used in the
cultivation so far were detached by tapping the flask several times
lightly and then the cell suspension was transferred. Furthermore,
50 ml of new culture medium was put into the used flask to suspend
the remained cells, and it was transferred to a new flask. The new
flask was put in the CO.sub.2 incubator at 37.degree. C. to
continue the cultivation.
(9)<Cryopreservation of CD4+ Cells>
[0146] A part of the cells cultivated in the above item (8) were
cryopreserved once on the sixth day of the cultivation. In a method
for preservation, the cells adhere to the bottom of the flask were
detached by tapping the flask several times, then 500 .mu.l of the
culture medium in the flask was taken out and spread onto a tryptic
soy agar (TSA) medium, and then incubated in the CO.sub.2 incubator
at 37.degree. C. to carry out an sterility testing. Furthermore, 10
.mu.l of the culture medium in the flask was taken out and the cell
number was counted similarly to the above item (3).
[0147] Next, 40 ml of the cultured cell solution was transferred to
each of two 50 ml tubes, and they were centrifuged at 1,000 rpm at
20.degree. C. for 5 minutes. In every tube, the supernatant was
discarded and the cell pellet was vortexed, the resulting cell
suspension and cell preservation solution [prepared by mixing 5 ml
of human serum, 5 ml of dimethyl sulfoxide (hereinafter,
abbreviated to DMSO) and a 40 ml of medium (RPMI1640+7)] were
cooled down on ice for 5 minutes, the cell suspension of every tube
was brought together to one tube with 4.5 ml of the cell
preservation solution, the cells in the tube were homogenized by
pipetting lightly, and then the resulting matter was divided into
three cryopreservation tubes (2.0 ml), respectively. Furthermore,
the tubes were put in a BICELL (Nihon Freezer co. Ltd., Tokyo
Japan), a freezing container, and preserved at -80.degree. C. for
several days, and then preserved in a liquid nitrogen tank. The
cell concentration at the culture was 1.9.times.10.sup.6/ml, and
the preserved cell number was 5.1.times.10.sup.7/tube.
(10)<Taking Out and Revival of Preserved CD4 Positive
Cells>
[0148] One of the tube containing CD4+ cells cryopreserved in the
above item (9) was taken out and then warmed up by a heat block at
37.degree. C. for 4 minutes. Ten ml of washing medium (RPMI1640+6)
was poured into a 15 ml centriigation tube aseptically, and then
the preserved lymphocyte were put into the medium with a transfer
pipette. Furthermore, after centrifugation at 1,000 rpm at
20.degree. C. for 5 minutes, the supernatant of it was discarded
and tapped lightly, then the cell pellet was suspended to 50 ml of
culture medium, and then the resulting cell suspension was
transferred to a cloning plate.
[0149] After observing the cells by a microscope, the plate was
incubated in the CO.sub.2 incubator at 37.degree. C. and the
cultivation started again (Cultivation 0 day). On the second day of
the cultivation, the vigorous growths of the cells all over the
plate were confirmed under the microscope and then the cells in the
cloning plate were transferred to a flask (225 cm, CELL
CULTUREFLASK). Furthermore, after washing the cloning plate with 50
ml of fresh culture medium to recover the remaining cells, the
cells were transferred to a flask and incubated in the CO.sub.2
incubator at 37.degree. C. to continue the cultivation.
[0150] On the fourth day of the cultivation, the OKT3 solution in
the OKT3 solidified flask (large) produced similarly to the above
item (5) was aspirated by suction. Fifty ml of PBS (-) was poured
into the flask, then the flask was thoroughly agitated with a cap
of the flask closed, the cap was then opened and the solution was
discarded. Next, fifty ml of PBS (-) was poured into the flask
aseptically, then the flask was thoroughly agitated with a cap of
the flask closed, the cap was then opened and the solution was
discarded by decanting. The liquid remaining inside the flask and
on the cap was aspirated thoroughly by suction.
[0151] The cells adhere to the bottom of the used flask (middle)
were detached by tapping the flask several times, and the cell
suspension was transferred to the new flask (large). Furthermore,
150 ml of fresh culture medium was added to the used flask (middle)
to suspend the remaining cells, and then the resulting suspension
was transferred to a new flask (large). The new flask (large) was
incubated put in the CO.sub.2 incubator at 37.degree. C. to
continue the cultivation.
(11)<Cultivation of CD4 Positive Cells in a Bag for
Cultivation>
[0152] On the sixth day of the cultivation, the cells adhered to
the bottom of the flask used in the cultivation in the above item
(10) were detached by tapping the flask several times, 1 ml of the
culture medium in the flask was taken out and was spread onto a
sabouraud dextrose (with chloramphenicol) agar plate and a sheep
blood agar plate equally, and the plates were incubated in the
CO.sub.2 incubator at 37.degree. C. to carry out an sterility
testing.
[0153] Ten .mu.l of the culture medium in the flask was taken out
to count the cell number similarly to the above item (7). A culture
bag A-1000 (a bag for cell cultivation) containing 750 ml medium in
which is 1:1 mixture of AIM-V medium prepared for bag cultivation
[which is that IL-2, human serum and oxaloacetic acid are added to
the medium of 10 L so that the final concentrations of them become
200 U/ml, 1% and 1 mM, respectively] and CP-2 (LL-7.1) medium (1 L)
was warmed up at 37.degree. C. The bag and a 50 ml syringe were
connected each other aseptically and the whole of the culture
medium in the flask was poured into the bag with the syringe. A
tube from the bag was clamped by a forceps, an end of the tube was
sealed, and the bag was put in the CO.sub.2 incubator for
cultivation at 37.degree. C. to continue the cultivation.
(12)<Sterility Testing and Endotoxin Assay of CD4 Cell>
[0154] On the Seventh day of the cultivation (the day before
administration), about 1 ml of the culture medium was sampled from
a sampling port of the culture bag cultivated in the above item (7)
by a 1 ml syringe with 24G injection needle aseptically. The first
solution was discarded, about 1 ml of the solution was sampled by
the same syringe again, about 200 .mu.l of the solution was plated
onto the sheep blood agar plate and the rest of the solution was
put into a dry heat sterilized conical test tube.
[0155] After cleaning the sampling port of the bag after sampling
with an alcohol cotton swab clamped by a forceps, the bag was put
in the CO.sub.2 incubator for cultivation at 37.degree. C. to
continue the cultivation. The agar plate was put in the CO.sub.2
incubator at 37.degree. C. for cultivation to carry out an
sterility testing, and the dry heat sterilized conical test tube
was centrifuged at 1,500 rpm at 20.degree. for 5 minutes.
[0156] One hundred .mu.l of prepared main solution was put into
three dry heat sterilized test tubes with dry heat sterilized
disposable capillary tip installed micro pipette, respectively. 100
.mu.l of water for injection is put into the test tube as negative
control and 100 .mu.l of standard solution was put into the test
tube as positive control with dry heat sterilized disposable
capillary tip installed micro pipette, respectively. 80 .mu.l of
water for injection and 20 .mu.l of the centrifuged culture
supernatant were put into the test tube for sample with toxipett
sampler. The three tubes were incubated in a water bath incubator
at 37.degree. C. for 30 minutes.
[0157] 30 minutes later, 400 .mu.l of 0.8M acetic acid solution was
added to each of three test tubes, the test tubes were stirred with
vortex mixer, and then 400 .mu.l of the reaction solution of each
test tube was transferred to 96 wells plate to measure values in
absorbance 405 nm (reference 630 nm) with a microplate reader. It
was confirmed that five times as much as the value of the
absorbance of the sample did not exceed to a value of the positive
control.
(13)<FACS Analysis of CD4+ Cell for Administration Before
Harvest Collection>
[0158] Before harvesting the CD4+ cells, a part of the culture
medium cultivated in the bag was sampled, the cell number was
measured by a method similar to the above item (7) and the content
of CD8+ cell was measured by a method similar to the item (4).
Consequently, the total cell number was 4.0.times.10.sup.9
including 0.8% of the CD8 cells. Due to the total cell number and
the ratio that the FACS analysis CD8+ cell count by percentage,
there were 2.0.times.10.sup.6 CD8+ cells.
(14)<Harvesting of CD4+ Cells>
[0159] 1000 ml of the culture medium cultivated in the bag in the
above item (12) was transferred in 250 ml centrifugation tubes
aseptically, and the tubes were centrifuged at 1,500 rpm at
20.degree. C. for 8 minutes. After the centrifugation, the
supernatant was discarded, the cell pellet was loosened with vortex
mixer, 250 ml of the cell washing solution was put into the
centrifugation tube, and then it was centrifuged at 1,800 rpm at
20.degree. C. for 8 minutes. After the centrifugation, the
supernatant of it was discarded and the cell precipitate was
loosened by vortexing. 100 ml of the cell washing solution was put
into the sedimentation tube, each of the cell suspension was
transferred into two 250 ml tubes, and then they were centrifuged
at 1,800 rpm at 20.degree. C. for 8 minutes. After the
centrifugation, the supernatant was discarded, the cell precipitate
was loosened by vortexing, and then 50 ml of the cell washing
solution was put into the two 250 ml tubes respectively.
[0160] After the centrifugation, the supernatant was discarded and
the cell pellet was loosened well with vortex mixer. The cells were
suspended in a final solution (a solution for administering
lymphocyte was made by putting 100 ml of saline and 5 ml of 20%
human serum albumin solution into a 250 ml centrifugation tube to
be a final solution) sufficiently, it was passed through a filter
with 100 micro-mesh and then fluid volume of the cell suspension
was measured.
[0161] Besides, the cell suspension was sampled in order to count
the cell number and the content of CD4. 50 ml syringe was connected
with a separation bag (300 ml) to pour the cell suspension through
the syringe into the separation bag. When the pouring was
completed, liquid remained in the syringe was pushed out by an
inner cylinder, and finally the tube was clamped by a forceps. The
tube was sealed and syringe was cut off by scissors. As a result of
the measurement, the liquid volume was 100 ml and the total cell
number was 3.7.times.10.sup.9.
(15)<FACS Analysis of CD4+ Cell for Administration after
Harvest>
[0162] FACS analysis of CD4+ cell for administration after harvest
was performed by a method similar to the above item (4) of the
embodiment No. 1. Consequently, the content by percentage of CD4+
cells was 99.75% and that of CD8+ cells was 0.25%.
(16)<Administering CD4+ Cells to a Patient>
[0163] CD4+ cells prepared in the above item (14) were administered
intravenously to the patient at ratio of 3.times.10.sup.7/Kg body
weight over one hour. Administration after that was carried out
three times a week. One dose was 3.times.10.sup.6 to
9.times.10.sup.7/Kg body weight and was administered intravenously
to the patient during one hour.
Embodiment No. 3
(1)<Measuring Immunogloblin Level in Blood>
[0164] As a result of measuring the level of immunogloblin in a
patient's peripheral blood, the immunogloblin level was increased
from the early stage of clinical course. Besides, relapse was
recognized in a patient in two months after the transplantation. In
this case, as the blast cells became the majority of blood cells, a
number of lymphocytes was decreased, but the immunogloblin level
remained within a normal range, so that immunoglobulin replacement
therapy was not necessary.
[0165] In spite of decreasing neutrophils or lymphocytes
remarkably, the patient did not suffered with serious infectious
disease. Thus, it was found that ability of immune response of the
patient was increased remarkably by administering the CD4+ cells.
Besides, according to production of antibodies from early stage
after transplantation in umbilical cord blood transplanted patient,
it is clear that donor derived activated lymphocyte promoted
survival of hematopoietic cells and promoted antibody
production.
* * * * *